GENETICALLY ENGINEERED C1-UTILIZING MICROORGANISMS AND PROCESSES FOR THEIR PRODUCTION AND USE
20180087024 ยท 2018-03-29
Assignee
Inventors
- Martin G. Lamarche (Laval, CA)
- Jonathan Perreault (Montreal, CA)
- Carlos Miguez (Beaconsfield, CA)
- Melanie Arbour (Ste-Marthe-sur-le-Lac, CA)
- Young Jun Choi (Pointe-Claire, CA)
Cpc classification
C12N1/32
CHEMISTRY; METALLURGY
C12P7/46
CHEMISTRY; METALLURGY
C12Y602/01005
CHEMISTRY; METALLURGY
C12Y402/01011
CHEMISTRY; METALLURGY
C12N9/1029
CHEMISTRY; METALLURGY
C12Y203/01
CHEMISTRY; METALLURGY
C12Y103/05001
CHEMISTRY; METALLURGY
International classification
C12N1/32
CHEMISTRY; METALLURGY
Abstract
Described are genetically engineered C1-utilizing bacteria for the preparation of dicarboxylic acids, e.g. succinic acid. For instance, the bacteria comprise a mutation in a gene encoding a tricarboxylic acid cycle (TCA) succinate dehydrogenase (Sdh), preferably a mutation which inactivates or reduces Sdh's activity. Processes for the production of the modified bacteria as well as their use in the preparation of succinic acid on a C1-compound as carbon source are also discussed.
Claims
1.-50. (canceled)
51. A method for producing a succinate-containing fermentation broth, said method comprising growing a genetically engineered serine cycle methylotroph or methanotroph in the presence of one or more C1-compound(s) under conditions enabling the production of succinate, wherein the methylotroph or methanotroph is genetically modified to disrupt a gene encoding a tricarboxylic acid (TCA) cycle succinate dehydrogenase or a subunit thereof, to overexpress a TCA cycle succinyl-CoA synthetase, and to disrupt poly--hydroxybutyric acid (PHB) biosynthesis in the methylotroph or methanotroph.
52. The method of claim 51, wherein the disruption of PHB biosynthesis comprises disruption of one or more enzymes in the Ethyl-Malonyl-CoA (EMC) pathway.
53. A genetically engineered C1-utilizing microorganism that produces succinate from a C1-carbon source, the microorganism being genetically modified to disrupt a gene encoding a tricarboxylic acid (TCA) cycle succinate dehydrogenase (Sdh) or a subunit thereof.
54. The microorganism of claim 53, wherein said microorganism is: (i) a serine cycle methylotroph from the genera Burkholderia, Fulvimarina, Granulibacter, Hyphomicrobium, Methylibium, Methylobacterium, Ruegeria, or Methylobacterium; (ii) a serine cycle methylotroph not mentioned in (i); (iii) a serine cycle methanotroph from the genera Methanomonas, Methylocystis, Methylocapsa, Methylocella, Methylococcus, Methylosinus, or Methylosinus; or (iv) a serine cycle methanotroph not mentioned in (iii).
55. The microorganism of claim 53, which is genetically modified: (a) by the knockout, knockdown or deletion of an sdh gene; (b) to inhibit, reduce or eliminate the activity of a protein involved in polyhydroxyalkanoate or poly--hydroxybutyric acid (PHB) biosynthesis and/or granule homeostasis; (c) to overexpress a TCA cycle succinyl-CoA synthetase; (d) to express or overexpress a isocitrate lyase and/or a protein involved in isocitrate synthesis; (e) to inhibit, reduce, or eliminate the activity of a protein involved in the Ethyl-Malonyl-CoA (EMC) pathway; or (f) any combination of (a) to (e).
56. The microorganism of claim 55, wherein: (a) the sdh gene is an sdhA gene; (b) the protein involved in polyhydroxyalkanoate or PHB biosynthesis and/or granule homeostasis comprises a Granule-Associated Protein (GAP), a phasin, a PHB synthase, Gap11, Gap 20, PhaC, or PhaR; (c) the succinyl-CoA synthetase comprises SucC and/or SucD; (d) the protein involved in isocitrate synthesis comprises a citrate synthase, an aconitase, or both a citrate synthase and an aconitase; or (e) the protein involved in the EMC pathway comprises: a protein that catalyzes the synthesis of acetoacetyl-CoA from acetyl-CoA, a protein that catalyzes the synthesis of hydoxybutyryl-CoA (OHB-CoA) from acetoacetyl-CoA, a beta-ketothiolase, an acetoacetyl-CoA reductase, an NADPH-linked acetoacetyl-CoA reductase, or any combination thereof.
57. The microorganism of claim 56, wherein: (d) said citrate synthase is gltA and/or said aconitase is acnA; or (e) said beta-ketothiolase is PhaA and/or said acetoacetyl-CoA reductase is PhaB.
58. The microorganism of claim 53, further comprising one or more of the following: (1) overexpression of one or more serine-cycle enzymes through modifications to their corresponding genes; (2) heterologous expression of one or more genes involved in succinic acid production; (3) incorporation of genetic switch(es); (4) modifications allowing accumulated PHB carbon to be made available for succinic acid production; and (5) inhibition/inactivation of one or more gene(s) encoding succinate dehydrogenase paralogues and/or orthologues.
59. The microorganism of claim 58, wherein: (1) the serine-cycle enzymes comprise a hydroxymethyltransferase, an enolase, and/or a malate dehydrogenase; (2) the one or more genes involved in succinic acid production encode a pyruvate carboxylase, a phosphoenol pyruvate carboxylase, and/or an isoctrate lyase; (3) the genetic switch(es) comprise sRNAs-, cumate-, CymR- and/or cTA-dependent genetic switch(es); (4) the modifications allowing accumulated PHB carbon to be made available for succinic acid production comprise genes encoding PHB depolymerases and/or recycling enzymes; or (5) the succinate dehydrogenase paralogues and/or orthologues comprise L-aspartate oxidase and/or a succinate dehydrogenase flavoprotein subunit.
60. A method for preparing succinic acid or a salt thereof, said method comprising growing the microorganism as defined claim 53 in the presence of one or more C1-compound(s) under conditions enabling the production of succinic acid or a salt thereof.
61. The method of claim 60, wherein said C1-compound comprises methane or methanol.
62. The method of claim 60, further comprising supplementation with malic acid or a salt thereof during cultivation.
63. The method of claim 60, wherein the microorganism is grown without additional supplementation with malic acid or a salt thereof during cultivation, other than malic acid added initially to the culture media.
Description
DESCRIPTION OF THE DRAWINGS
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SEQUENCE LISTING
[0088] The present application includes a sequence listing which lists the following sequences:
TABLE-US-00001 SEQ ID NO: Description 1 pCHOI2 vector nucleic acid sequence 2 Nucleic acid sequence of the sdh operon of Methylobacterium extorquens ATCC 55366, which includes: the sdhC gene from residues 942 to 1343; the sdhD gene from residues 1352 to 1771; the sdhA gene from residues 1801 to 3618; and the sdhB gene from residues 4280 to 5104 3 Nucleic acid sequence comprising the gap20 gene from Methylobacterium extorquens ATCC 55366, which includes coding residues 501 to 941 4 Nucleic acid sequence including the sucC (residues 1 to 1197) and sucD (residues 1206 to 2093) genes from Methylobacterium extorquens ATCC 55366 5 the sdhA-up-F primer used in Example 1.1. 6 the sdhA-up-R primer used in Example 1.1. 7 the sdhA-down-F primer used in Example 1.1. 8 the sdhA-down-R primer used in Example 1.1. 9 the 5-Forward primer of Example 1.3. 10 the 5-Reverse primer of Example 1.3. 11 the sucC-BamHI-F primer of Example 1.4 12 the sucD-Kpn1-R primer (Example 1.4). 13 the glmS-F primer of Example 1.4. 14 the dhaT-R primer of Example 1.4. 15 Eno-BamHI-F primer (Example 1.3) 16 Eno-Nhel-R primer (Example 1.3) 17 upPhaC-F primer (Example 1.5) 18 downPhaC-R primer (Example 1.5) 19 upPhaC-R primer (Example 1.5) 20 downPhaC-F primer (Example 1.5) 21 loxP-BamHI-F primer (Example 1.5) 22 loxP-BamHI-R primer (Example 1.5)
DETAILED DESCRIPTION
[0089] All technical and scientific terms used herein have the same meaning as commonly understood by one ordinary skilled in the art to which the invention pertains. For convenience, the meaning of certain terms and phrases used herein are provided below.
[0090] To the extent the definitions of terms in the publications, patents, and patent applications incorporated herein by reference are contrary to the definitions set forth in this specification, the definitions in this specification control. The section headings used herein are for organizational purposes only, and are not to be construed as limiting the subject matter disclosed.
[0091] The term succinic acid as used herein defines, 1,4-butanedioic acid, including its free acid or anionic forms like succinate salts.
[0092] The terms C1, C1-compound, C1-carbon source and equivalent expressions designate a molecule containing one carbon atom or containing two or more 1-carbon groups (e.g. methyl) not directly linked to each other. Examples of C1-compounds include, without limitation, methane, methanol, formaldehyde, formic acid, carbon monoxide, carbon dioxide, dimethyl ether, methyl formate, methylamine, dimethylamine, trimethylamine, and the like.
C1-Utilizing Microorganisms
[0093] In some aspects, the present description relates to a C1-utilizing microorganism. More specifically, the present description relates to a C1-utilizing microorganism which is capable of accumulating a dicarboxylic acid (e.g., succinic acid) when growing on a C1-compound as a carbon source.
[0094] The expression C1-utilizing microorganism or similar expressions, as used herein, designates a microorganism like a bacteria or yeast, which assimilates and/or dissimilates C1-compounds as above-defined, and/or uses C1-compounds as carbon sources. These include, for example, methylotroph and methanotroph microorganisms.
[0095] In some embodiments, the C1-utilizing microorganism may be a methylotroph or a methanotroph. As used herein, the term methylotroph defines a group of microorganisms that can use C1-compounds, such as methanol, as the carbon source for their growth. Examples of methylotrophs include, without limitation, bacteria within the genera Burkholderia, Fulvimarina, Granulibacter, Hyphomicrobium, Methylibium, Methylobacterium, Ruegeria. In contrast, the terms methanotroph or methanophile define a group of microorganisms able to metabolize methane as their source of carbon. Methanotrophs include type I methanotrophs which use the ribulose monophosphate (RuMP) pathway, and type II methanotrophs which use the serine pathway for carbon assimilation. Examples of type I methanotrophs include, without limitation, bacteria within the genera Methylobacillus, Methylobacter, Methylococcus, Methylomonas, Methylophaga, Methylotenera, Methylophilales. Examples of type II methanotrophs include, without limitation, bacteria within the genera Methanomonas, Methylocapsa, Methylocella, Methylocystis and Methylosinus.
[0096] In some embodiments, the C1-utilizing microorganism may be a serine-cycle C1-utilizing microorganism. Serine cycle methylotrophs have the ability to consume methanol for their growth, and can therefore convert methanol to succinic acid through their one-carbon metabolism and tricarboxylic acid (TCA) cycles. Although the methanol assimilation pathway of a serine-cycle methylotrophic bacteria is illustrated in
[0097] As an example, Methylosinus trichosporium, a serine cycle methanotroph has been intensively studied.sup.7 for its capacity to use methane as the sole source of carbon and energy, and could be modified as herein described and used to produce succinate from methane. Furthermore, this bacterium has also been recently used as a biocatalyst for the oxidation of methane to methanol.sup.8.
[0098] Methylobacterium extorquens is also a suitable C1-utilizing model in the present bioprocess to produce succinic acid. M. extorquens is a pink pigmented, non-pathogenic, Gram-negative serine-cycle methylotroph bacterium ubiquitous in the environment, and particularly associated with plants.sup.4,5. M. extorquens can also be grown to very high cell densities using a controlled methanol supplied bioprocess.sup.23.
[0099] M. extorquens' genes involved in methanol dissimilation and assimilation have been extensively studied since the 1960s.sup.6,9,10-21. The dissimilation of methanol begins in the periplasm by its oxidation, forming formaldehyde (see
[0100] Condensation of methylene-H.sub.4F with glycine and water produces serine, thereby beginning the serine cycle. Acetyl-CoA supplied by the serine cycle is a branching point molecule with the Ethyl-Malonyl-CoA (EMC) pathway and poly--hydroxybutyrate (PHB) cycles. As depicted in
[0101] The EMC pathway also shares its two first steps with the PHB cyclei.e., the successive synthesis of acetoacetyl-CoA and hydoxybutyryl-CoA (OHB-CoA) from acetyl-CoA, achieved by PhaA, a -ketothiolase, and PhaB, a NADPH-linked acetoacetyl-CoA reductase, respectively. The final step of PHB synthesis is performed by the PHB synthase PhaC. The genes depA, depB, hbd and atoAD are responsible for its depolymerisation into acetoacetyl-CoA. PHB belongs to the polyester family of polyhydroxyalkanoate (PHA) and is synthesized by M. extorquens and some other bacteria during nutrient and oxygen limitation.sup.22.
[0102] PHB producing bacteria such as M. extorquens accumulate PHB in their cytoplasm as granules which can account easily for 40% of the dry biomass.sup.23-25. Moreover, Granule-Associated Proteins (GAP) are important players of PHB granules homeostasis. Among GAPs, phasins are implicated in the regulation of granule size, stability, localization, number, and their segregation during cell division.sup.22,26,27. Although their mechanisms of action are not fully understood, it has been shown that some phasins bind PHB synthases and depolymerases.sup.28-30. Other regulators such as PhaR, which controls acetyl-CoA flux and PHB synthesis, could also be associated to phasins in M. extorquens.sup.25,31. For instance, at least two phasins have been identified in M. extorquens: Gap11 and Gap20.sup.24,25,32.
[0103] Examples of challenges faced when producing succinic acid in C1-utilizing serine-cycle microorganisms, include the following: (i) the genes from the TCA cycle are poorly expressed during growth on methanol; (ii) an inactivating mutation within the TCA cycle was found lethal to the bacteria when grown on methanol as the sole carbon source; and (iii) PHB accumulated during growth on methanol. For instance, the bacterial strains and/or methods herein described were found to solve one or more of these issues as explained in more detail below.
[0104] (i) M. extorquens can use simultaneously both methanol and succinic acid for growth but the latter is preferred and more rapidly consumed than methanol.sup.14. Consequently, methanol may not be assimilated efficiently in sdh null mutants or sdh knockdown backgrounds, considering regulatory effects of succinic acid accumulation on TCA and EMC gene expression. Indeed, genes belonging to the TCA are poorly expressed during methylotrophic growth, with a noticeably weak aconitase (Acn) activity, reducing the oxidative TCA flux from citrate. Thus, in contrast to what is observed during growth on succinate, the TCA cycle is expressed at a weak basal level while the EMC is up-regulated during growth on methanol, thereby favoring methanol assimilation.sup.16. Similarly, feedback inhibition could also occur, thus down-regulating genes needed for succinic acid production. Nevertheless, as presented in Example 3.1, the inactivation of an sdh gene was sufficient to allow succinic acid accumulation in this bacterium when grown on a C1-compound.
[0105] (ii) Some bacterial species, such as Escherichia coli, can produce succinic acid as an electron sink, in rich media, when shifting from aerobic to anaerobic conditions.sup.33. However, as M. extorquens is a strictly aerobic microbe. As such, one way of enhancing succinic acid production would be through metabolic engineering in the TCA cycle, for instance, by blocking the enzymatic conversion of succinate to fumarate. Unfortunately, an inactivating mutation within the succinate dehydrogenase operon sdhCDAhB, responsible for this step, is lethal when grown on methanol alone because it interrupts the TCA and thus, glyoxylate regeneration achieved by the EMC.
[0106] The TCA enzymes succinyl-CoA synthetase SucCD, succinate dehydrogenase SdhCDAB, and fumarate dehydrogenase FumC, complete the EMC flux.sup.10 and this allows for the formation of two molecules of glyoxylate per round of EMC and serine cycles. The TCA cycle supplements the serine cycle with malate, which is also essential for central metabolism. However, as shown in Example 3, succinic acid accumulation is possible with the sdh operon mutants if the growth media is supplemented with malate, which complements the incomplete TCA cycle.
[0107] (iii) M. extorquens accumulates PHB during growth on methanol and growth to high density obviously creates a nutrient limited environment also in favor of PHB synthesis.sup.23-25. As described in Example 4, succinic acid production by M. extorquens, using methanol as the source of carbon and energy, is further improved by modulating PHB reserves to promote succinic acid accumulation. In fact, the sdhA gap20 double mutant produced 4.76 fold less PHB than the sdhA mutant.
Genetic Modifications
[0108] While naturally-occurring C1-utilizing microorganisms have the ability to produce succinic acid as a TCA cycle metabolite, they generally do not accumulate significant amounts of succinic acid when grown on methanol. In fact, no accumulation of succinic acid was detected when the wild-type strain of the methylotrophic bacterium M. extorquens was cultured using methanol as the carbon source (Example 3.1). Accordingly, in some aspects, the present description relates to a C1-utilizing bacterium that has been genetically engineered to accumulate succinic acid (e.g., via the oxidative TCA pathway).
[0109] As used herein, the expression modified, genetically modified, genetically engineered or similar expressions associated with term microorganism or bacterium, refer to a microorganism or bacterium whose genome has been modified, for instance, by the addition, substitution and/or deletion of genetic material. Methods for modifying organisms are known and include, without limitation, random mutagenesis, point mutations, including insertions, deletions and substitutions, knockouts, transformations using recombinant nucleic acid sequences, including both stable and transient transformants.
[0110] Accordingly, in some aspects, the present description relates to a genetically engineered C1-utilizing bacterium that has been modified to disrupt a gene encoding a TCA cycle succinate dehydrogenase (Sdh) or a subunit thereof, thereby accumulating succinic acid from the oxidative TCA pathway. In some embodiments, the gene encoding the TCA cycle succinate dehydrogenase may be sdhA, sdhB, sdhC, sdhD, or any combination thereof.
[0111] As used herein, the expression gene disruption and equivalent expressions designate a genetic alteration that renders the encoded gene product inactive. The genetic alteration can be, for example, deletion of the entire gene, deletion of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product, or by any other mutation which inactivates the encoded gene product, for example via knockout or knockdown of the gene, or via one or more amino acid substitutions or deletions at residues critical for activity of the encoded protein. In some embodiments, where one or more genes are to be disrupted in accordance with the present description, one or more small RNAs (sRNAs) may be used to knockdown their expression. In some embodiments, a modified CRISPR system may also be used in a very similar way, for example using a catalytically inactive CRISPR endonuclease (e.g., a catalytically inactive Cas9).
[0112] In addition to the disruption of an sdh gene, the genetically engineered C1-utilizing microorganisms of the present description may be further modified, for example, to improve one or more of the following aspects: increasing succinic acid production, reducing PHB production or rendering PHB available as a carbon source for succinic acid production, and/or decreasing the need for malate supplementation.
[0113] PHB formation/accumulation can be reduced, for example, by blocking or reducing PHB synthesis directly, or by over-expressing PHB depolymerases. For instance, phasins are GAPs (granules-associated proteins) implicated in the regulation of granule size, stability, localization, number and their segregation during cell division. As such, inactivation (e.g., by gene deletion, knockout or knockdown) of one or more phasins such as gap11 or gap20 in M. extorquens, reduces PHB production during growth on methanol.
[0114] Other proteins involved in the PHB pathway could also be modulated. For instance, an sdhA phaC double mutant, that produces no PHB, but grows normally on methanol, could be obtained. On the other hand, modifications within the PHB pathway could also allow biomass accumulated in the form of PHB to be converted to succinic acid. For instance, this could be achieved by cloning genes encoding PHB depolymerases and recycling enzymes, alone or in combination, under an inducible promoter (see also Example 8).
[0115] Accordingly, in some embodiments, the genetically engineered C1-utilizing microorganism may further be modified to inhibit, reduce or eliminate the activity of a protein such as Granule-Associated Protein (GAP), a phasin, a PHB synthase, Gap11, Gap 20, PhaC, PhaR, or any combination thereof.
[0116] In some embodiments, the genetically engineered C1-utilizing microorganism may further be modified to overexpress PHB depolymerases and/or PHB recycling enzymes. In some embodiments, the genetically engineered C1-utilizing microorganism may further be modified to overexpress the gene depA, depB, hbd, atoAD, or any combination thereof, which are responsible for PHB depolymerisation into aceto-acetyl-CoA.
[0117] As used herein, the term overexpression and equivalent terms indicate that a particular gene product is produced at higher levels in a modified microorganism compared to its unmodified version. For example, a microorganism that includes a recombinant nucleic acid configured to overexpress an enzyme produces the enzyme at a greater amount than a microorganism that does not include the recombinant nucleic acid. The term overexpression when associated with a gene means an increased expression of such gene in a modified microorganism compared to its unmodified version. Gene overexpression, for instance, also results in the overexpression of its encoded gene product. Overexpression may be done by any means known in the art, such as by integration of additional copies of the target gene in the cell's genome, expression of the gene from an episomal expression vector, introduction of an episomal expression vector which comprises multiple copies of the gene, or by the use of a promoter heterologous to the coding sequence to which it is operably linked, i.e. the sequence coding for the gene product to be overexpressed.
[0118] Enzymes upstream of the Sdh protein in the TCA cycle may also be overexpressed through genetic modifications in order to improve succinic acid production and/or reduce the need for malate supplementation, preferably an enzyme common to both the TCA cycle and EMC pathway, e.g., overexpression of a succinyl-CoA synthetase.
[0119] Accordingly, in some embodiments, the genetically engineered C1-utilizing microorganism may further be modified to overexpress of a succinyl-CoA synthetase (e.g., a TCA cycle succinyl-CoA synthetase). In some embodiments, the succinyl-CoA synthetase may be SucC and/or SucD. In some embodiments, the succinyl-CoA synthetase may be inserted into the genome of the C1-utilizing microorganism (e.g., using a strong promoter such as the mxaF promoter).
[0120] Based on transcriptomic analysis, some genes belonging to the methanol dissimilation/assimilation pathway were found to be up-regulated (mtdA, fch and most serine cycle genes) in the sdhA mutant model. Without wishing to be bound by theory, it can be deduced that the sdhA mutation acts in synergy with methanol and further increases expression of methanol assimilation genes.
[0121] The transcriptomic analysis showed that gck and mtk expression was up-regulated, whereas eno and mdh genes were not differentially expressed, when comparing the sdhA mutant to the wild-type ATCC55366 strain (see Example 3.2). Overexpression of proteins encoded by the glyA (serine hydroxymethyltransferase), eno (enolase), and mdh (malate dehydrogenase enzyme) genes within the sdhA mutant is expected to promote the continuous flow of the serine cycle as well as the synthesis of acetyl-CoA.
[0122] Accordingly, in some embodiments, the genetically engineered C1-utilizing microorganism may further be modified to overexpress a serine hydroxymethyltransferase, an enolase, a malate dehydrogenase, or any combination thereof.
[0123] Some succinate dehydrogenase activity may still be present within the modified strain, e.g. through sdh paralogues and/or orthologues. If it would be the case, succinic acid accumulation would be slowed down and eventually consumption would overtake synthesis. As such, one or more genes encoding sdh paralogues and/or orthologues may also be inactivated. Thus, in some embodiments, the genetically engineered C1-utilizing microorganism may further be modified to disrupt sdh paralogues and/or orthologues. In some embodiments, the genetically engineered C1-utilizing microorganism may be further modified to disrupt an L-aspartate oxidase and/or a succinate dehydrogenase flavoprotein subunit.
[0124] In some embodiments, the genetically engineered C1-utilizing microorganism (e.g., an sdhA mutant) may also be complemented using genetic switches, such as described in Example 9. Such switches may be employed for example to eliminate the need for initial malate addition for growth on methanol to produce succinic acid, by controlling the expression of a TCA cycle succinate dehydrogenase (Sdh) or a subunit thereof (e.g., an sdh operon). Sdh proteins produced from such switches are expected to be exhausted later on during growth and succinic acid would then accumulate. In some embodiments, the genetic switch may be a cumate-dependent genetic switch. In some embodiments, the genetically engineered C1-utilizing microorganism may comprise one or more genetic switch(es) such as sRNAs-, cumate-, CymR- and/or cTA-dependent genetic switch(es).
[0125] In some embodiments, the genetically engineered C1-utilizing microorganism may also be further modified through heterologous gene expression. More specifically, in some embodiments, the genetically engineered C1-utilizing microorganism may be further modified to overexpress enzymes responsible for the conversion of pyruvate and PEP into OAA. In some embodiments, such enzymes may be a pyruvate carboxylase (e.g., encoded by the pyc gene) and/or a phosphoenolpyruvate (PEP) carboxylase (e.g., encoded by the ppc gene). The overexpression of such proteins has been shown to improve aerobic succinate production in some bacteria.sup.54,55. The increase of the OAA pool within M. extorquens cells is expected to provide more carbon input into the EMC, especially if the mdh gene is also functionally overexpressed.
[0126] In some embodiments, the above mentioned pyc gene may be from Rhodopseudomonas palustris BisA53, which is an environmental non-pathogenic bacteria belonging to the rhizobiale group of alphaproteobacteria.sup.56.
[0127] In some embodiments, the genetically engineered C1-utilizing microorganism may also be further modified to overexpress an enzyme that catalyzes the formation of glyoxylate and succinate from isocitrate (e.g., an isocitrate lyase).sup.57,58. Isocitrate lyase is a key enzyme of the glyoxylate regeneration pathway and is absent from the M. extorquens genome, which uses the EMC pathway. For example, isocitrate lyase may be used to increase the oxidative flux from citrate within the TCA, which may occur as succinic acid accumulates in a genetic switch complemented sdhA mutant. Heterologous overexpression of an isocitrate lyase within a genetically engineered C1-utilizing microorganism of the present description could also allow subsequent inactivation of the EMC pathway, which theoretically would result in a larger amount of carbon available for succinic acid production. This would involve introducing a heterologous glyoxylate shunt, as described in more detail in Example 13. Briefly, isocitrate produced by the TCA cycle can be converted by the heterologous isocitrate lyase to form glyoxylate and succinate, instead of the isocitrate being further decarboxylated (by isocitrate dehydrogenase). The glyoxylate can then be used together with acetyl-CoA to produce malate (e.g., by malate synthase), making the missing carbon to enter the central metabolism (and thus potentially reducing the need for malate).
[0128] Accordingly, in some embodiments, the genetically engineered C1-utilizing microorganism (e.g., expressing heterologous isocitrate lyase) may also be further modified to overexpress of a protein involved in isocitrate synthesis (e.g., a citrate synthase (e.g., gltA), an aconitase (e.g., acnA), or both a citrate synthase and an aconitase). In some embodiments, the genetically engineered C1-utilizing microorganism (e.g., expressing heterologous isocitrate lyase) may also be further modified to overexpress a malate synthase, and/or to disrupt a gene encoding an isocitrate dehydrogenase.
[0129] In some embodiments, the genetically engineered C1-utilizing microorganism (e.g., expressing heterologous isocitrate lyase) may also be further modified to inhibit, reduce, or eliminate the activity of a protein involved in the EMC pathway. In some embodiments, the protein involved in the EMC pathway may be: (a) a protein that catalyzes the synthesis of acetoacetyl-CoA from acetyl-CoA; (b) a protein that catalyzes the synthesis of hydoxybutyryl-CoA (OHB-CoA) from acetoacetyl-CoA; or (c) both (a) and (b). In some embodiments, the protein involved in the EMC pathway may be a beta-ketothiolase, an acetoacetyl-CoA reductase, an NADPH-linked acetoacetyl-CoA reductase, or any combination thereof. In some embodiments, (i) the beta-ketothiolase may be PhaA; (ii) the acetoacetyl-CoA reductase may be PhaB; or both (i) and (ii).
[0130] In some embodiments, where one or more genes are to be disrupted in accordance with the present description, one or more small RNAs (sRNAs) may be used to knockdown their expression, as described in Example 12. In some embodiments, a modified CRISPR system may also be used in a very similar way, for example using a catalytically inactive CRISPR endonuclease (e.g., a catalytically inactive Cas9).
Methods
[0131] In some aspects, the present description relates to a method for preparing succinic acid or a salt thereof. The method generally comprises growing a genetically engineered C1-utilizing microorganism as defined herein in the presence of one or more C1-compound(s). In some embodiments, the C1-compound may comprise methane and/or methanol.
[0132] In some embodiments, the method may comprise supplementing the culture with malic acid or a salt thereof. In some embodiments, the genetically engineered C1-utilizing microorganism may be grown without additional supplementation with malic acid or a salt thereof during cultivation, other than malic acid added initially to the culture media. In some embodiments, the genetically engineered C1-utilizing microorganism may be grown without the addition malate during culture, or may require less malate during culture (e.g., for genetically engineered C1-utilizing microorganisms comprising genetic switches to control TCA cycle metabolism, and/or for genetically engineered C1-utilizing microorganisms comprising an operative glyoxylate shunt pathway).
EXAMPLES
[0133] The following examples are for illustrative purposes and should not be construed as further limiting the invention as herein described. [0134] Bacterial strains, plasmids, media and cultures: Bacterial strains and plasmids are listed in Table 1. Escherichia coli strains were grown using Tryptic Soy Broth (TSB) and Agar (TSA). Methylobacterium extorquens was grown using the CHOI4 medium (see Table 2), which was developed to yield high cell density fermentation.sup.23. M. extorquens cultures were carried out using 250 or 3000 mL baffled Erlenmeyer flasks containing 35 or 300 mL of CHOI4 medium, respectively. Strains were grown at 30 C., 250 rpm and cultures were supplemented initially with 0.5% methanol and 0.5% every 24 h, unless indicated otherwise. Malic acid was added as indicated. Each time point corresponds to 24 h. Antibiotics were used at the following final concentrations: carbenicillin, 100 g/mL; kanamycin, 40 g/mL; tetracyclin, 10 g/mL; and streptomycin, 50 g/mL.
TABLE-US-00002 TABLE 1 Bacterial strains and plasmids Bacterial strain or plasmid Relevant characteristics Reference or source Escherichia coli strains SM10pir thi-1, leu, tonA, lacY, supE, recA::RP4-2-Tc::Mu, pir, Km.sup.r Miller and Mekalanos, 1988, J. Bacteriol 170:6, 2575-83 7213 SM10pir asd Reference 35 Methylobacterium extorquens strains ATCC55366 Wild-type Bourque et al, 1992, App Microbiol Biotechnol, 37:7-12 sdhA Polar mutation, marker less See Example 1.1 gap20 May also be referred to as gap20::145, 145 bp insertion (scar See Example 4 from Cre-Lox), Marker less sdhA gap20 Derived from sdhA and gap20 strains, marker less See Example 4 sdhA gap20 phaC Derived from the sdhA gap20 strain, phaC refers to the See Example 5 complete deletion of phaC together with the 5 end of its upstream gene, marker less, PHB negative sdhA gap20 Triple mutant described above before the Cre-Lox See Example 7 phaC::Km.sup.R recombination, Km.sup.r, PHB negative, with chromosomal Tn7::sucCD insertion of sucCD genes under the methanol dehydrogenase promoter P .sub.mxaF, Tet.sup.r sdhA gap20 phaC The marker less triple mutant described above overexpressing See Example 8 pCHOI2::eno (Km.sup.R) the eno gene onto a plasmid (see below) Plasmids pGEM-T easy TA cloning vector Promega Madison, WI, USA pCHOI2::sucCD pCM110 derived, P.sub.mxaF, Tet.sup.r Reference 36, and Example 1.4 pUC18T mini- FRT, Tet.sup.r Reference 39, and Tn7T::P.sub.mxaFsucCD Example 1.4 pCHOI2::eno (Km.sup.R) P.sub.mxaF, Tet.sup.r Examples 1.3 and 8 Km.sup.r: Kanamycin; Tet.sup.r: Tetracycline
TABLE-US-00003 TABLE 2 CHOI4 Medium for M. extorquens high cell density fermentation Chemicals Final concentration (mM) Solution (NH.sub.4).sub.2SO.sub.4 11.35 1 KH.sub.2PO.sub.4 9.57 Na.sub.2HPO.sub.4 15 MgSO.sub.4 5.48 Final concentration (M) Solution MnSO.sub.4 87 2 ZnSO.sub.4 27 CuSO.sub.4 10 Na.sub.2MoO.sub.4 10 CoCl.sub.2 10 H.sub.3BO.sub.3 29 FeSO.sub.4 216 CaCl.sub.2 408
Example 1
Construction of Mutant Strains and Vectors
[0135] 1.1Construction of the sdhA Mutant
[0136] In general terms, the sdhA gene is deleted using the pCM184 allelic exchange vector technology. This technology is described in
[0137] More specifically, Phusion High fidelity DNA polymerase (New England BioLabs, Inc., Ipswich, Mass., USA) was used for all DNA amplifications. All restriction enzymes used herein were from NEB as well. Linear fragments were circularized using the T4 DNA ligase from NEB. Genomic regions located upstream and downstream of the M. extorquens ATCC55366 sdhA gene were amplified using the following two primer pairs:
TABLE-US-00004 sdhA-up-F: (SEQIDNO:5) 5-GAATTCCTGATGCTCGCCTTCGTC-3/ sdhA-up-R: (SEQIDNO:6) 5-GCGGCCGCTGCTCGAGTTCGTAGAC-3, containingtheEcoRIandNotIrestrictionsites respectively(underlined); and sdhA-down-F: (SEQIDNO:7) 5-GGGCCCGTCGTGACCATGGAATC-3/ sdhA-down-R: (SEQIDNO:8) 5-GAGCTCGCTGCCGCGGTAGA-3, containingtheApaIandSacIrestrictionsites respectively.
[0138] Each fragment was cloned into the TA cloning vector pCRII (Life Technologies). The E. coli DH5 strain (Life Technologies) was used for propagation. Then, each fragment was excised from pCRII using the corresponding restriction enzymes and successively cloned into the allelic exchange vector pCM184.sup.34. The resulting pCM184::sdhA-loxP-Km-loxP-sdhA vector was mobilized into M. extorquens recipient strains using the asd Sm10pir strain 7213.sup.35. On-filter conjugation was allowed to occur during 16 h at 37 C. on Luria plates containing diaminopimelate (DAP). Filters were transferred onto CHOI4 agar plates and incubated at 30 C. for 24 hours.
[0139] Growing clones were then diluted in PBS and different volumes were spread out on CHOI4 agar plates containing kanamycin. The kanamycin marker was removed from the sdhA mutants using the cre-lox system.sup.34. Mutants were transformed with the Cre recombinase positive vector pCM157 and grown in CHOI4 medium containing tetracycline. Then, kanamycin negative clones were selected and further grown in CHOI4 medium without any antibiotic selective pressure, to promote the loss of pCM157. Kanamycin and tetracycline negative clones were screened by PCR for the marker less sdhA mutation, using the sdhA-up-F and sdhA-down-R primers. A positive clone was selected and the sdhA mutation was confirmed by sequencing.
1.2Construction of the sdhA gap20 Double Mutant
[0140] A 910 bp fragment containing gap20 and its flanking regions was amplified by PCR and cloned into the pCRII vector, giving pCRII::gap20. The gentamycin resistance marker (Gm) together with its loxP flanking sites was amplified from pCM351.sup.34 using primers containing either HincII or BpII restriction site. The resulting fragment was cut with HincII and BpII and cloned into pCRII::gap20 linearized using the same enzymes, giving pCRII:gap20Gm.sup.r. The gap20Gm.sup.r fragment was amplified by PCR and used to transform by electroporation the marker sdhA mutant strain from Example 1.1. Clones were selected on CHOI4 agar plates containing gentamycin. The gentamycin marker was removed from the sdhA gap20 double mutant using the cre-lox system as described above.sup.34.
1.3Construction of the pCHOI2 Vector and pCHOI2::eno
[0141] The pCHOI2 vector was constructed from the pCM110 vector.sup.36. Km resistance gene was amplified using the pNEW vector.sup.37 as a template with primers 5-Forward-CTGCAGATGATTGAACAAGATGG-3 (SEQ ID NO: 9) and 5-Reverse-CTGCAGTCAGAAGAACTCGTCAAGAA-3 (SEQ ID NO: 10), each containing the PstI restriction site in 5. PCR product was introduced into pCM110 digested with PstI and the positive colonies were selected on plates containing kanamycin. Then, tetA and tetR genes were removed by double digestion with AfeI and FspI. MCS from pSL1190.sup.38 (Genbank accession #U13866) was introduced into the blunt ended vector to complete pCHOI2.
[0142] The eno gene was amplified using the following primers:
TABLE-US-00005 Eno-BamHI-F: (SEQIDNO:15) AAAAAA-GGATCC-ATGACCGCGATCACCAATATC and Eno-NheI-R: (SEQIDNO:16) AAAAAA-GCTAGC-atgcttcaggtgcgaTCAGC,
giving a PCR fragment of 1305 bp. The fragment was cut with BamHI and NheI and cloned into pCHOI2 cut with the same enzymes and propagated in E. coli DH5. The resulting pCHOI2::eno was introduced in M. extorquens strains by electroporation using a Biorad apparatus (2.5 Kv, 200).
1.4Construction of pCHOI2::sucCD and Tn7::sucCD
[0143] The M. extorquens ATCC55366 sucCD genes were amplified using the primers sucC-BamHI-F: 5-GGATCCATGAACATCCACGAATACCA-3 (SEQ ID NO: 11) and sucD-Kpn1-R 5-GGTACCTCACCTGGACTTCAGCAC-3 (SEQ ID NO: 12). The resulting PCR fragment was cloned into the TA cloning vector pGEM-T easy (Promega) and propagated in E. coli DH5. The sucCD genes were then excised using BamIH and SacI and introduced downstream of the mxaF promoter (P.sub.mxaF), in the pCHOI2 vector linearized with the same enzymes. The resulting pCHOI2::sucCD was introduced in M. extorquens strains by electroporation using a Biorad apparatus (2.5 Kv, 200).
[0144] For chromosome insertion, a modified version of pUC18T-miniTn7T-Gm.sup.39, carrying a tetracycline marker within SacI of the MCS, was used. Briefly, the P.sub.mxaFsucCD fragment was excised from the pCHOI2::sucCD vector using the HindIII and KpnI restriction enzymes and introduced in the pUC18T-miniTn7T vector. Conjugation was performed as described above and clones were selected on CHOI4 agar plates containing tetracycline.
[0145] Insertion of the Tn7 into the glmS-dhaT integration site was confirmed by PCR using the glmS-F: 5-CGAGAAGACTGTCTCGAAC-3 (SEQ ID NO: 13) and dhaT-R: 5-CATCGCGATTGTCGATTCG-3 (SEQ ID NO: 14) primers. Integration occurs within a noncoding region of the chromosome, making the insert stable and silent in regard of the surrounding genes.sup.40. [0146] Antibiotic markers are removed from the different genetic constructs using Cre-Lox or flipase technologies, making the final M. extorquens engineered strain suitable for bioprocesses purpose.sup.34.
1.5Construction of the sdhA gap20 phaC Triple Mutant
[0147] A 3719 bp fragment containing phaC and its flanking regions was amplified by PCR using the following primers: upPhaC-F: 5-ATGTTGGCGAAGCCCTCCTTC-3 (SEQ ID NO: 17) and downPhaC-R: 5-GATTCGGCGAGCACCATTCC-3 (SEQ ID NO: 18). The resulting fragment was cloned into the pGEM-T easy vector (Promega), giving pGEM-T easy::phaC. Then, the phaC gene was deleted by performing an inverse PCR using the following BamHI containing primers: upPhaC-R: 5-GGATCCACACGTCCTCCCAAAGGT-3 (SEQ ID NO: 19) and downPhaC-F: 5-GGATCCTGAAGGTGTGAGGGATCG-3 (SEQ ID NO: 20); giving the linear pGEM-T easy::phac fragment.
[0148] A 1340 bp fragment containing a kanamycin resistance cassette flanked on both sides by the loxP recombination recognition sequence was amplified from pCM184 (Marx and Lidstrom, 2002) using the following BamHI containing primers: loxP-BamHI-F: 5-GGATCCGCATAACTTCGTATAGCATAC-3 (SEQ ID NO: 21) and loxP-BamHI-R: 5-GATAAGCTGGATCCATAACTTCG-3 (SEQ ID NO: 22); giving the loxP-Km.sup.R-loxP fragment. The pGEM-T easy::phaC fragment cut with BamHI was then resolved with the loxP-Km.sup.R-loxP fragment cut with the same enzyme. The phaC::Km.sup.R fragment was finally cloned into the suicide vector pCM433 (Marx, 2008). Conjugation was performed as described for the sdhA mutant, using the sdhA gap20 double mutant as recipient strain. Then, to select the double-crossover allele replacement, a kanamycin resistant clone was grown in CHOI4 medium without antibiotic for 3 days and spread out on Luria plates containing 7% sucrose. Kanamycin resistant and tetracycline sensitive clones were kept. The kanamycin marker was removed using the cre-lox system as described above. The phaC mutation was confirmed by PCR and sequencing, which also revealed an additional deletion of the 5 end of a small hypothetical gene, just upstream phaC.
Example 2
Analytical Methods
[0149] 2.1Poly--hydroxybutyrate (PHB) Analysis
[0150] PHB was quantified using the Braunegg, Sonnleitner and Lafferty method (1978) with slight modifications.sup.41-43. Briefly, each bacterial cell culture was centrifuged at 4 C., 4000 rpm for 20 minutes. Pellets were then washed once with ice-cold water, centrifuged and lyophilised. Dry cells were resuspended using a methanolysis solution (methanol, sulfuric acid 3% and methyl benzoate 16 mM as internal standard) to obtain 5 mg of dry cells/mL. Then, 2 mL were transferred into screw cap Pyrex glass tubes containing 2 mL of chloroform, vortexed briefly and incubated at 100 C. during 140 minutes, to allow the formation of methyl esters. During that time, tubes were vortexed occasionally. Tubes were chilled on ice and 1 mL of water was added to each reaction. Tubes were vortexed during 30 seconds and Bligh-Dyer phases were allowed to separate. The lower chloroform phases were withdrawn and PHB content was measured by gas chromatography.
2.2Organic Acids Quantification
[0151] Detection and quantification of succinic acid and other carboxylic acids from the TCA cycle were performed by HPLC-UV using ICSep ICE-ION-300 column (Transgenomic), a cation-exchange polymer in the hydrogen ionic form, at a temperature of 40 C. Acidified HPLC grade water (H.sub.2SO.sub.4; 0.008 N) was used as the mobile phase at a flow rate of 0.4 mL/min. The analytical method used is similar to previously described methods, with slight modifications.sup.44.
2.3Microarrays
[0152] For microarrays, M. extorquens ATCC55366 and the sdhA mutant were cultivated for 18-24 hours at 30 C., 250 rpm, in 50 mL of CHOI4 medium supplemented with 18.5 mM malate in the presence or absence of 0.5% (v/v) methanol. Samples were prepared as described previously.sup.45. Briefly, immediately after cultivation, culture aliquots equivalent to 10 OD were mixed with 1/10.sup.th the culture volume of cold stop solution (5% water saturated phenol, pH 7.0, 95% ethanol) and harvested at 4 C.
[0153] The cells were resuspended in 0.5 mL fresh lysosyme (3 mg/mL prepared in 10 mM Tris, 1 mM EDTA, pH 8.0) and 80 L of 10% SDS was added. The tubes were incubated at 64 C. for 5 minutes then 88 L 3M sodium acetate, pH 5.2 was added. Each tubes were supplemented with 800 L prewarmed phenol:chloroform (Ambion, Burlington, Ontario), mixed by inverting the tubes and incubated at 64 C. for 6 minutes.
[0154] After cooling the tubes on ice, the samples were centrifuged at 16,000g for 10 minutes at 4 C. to separate the phases. The aqueous phase was then mixed with the same volume of chloroform and centrifuged. The total RNA was finally precipitated with ethanol, resuspended in nuclease-free water, treated with DNase I and cleaned with RNeasy Plus Mini kit (Qiagen, Toronto, Ontario). The preparation of labeled cDNA and microarray hybridization were done exactly as described in Okubo, Y. et al (2007).sup.46.
[0155] Arrays were scanned using the ScanArray Express microarray analysis system (Perkin Elmer Life Sciences, Waltham, Mass.), and the data extracted using the ImaGene software (BioDiscovery Inc. Hawthorne, Calif.). Microarray data were normalized using the Lowess algorithm. Gene expression patterns were determined with GeneSpring visualization software version GX11 (Agilent Technologies, Santa Clara, Calif.). Gene expression levels were considered significant (p<0.05) when the fold change between strains and or conditions was more than two.
Example 3
The sdhA Mutant
[0156] 3.1Organic Acids and PHB in the sdhA Mutant
[0157] The M. extorquens wild-type strain ATCC55366 was tested and did not accumulate succinic acid when grown on methanol (data not shown). Thus, in order to achieve succinic acid build-up in cultures of M. extorquens, the sdhA gene was first knocked out as described in Example 1.1. As expected, the sdhA mutant did not grow on methanol as the sole source of carbon and energy. Nevertheless, it was capable of growing in the presence of malate which rescued the TCA cycle, thereby achieving succinic acid production.
[0158] When the sdhA mutant was fed only initially with 0.5% methanol, growth of the sdhA mutant ceased when malic acid was completely consumed which occurred rapidly (data not shown). However, supplementing the culture with methanol throughout the course of the experiment allowed for continued growth without the addition of further malic acid. Consequently, succinic acid production by sdhA mutant strain was measured while supplementing with methanol during the course of the experiment (
[0159] The level of competition between PHB synthesis and succinic acid production was also determined by quantifying PHB in the sdhA mutant and in the wild-type strain. As shown in
3.1.1sdhA Mutant Fed Only Initially with 0.5% Methanol
[0160] In order to learn more about malic acid consumption, the sdhA mutant was cultured for 7 days (168 h) as described above, except that 0.5% v/v methanol was added only initially without further supplementation. Cell optical density (600 nm), as well as the concentrations of succinic acid, malic acid, and methanol in the culture media were monitored over the course of the experiment.
[0161] As shown in
[0162] Malic acid was rapidly consumed during the first 24 hours, and was consumed more slowly afterwards. A total of 1.77 g/L of malic acid was consumed throughout the 7-day experiment, which is a concentration greater than the concentration of succinic acid that was synthesized (
3.1.2sdhA Mutant Cultured with Periodic Methanol Supplementation
3.1.2.1Succinic Acid Concentrations Produced by sdhA Mutant
[0163] The sdhA mutant was cultured for 5 days as described above in CHOI4 medium with 3 g/L (22.37 mM) of malate, while supplementing with methanol (0.5% v/v) throughout the course of the experiment. Cell optical density (600 nm), as well as the concentrations of succinic acid, malic acid, and methanol were monitored over the course of the experiment. Results are shown numerically in Table 3A and graphically in
TABLE-US-00006 TABLE 3A sdhA mutant: Cumulative data from succinic acid production kinetics Days (cumulative) Parameter 1 2 3 4 5 Optical density (600 nm) 0.54 2.76 3.98 5.92 6.94 Succinic acid yield (mg*L.sup.1*h.sup.1*ODu.sup.1) 2.13 2.56 3.74 3.18 2.94 Succinic acid yield (mg*L.sup.1*h.sup.1*ODu.sup.1*gMeOH.sup.1) 4.39 0.52 0.43 0.25 0.18 Succinic acid concentration (g/L) 0.03 0.34 1.07 1.81 2.45 Consumed Malic acid (g/L) 1.28 1.68 1.77 1.92 2.04 Consumed Methanol (g/L) 2.07 8.88 12.60 16.57 16.79 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
[0164] As shown in Table 3A, the sdhA mutant achieved a succinic acid concentration of 1.07 g/L (9.06 mM) at an OD of 3.98 (reached in 3 days). This point was chosen as an optical density reference to compare with subsequent experiments.
[0165] Malic acid was rapidly consumed from 3.11 g/L (23.2 mM) to 1.84 g/L (13.7 mM) in the first 24 hours, and was consumed more slowly afterwards, as observed when methanol was added only initially (Example 3.1.1). At the end of the 5-day experiment, 2.04 g/L (15.2 mM) of malic acid was consumed, which is slightly higher than the malic acid consumed without periodic methanol supplementation (1.67 g/L after 5 days; see Example 3.1.1 and
[0166] Consequently, when considering the concentration of consumed malic acid, it can be deduced that at least 0.7 g/L (5.6 mM) of succinic acid must have been synthesized from methanol. Interestingly, while the periodic additions of methanol significantly improved the growth of the sdhA mutant, malic acid consumption was not significantly affected in this experiment.
3.1.2.2Cumulative Yield of Succinic Acid Produced by the sdhA Mutant
[0167] At the reference optical density (4), the cumulative yield of succinic acid was 3.7 mg *L.sup.1*h.sup.1*ODu.sup.1 (Table 3A and
[0168] Because of technical limitations, methanol loss due to evaporation was not quantified. Nevertheless, making the hypothesis that evaporative methanol loss was low and constant between experiments, the specific succinic acid yield per gram of consumed methanol (gMeOH) was also calculated at each time point. It was estimated at 4.39 mg of succinic acid *L.sup.1*h.sup.1*ODu.sup.1*gMeOH.sup.1 after one day (Table 3A). Yields for the following two days were estimated at 0.52 mg and 0.43 mg of succinic acid *L.sup.1*h.sup.1*ODu.sup.1*gMeOH.sup.1, respectively. Then, for the remaining days, specific yields diminished below 0.25 mg of succinic acid *L.sup.1*h.sup.1*ODu.sup.1*gMeOH.sup.1.
3.1.2.3Yield for Each Period of 24 Hours Produced by sdhA Mutant
[0169] To determine whether the sdhA mutant cultures had gradually lost the capacity to produce succinic acid, as suggested by the above results, the amount of methanol consumed periodically was estimated and used to calculate the specific yield for each individual segment of 24 hours. Results are shown numerically in Table 3B and graphically in
TABLE-US-00007 TABLE 3B sdhA mutant: Data from succinic acid production kinetics for each period of 24 hours Days (period of 24 h) Parameter 1 2 3 4 5 Optical density ( 600 nm) 0.54 2.76 3.98 5.92 6.94 Succinic acid yield (mg*L.sup.1*h.sup.1*ODu.sup.1) 2.13 4.71 7.66 5.18 3.86 Succinic acid yield (mg*L.sup.1*h.sup.1*ODu.sup.1*gMeOH.sup.1) 4.39 1.06 2.06 1.30 0.93 Succinic acid concentration (g/L) 0.03 0.31 0.73 0.74 0.64 Consumed Malic acid (g/L) 1.28 0.40 0.09 0.15 0.12 Consumed Methanol (g/L) 0.49 4.45 3.72 3.97 4.16
[0170] The overall succinic acid yield was found to be relatively stable between periods, with only a slight decrease over time, when compared to that obtained from cumulative data (Table 3A vs Table 3B,
3.2sdhA Mutant Transcriptomic Analysis
[0171] Transcriptomic analyses were performed on the sdhA mutant strain using microarrays. Bacteria were grown in CHOI4 medium containing malate, or both malate and methanol as carbon and energy sources. Results of growth on malate confirmed the succinate dehydrogenase null phenotype of our mutant, as the sdhA and sdhB transcripts were barely detected when compared to that of the wild-type strain. In contrast, sdhC and sdhD genes were up-regulated in the mutant. Thus, in these conditions, the sdhA mutation had a polar effect on the downstream genes of the operon while having a positive feedback effect on its expression. This phenomenon was not observed when the strains were grown in media supplemented with methanol, due to the weak expression of the TCA cycle genes during methylotrophic growth.
[0172] The microarray analyses also revealed that an important nutrient stress response is induced by the inactivation of the succinate dehydrogenase. Importantly, chemotaxis and flagellar genes are modulated and this is known to occur because of the fumarate concentration fluctuation.sup.47-49. These microarray results are also in accordance with stimuli known to induce PHB polymerisation.sup.22,27.
[0173] When considering specifically the genes involved in methanol dissimilation and assimilation, up-regulated genes included the NADP-dependent methylene-tetrahydromethanopterin/methylene-tetrahydrofolate dehydrogenase MtdA, the methenyltetrahydrofolate cyclohydrolase Fch, and the subunit C of the formyltransferase/hydrolase complex Fhc.
[0174] Most serine cycle genes (e.g. gck, mtk) were also up-regulated, except glyA, eno and mdh, respectively encoding for serine hydroxymethyltransferase, enolase and malate dehydrogenase. The malate dehydrogenase mqo gene, however, was downregulated. HPLC tests showed that this phenomenon was not caused by oxaloacetate (OAA) accumulation in sdhA mutant cultures. PHB depolymerases DepB and HbdA were also down-regulated, which is in agreement with the higher PHB content of the sdhA mutant, compared to that of the wild-type strain.
[0175] No genes belonging to the EMC were found to be differentially expressed, suggesting that no feedback inhibition occurs on these genes, at the level of transcription, as a result of inactivation of sdhA and/or succinic acid accumulation. Except for sdh operon genes, no other TCA gene was differentially expressed.
Example 4
gap20 Mutation and PHB Synthesis
[0176] 4.1PHB Synthesis in gap20 Mutants
[0177] Even though the above results showed that the sdhA mutant was able to produce succinic acid, they also demonstrated that an important proportion of the available carbon was used by the sdhA mutant for the synthesis of PHB. By reducing PHB formation, carbon flux should flow through the EMC increasing succinic acid accumulation and glyoxylate synthesis, thereby reducing the need for malic acid supplementation. PHB formation/accumulation may be reduced, for example, by blocking or reducing PHB synthesis directly, or by over-expressing PHB depolymerases. For instance, inactivation or inhibition of a phasin protein or its encoding gene belongs to the first category.
[0178] As mentioned above, phasins Gap11 and Gap20 have previously been identified in M. extorquens.sup.25. A mutation was thus introduced within the phasin gene gap20 (see Example 1.2). Inactivation of the gap20 gene alone in the wild-type ATCC55366 strain (using the same method as described in Example 1.2) only slightly diminished PHB accumulation, i.e. from 24% to 20% compared to ATCC55366 (w/w;
[0179] Introduction of this mutation within the sdhA mutant (see Example 1.2) highly reduced its PHB content, i.e. from 81% to 17%, a 4.76 fold decrease. The sdhA gap20 double mutant thus produced PHB at levels comparable to the wild-type strain and the gap20 mutant (
4.2Organic Acid Synthesis in gap20 Mutants
[0180] Inactivation of the gap20 gene in the ATCC55366 strain did not result in any accumulation of succinic acid. On the other hand, as shown in
4.3sdhA gap20 Double Mutant Cultured with Periodic Methanol Supplementation
4.3.1Succinic Acid Concentrations Produced by sdhA gap20 Double Mutant
[0181] The sdhA gap20 double mutant was cultured for 5 days as described above, while supplementing with methanol (0.5% v/v) throughout the course of the experiment. Cell optical density (600 nm), as well as the concentrations of succinic acid, malic acid, and methanol were monitored over the course of the experiment. Results are shown numerically in Table 4A and graphically in
TABLE-US-00008 TABLE 4A sdhA gap 20 mutant: Cumulative data from succinic acid production kinetics Days (cumulative) Parameter 1 2 3 4 5 6 7 Optical density (600 nm) 0.73 2.05 2.93 3.93 4.93 5.63 6.15 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1) 7.35 4.08 3.84 3.70 3.56 3.42 3.31 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1 * gMeOH.sup.1) 6.19 0.90 0.49 0.31 0.22 0.17 0.13 Succinic acid concentration (g/L) 0.13 0.40 0.81 1.40 2.11 2.78 3.43 Consumed Malic acid (g/L) 1.56 1.78 1.92 2.07 2.17 2.26 2.34 Consumed Methanol (g/L) 1.19 4.55 7.89 11.99 16.46 20.66 24.61 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
[0182] As shown in Table 4A, when the sdhA gap20 double mutant reached an optical density of about 4, it produced 1.4 g/L (11.86 mM) of succinic acid. In contrast, when the sdhA mutant reached the same optical density, it produced 1.07 g/L (9.06 mM) of succinic acid (Table 3A). However, the sdhA mutant reached this optical density one day faster (3 days) and the sdhA gap20 double mutant (4 days).
[0183] After 5 days, the culture of the sdhA gap20 double mutant reached an OD.sub.600 of 4.93 and a succinic acid concentration of 2.11 g/L (17.8 mM) (Table 4A). In comparison, the culture of the sdhA mutant after 5 days reached a succinic acid concentration of 2.45 g/L (20.8 mM) (Table 3A). Also after 5 days, 2.17 g/L (16.27 mM) of malic acid was consumed in the culture of the sdhA gap20 double mutant. Consequently, at least 0.18 g/L (1.53 mM) of succinic acid must have been synthesized from methanol by the sdhA gap20 double mutant, compared to 0.7 g/L (5.6 mM) for the sdhA mutant (OD.sub.600 of 6.94).
[0184] At the end of the experiment (OD.sub.600=6.15; 7 days), 3.43 g/L (29 mM) of succinic acid was achieved for the sdhA gap20 double mutant, while the consumed amount of malic acid was 2.34 g/L (17.53 mM). Consequently, at least 1.36 g/L (11.5 mM) of succinic acid must have been synthesized from methanol.
[0185] In summary, the absolute concentrations of synthesized succinic acid that were measured at reference points (OD.sub.6004 and 5 days), are lower for the sdhA gap20 double mutant than for the sdhA mutant. However, in the conditions tested, growth of the double mutant was a little slower than the sdhA mutant and, as described below, this has had an impact on succinic acid yield measurements.
4.32Cumulative Yield of Succinic Acid Produced by the sdhA gap20 Double Mutant
[0186] At the reference optical density (OD.sub.600 3.93), the cumulative yield for the sdhA gap20 double mutant was 3.7 mg*L.sup.1*h.sup.1*ODu.sup.1 (Table 4A and
[0187] When considering the methanol consumption, cumulative yields were also higher in the sdhA gap20 double mutant than in the sdhA mutant (Table 4A and
4.33Yield for Each Period of 24 Hours Produced by the sdhA gap20 Double Mutant
[0188] When considering succinic acid production as well as methanol consumption for each segment of 24 hours, the overall succinic acid yield was found to be more stable between periods, with a slow constant decrease over time, when compared to that obtained from cumulative data (Table 4B and
TABLE-US-00009 TABLE 4B sdhA gap20 mutant: Data from succinic acid production kinetics for each period of 24 hours Days (period of 24 h) Parameter 1 2 3 4 5 6 7 Optical density (600 nm) 0.73 2.05 2.93 3.93 4.93 5.63 6.15 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1) 7.35 5.54 5.82 6.19 6.03 4.94 4.41 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1 * gMeOH.sup.1) 6.19 1.65 1.75 1.51 1.35 1.17 1.12 Succinic acid concentration (g/L) 0.13 0.27 0.41 0.58 0.71 0.67 0.65 Consumed Malic acid (g/L) 1.56 0.21 0.14 0.15 0.11 0.09 0.08 Consumed Methanol (g/L) 1.19 3.37 3.33 4.11 4.46 4.21 3.95 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
[0189] Except for one time point (3 days), all other specific succinic acid yields were higher in the sdhA gap20 double mutant (Table 4B), compared to the sdhA mutant (Table 3B). When omitting methanol consumption, yields were similar between cumulative and periodic data (Table 4A vs. Table 4B;
Example 5
The sdhA gap20 phaC Triple Mutant
[0190] 5.1PHB Synthesis in sdhA gap20 phaC Triple Mutant
[0191] A phaC::Km.sup.R mutation was introduced into the sdhA gap20 double mutant background and the genotype of the kanamycin sensitive derivative (after Cre-Lox excision of the Km marker) was confirmed by sequencing, as described in Example 1.5. The sdhA gap20 phaC::Km.sup.R triple mutant does not accumulate PHB, as determined by GC analyses (data not shown). The kanamycin sensitive triple mutant was used as a recipient strain for the pCHOI2 Km.sup.R plasmid, as further described below. Using these PHB null mutants as cell factories, it was hypothesized that more carbon would be available for succinic acid synthesis.
5.2sdhA gap20 phaC Triple Mutant with Periodic Methanol Supplementation
5.2.1Succinic Acid Concentrations Produced by sdhA gap20 phaC Triple Mutant
[0192] The sdhA gap20 phaC triple mutant was cultured for 10 days as described above in CHOI4 medium with 3 g/L (22.37 mM) of malate, while supplementing with methanol (0.5% v/v) throughout the course of the experiment. Cell optical density (600 nm), as well as the concentrations of succinic acid, malic acid, and methanol were monitored over the course of the experiment. Results are shown numerically in Table 5A and graphically in
TABLE-US-00010 TABLE 5A sdhA gap20 phaC mutant: Cumulative data from succinic acid production kinetics Days (cumulative) Parameter 1 2 3 4 5 6 7 8 9 10 Optical density (600 nm) 0.74 2.03 2.37 3.10 3.39 3.96 4.34 4.59 5.07 5.46 Succinic acid yield 11.44 9.41 8.17 6.93 6.76 5.98 5.36 4.85 4.54 4.27 (mg * L.sup.1 * h.sup.1 * ODu.sup.1) Succinic acid yield 10.28 1.92 0.95 0.54 0.40 0.29 0.22 0.18 0.14 0.12 (mg * L.sup.1 * h.sup.1 * ODu.sup.1 * gMeOH.sup.1) Succinic acid concentration (g/L) 0.20 0.92 1.39 2.06 2.75 3.41 3.91 4.28 4.98 5.60 Consumed Malic acid (g/L) 1.41 1.49 1.57 1.64 1.72 1.79 1.85 1.92 1.99 2.08 Consumed Methanol (g/L) 1.11 4.90 8.57 12.80 16.75 20.70 24.16 27.27 31.35 35.83 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
[0193] The triple mutant produced 3.41 g/L (28.9 mM) of succinic acid at an optical density of 3.96 (6 days), which is 2 g/L greater than the amount of succinic acid produced by the sdhA and sdhA gap20 mutants, grown at the same optical density (Table 5A and
[0194] Because growth of this triple mutant was even slower than that of the sdhA gap20 double mutant, cultures reached an average OD.sub.600 of only 3.39 after 5 days, whereas it achieved a succinic acid concentration of 2.75 g/L (23.3 mM), compared to 2.45 and 2.11 g/L for the sdhA and sdhA gap20 mutants, respectively. At this time point, 1.72 g/L (12.83 mM) of malic acid was consumed, giving a succinic acid concentration produced from methanol of at least 1.24 g/L (10.47 mM), which is more than that obtained with both sdhA and sdhA gap20 mutants (0.7 and 0.18 g/L respectively).
[0195] Likewise, after seven days, the average succinic acid concentration reached by the triple mutant cultures (3.91 g/L, 33.11 mM) was higher than that obtained with the sdhA gap20 mutant cultures (3.43 g/L, 29 mM), though it reached a lower optical density (4.34 vs 6.15). Also, 1.85 g/L of malic acid was consumed by the triple mutant, giving a succinic acid concentration synthesized from methanol of at least 2.28 g/L (19.31 mM) versus 1.36 g/L (11.15 mM) for the sdhA gap20 double mutant.
[0196] Furthermore, the triple mutant was able to produce succinic acid for a longer period of time than the sdhA and sdhA gap20 mutant strains, and at the end of the experiment, succinic acid concentration reached 5.60 g/L (47.47 mM) at an OD of 5.46 (10 days). Also, 2.04 g/L (15.22 mM) of malic acid was consumed. Consequently, at least 3.8 g/L (32.25 mM) of succinic acid must have been synthesized from methanol. Of note, succinic acid synthesis occurred while malic acid was only slightly consumed. Indeed, malic acid was rapidly consumed during the first 24 hours of the experiment, but was then barely consumed with an average of 0.076 g/L/24 h (Table 5A).
5.2.2Cumulative Yield of Succinic Acid Produced by the sdhA gap20 phaC Mutant
[0197] At the reference OD.sub.600 of 3.96, the triple mutant produced 5.98 mg of succinic acid *L.sup.1*h.sup.1*ODu.sup.1 of succinic acid, which is at least 2.2 mg more than the sdhA or sdhA gap20 mutants. At 5 days, 6.8 mg*L.sup.1*h.sup.1*ODu.sup.1 of succinic acid was produced, compared to 2.9 and 3.6 mg for the sdhA and sdhA gap20 mutants, respectively. After 10 days, 4.28 mg of succinic acid *L.sup.1*h.sup.1*ODu.sup.1 was achieved. Similar to results obtained with the other mutants, succinic acid yield diminished over time. However, succinic acid yields were found to be nearly two times higher in the PHB negative mutant compared to the sdhA gap20 double mutant. This was also true when taking into account the methanol consumption (Table 5A and
5.2.3Yield for Each Period of 24 Hours Produced by the sdhA gap20 phaC Mutant
[0198] Similar to what was observed for the sdhA gap20 double mutant, when considering 24 hour periods, the overall yield (including methanol consumption) was found to be relatively stable between periods, with a constant and slow decrease over time, when compared to that obtained from cumulative data (Table 5B;
TABLE-US-00011 TABLE 5B sdhA gap20 phaC mutant: Data from succinic acid production kinetics for each period of 24 hours Days (period of 24 h) Parameter 1 2 3 4 5 6 7 8 9 10 Optical density (600 nm) 0.74 2.03 2.37 3.10 3.39 3.96 4.34 4.59 5.07 5.46 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1) 11.44 14.68 8.37 8.98 8.44 6.94 4.82 3.32 5.72 4.78 Succinic acid yield 10.28 3.88 2.28 2.13 2.14 1.76 1.39 1.07 1.40 1.07 (mg * L.sup.1 * h.sup.1 * ODu.sup.1 * gMeOH.sup.1) Succinic acid concentration (g/L) 0.20 0.72 0.48 0.67 0.69 0.66 0.50 0.37 0.70 0.63 Consumed Malic acid (g/L) 1.36 0.08 0.08 0.07 0.08 0.07 0.07 0.07 0.07 0.09 Consumed Methanol (g/L) 1.11 3.79 3.67 4.23 3.95 3.95 3.46 3.11 4.08 4.48 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
5.3Culture of sdhA gap20 phaC Mutant Strain in 3 L Shake Flask
[0199] Succinic acid production in larger shake flasks was explored for the sdhA gap20 phaC mutant strain. The experiment in
Example 6
Effect of sucCD Overexpression
[0200] While the inactivation of the gap20 gene reduced utilisation of malic acid by the sdhA mutant, half of the amount of carbon is incorporated in succinic acid by the sdhA gap20 double mutant when compared to the sdhA mutant. It was found that one way of pulling even more carbon through the EMC is through the overexpression of the succinyl-CoA synthetase SucCD that oxidizes succinyl-CoA to succinate (see
[0201] The sucCD genes, which overexpresses alpha and beta subunit genes (sucCD) of the succinyl-CoA synthetase, were introduced in a plasmid or within the chromosome under the P.sub.mxaF promoter, using the M. extorquens sdhA gap20 mutant as the recipient strain (Example 1.4). In the sdhA gap20 double mutant, pCHOI2::sucCD confers a slight growth improvement as compared to the plasmid minus isogenic strain when cultured in 250 mL baffled Erlenmeyer flasks. Succinic acid production of the sdhA gap20 pCHOI2::sucCD mutant strain was tested using 3 L baffled Erlenmeyer flasks. Succinic acid concentration reached 2.7 g/L (22.86 mM) at an optical density of 4.16 and 7.48 g/L (63.34 mM) at an optical density of 6.99 while the malic acid consumption reached 2.19 g/L (16.33 mM) (
[0202] Overexpression of chromosome-integrated sucCD genes in the sdhA gap20 double mutant (i.e. giving the sdh gap20 Tn7::sucCD strain) resulted in increased malic acid consumption by this mutant. Succinic acid concentration reached 3.99 g/L (33.79 mM) at an optical density of 4.2 when malic acid was added ad libidum (1.5 g/L every 24 h), with a total consumption of 10.56 g/L 78.73 mM) (
[0203] Succinic acid production was also further tested in sdhA gap20 Tn7::sucCD cultures when supplemented with malic acid only at the start of the culture (
[0204] The Tn7::sucCD insertion is stable and the selection marker can be removed.sup.39. Unexpectedly, this genetic modification abolished completely the malic acid consumption phenotype of the sdhA gap20 double mutant, which consumes slowly the malic acid. Indeed, the malic acid initially added to media was completely consumed after only three days for the double mutant carrying the Tn7::sucCD fragment (
Example 7
The sdhA gap20 phaC::Km.SUP.R .Tn7::sucCD Mutant
[0205] 7.1Succinic Acid Concentrations Produced by the sdhA gap20 phaC::Km.sup.R Tn7::sucCD Mutant
[0206] The sdhA gap20 phaC::Km.sup.R Tn7::sucCD mutant was constructed as described in Example 6, except that the recipient strain was the sdhA gap20 phaC::Km.sup.R triple mutant. The resulting sdhA gap20 phaC::Km.sup.R Tn7::sucCD quadruple mutant was cultured for 8 days as described above in CHOI4 medium with 3 g/L (22.37 mM) of malate, while supplementing with methanol (0.5% v/v) throughout the course of the experiment. Cell optical density (600 nm), as well as the concentrations of succinic acid, malic acid, and methanol were monitored over the course of the experiment. Results are shown numerically in Table 6A and graphically in
TABLE-US-00012 TABLE 6A sdhA gap20 phaC::Km.sup.R Tn7::sucCD mutant: Cumulative data from succinic acid production kinetics Days (cumulative) Parameter 1 2 3 4 5 6 7 8 Optical density (600 nm) 0.86 2.62 3.03 3.94 4.10 4.32 4.82 5.24 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1) 22.98 9.15 8.67 7.01 6.49 6.04 5.64 5.28 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1 * gMeOH.sup.1) 14.37 3.44 2.22 1.08 0.74 0.58 0.43 0.34 Succinic acid concentration (g/L) 0.47 1.15 1.89 2.65 3.20 3.75 4.57 5.31 Consumed Malic acid (g/L) 1.16 1.30 1.38 1.50 1.58 1.65 1.72 1.80 Consumed Methanol (g/L) 1.60 2.66 3.90 6.48 8.74 10.35 13.10 15.32 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
[0207] The sdhA gap20 phaC::Km.sup.R Tn7::sucCD quadruple mutant, which overexpresses alpha and beta subunit genes (sucCD) of the succinyl-CoA synthetase, produced 2.65 g/L (22.44 mM) of succinic acid at an optical density of 3.94 (4 days), which is less than the amount produced by the sdhA gap20 phaC triple mutant alone (3.41 g/L; 28.9 mM) grown at the same optical density (Table 6A and
[0208] Thus, after 5 days, the culture of the quadruple mutant reached an OD.sub.600 of 4.1 and a succinic acid concentration 3.2 g/L (27.11 mM), compared to 2.75 g/L (23.3 mM; OD.sub.600 3.39) for the triple mutant. At this point, 1.72 g/L of malic acid was consumed, giving a synthesized succinic acid concentration from methanol of at least 1.69 g/L (14.29 mM), which is more than with the triple mutant at the same time point (1.24 g/L; 10.47 mM).
[0209] At the end of the experiment (8 days), succinic acid concentration reached 5.31 g/L (44.97 mM) at an OD.sub.600 of 5.24, compared to 4.28 g/L (36.25 mM; OD600 of 4.59) for the parent triple mutant strain. The consumed amount of malic acid was 1.8 g/L (14.42 mM), giving a concentration of succinic acid that must come from methanol carbon of 3.6 g/L (30.55 mM), compared to 2.59 g/L (21.93 mM) for its parent triple mutant strain, at the same time point. Again, succinic acid synthesis occurred while malic acid was only slightly consumed (Table 6A).
7.2Cumulative Yield of Succinic Acid Produced by the sdhA gap20 phaC::Km.sup.R Tn7::sucCD Quadruple Mutant
[0210] At the reference OD.sub.600 of about 4, 7.01 mg of succinic acid *L.sup.1*h.sup.1*ODu.sup.1 was produced (Table 6 and
7.3Yield for Each Period of 24 h Produced by the sdhA gap20 phaC::Km.sup.R Tn7::sucCD Quadruple Mutant
[0211] Strikingly, considering the methanol consumption, succinic acid yields were found to be much higher than those of the parent strain (Table 6B and
TABLE-US-00013 TABLE 6B sdhA gap20 phaC::Km.sup.R Tn7::sucCD mutant: Cumulative data from succinic acid production kinetics Days (period of 24 h) Parameter 1 2 3 4 5 6 7 8 Optical density (600 nm) 0.86 2.62 3.03 3.94 4.10 4.32 4.82 5.24 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1) 22.98 10.79 10.20 8.06 5.51 5.38 7.10 5.85 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1 * gMeOH.sup.1) 14.37 10.18 8.19 3.13 2.44 3.34 2.59 2.63 Succinic acid concentration (g/L) 0.47 0.68 0.74 0.76 0.54 0.56 0.82 0.74 Consumed Malic acid (g/L) 1.16 0.13 0.09 0.12 0.08 0.07 0.07 0.08 Consumed Methanol (g/L) 1.60 1.06 1.25 2.57 2.26 1.61 2.74 2.22 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
Example 8
Effect of Overexpression of the Enolase Gene eno
[0212] The construction of the plasmid containing the enolase gene eno is described in Example 1.3, and was used to overexpress the eno gene on the background of the sdhA gap20 phaC::Km.sup.R triple mutant, giving the strain designated as sdhA gap20 phaC pCHOI2::eno (Km.sup.R).
8.2.1Succinic Acid Concentrations Produced by sdhA gap20 phaC pCHOI2::eno (Km.sup.R)
[0213] The sdhA gap20 phaC pCHOI2::eno (Km.sup.R) mutant overexpressing the enolase gene eno was cultured for 8 days as described above in CHOI4 medium with 3 g/L (22.37 mM) of malate, while supplementing with methanol (0.5% v/v) throughout the course of the experiment. Cell optical density (600 nm), as well as the concentrations of succinic acid, malic acid, and methanol were monitored over the course of the experiment. Results are shown numerically in Table 7A and graphically in
TABLE-US-00014 TABLE 7A sdhA gap20 phaC pCHOl2::eno (Km.sup.R) mutant: Cumulative data from succinic acid production kinetics Days (cumulative) Parameter 1 2 3 4 5 6 7 8 Optical density (600 nm) 0.48 2.64 3.90 4.16 4.64 5.16 5.67 5.93 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1) 15.33 9.94 8.08 7.46 6.68 5.82 5.07 4.77 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1 * gMeOH.sup.1) 17.37 1.75 0.84 0.55 0.38 0.27 0.20 0.16 Succinic acid concentration (g/L) 0.18 1.26 2.27 2.98 3.72 4.33 4.82 5.44 Consumed Malic acid (g/L) 1.51 1.56 1.73 1.87 1.95 2.05 2.15 2.26 Consumed Methanol (g/L) 0.88 5.69 9.64 13.59 17.54 21.49 25.44 29.39 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
[0214] The sdhA gap20 phaC pCHOI2::eno (Km.sup.R) mutant produced 2.27 g/L (19.22 mM) of succinic acid at an optical density of 3.90 (3 days), which is less than the amount produced by the sdhA gap20 phaC::Km.sup.R mutant (3.41 g/L; 28.9 mM; 6 days), grown at the same optical density (Table 7A and
[0215] After 5 days, the culture reached an OD.sub.600 of 4.64 and a succinic acid concentration 3.72 g/L (31.5 mM). The consumed amount of malic acid was 1.95 g/L (14.54 mM), giving an amount of succinic acid synthesized from methanol of at least 2 g/L (16.94). This was more than with both the triple mutant and its isogenic derivative overexpressing sucCD (1.24 and 1.69 g/L respectively).
[0216] At the end of the experiment (8 days), succinic acid concentration reached 5.44 g/L (46.07 mM) at an OD.sub.600 of 5.93 (8 days). The amount of consumed malic acid was 2.26 g/L (16.85 mM). Consequently, at least 3.45 g/L (29.22 mM) of succinic acid must have been synthesized from methanol. This is less than for the sucCD overexpressing strain (3.6 g/L), but more than the triple mutant alone (2.59 g/L).
8.2.2Cumulative Yield of Succinic Acid Produced by the sdhA gap20 phaC pCHOI2::eno (Km.sup.R) Mutant
[0217] At the reference OD (3 days), the triple mutant produced 8.08 mg of succinic acid *L.sup.1*h.sup.1*ODu.sup.1 (Table 8 and
8.2.3Yield for Each Period of 24 Hours Produced by the sdhA gap20 phaC pCHOI2::eno (Km.sup.R) Mutant
[0218] General observations from the triple mutant described above, were also true for its isogenic mutant overexpressing eno. Looking closer at days 2 and 3, without considering methanol consumption, periodic yields were found to be the highest of all experiments (Table 9 and
TABLE-US-00015 TABLE 7B sdhA gap20 phaC pCHOl2::eno (Km.sup.R) mutant: Data from succinic acid production kinetics for each period of 24 hours Days (period of 24 h) Parameter 1 2 3 4 5 6 7 8 Optical density (600 nm) 0.48 2.64 3.90 4.16 4.64 5.16 5.67 5.93 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1) 15.33 17.07 10.77 7.07 6.65 4.93 3.66 4.31 Succinic acid yield (mg * L.sup.1 * h.sup.1 * ODu.sup.1 * gMeOH.sup.1) 17.37 3.55 2.73 1.79 1.68 1.25 0.93 1.09 Succinic acid concentration (g/L) 0.18 1.08 1.01 0.71 0.74 0.61 0.50 0.61 Consumed Malic acid (g/L) 1.51 0.04 0.18 0.14 0.08 0.10 0.10 0.11 Consumed Methanol (g/L) 0.88 4.81 3.95 3.95 3.95 3.95 3.95 3.95 ODu.sup.1: per optical density unit; h.sup.1: per hour; gMeOH.sup.1: specific yield succinic acid per gram of consumed methanol
Summary from Examples 3-8
[0219] Table 10A below compiles the cumulative data from succinic acid production kinetics from Tables 3A, 4A, 5A, 6A, 7A and 8A, for the different mutants tested. Table 10B below compiles the data from succinic acid production kinetics for each period of 24 hours from Tables 3B, 4B, 5B, 6B, 7B and 8B, for the different mutants tested. The different mutants shown in Tables 10A and 10B are as follows:
[0220] A: sdhA D: sdhA gap20 phaC::Km.sup.R Tn7::sucCD (Tet.sup.R)
[0221] B: sdhA gap20 E: sdhA gap20 phaC pCHOI2::eno (Km.sup.R)
[0222] C: sdhA gap20 phaC Km.sup.R
[0223] For ease of comparison, the results after Day 5 of culture, which was the end-point of the experiment with the sdhA single mutant, are shown in bold.
TABLE-US-00016 TABLE 10A Complied data from cumulative succinic acid production kinetics Day A B C D E A B C D E A B C D E Optical Density Yield (mg * L.sup.1 * Yield (mg * L.sup.1 * H.sup.1 * ( = 600 nm) H.sup.1 * ODu.sup.1) ODu.sup.1 * gMeOH.sup.1) 1 0.54 0.73 0.74 0.86 0.48 2.13 7.35 11.44 22.98 15.33 4.39 6.19 10.28 14.37 17.37 2 2.76 2.05 2.03 2.62 2.64 2.56 4.08 9.41 9.15 9.94 0.52 0.90 1.92 3.44 1.75 3 3.98 2.93 2.37 3.03 3.90 3.74 3.84 8.17 8.67 8.08 0.43 0.49 0.95 2.22 0.84 4 5.92 3.93 3.10 3.94 4.16 3.18 3.70 6.93 7.01 7.46 0.25 0.31 0.54 1.08 0.55 5 6.94 4.93 3.39 4.10 4.64 2.94 3.56 6.76 6.49 6.68 0.18 0.22 0.40 0.74 0.38 6 5.63 3.96 4.32 5.16 3.42 5.98 6.04 5.82 0.17 0.29 0.58 0.27 7 6.15 4.34 4.82 5.67 3.31 5.36 5.64 5.07 0.13 0.22 0.43 0.20 8 4.59 5.24 5.93 4.85 5.28 4.77 0.18 0.34 0.16 9 5.07 4.54 0.14 10 5.46 4.27 0.12 Succinic acid Consumed Malic Consumed concentration (g/L) acid (g/L) Methanol (g/L) 1 0.03 0.13 0.20 0.47 0.18 1.28 1.56 1.41 1.16 1.51 2.07 1.19 1.11 1.60 0.88 2 0.34 0.40 0.92 1.15 1.26 1.68 1.78 1.49 1.30 1.56 8.88 4.55 4.90 2.66 5.69 3 1.07 0.81 1.39 1.89 2.27 1.77 1.92 1.57 1.38 1.73 12.60 7.89 8.57 3.90 9.64 4 1.81 1.40 2.06 2.65 2.98 1.92 2.07 1.64 1.50 1.87 16.57 11.99 12.80 6.48 13.59 5 2.45 2.11 2.75 3.20 3.72 2.04 2.17 1.72 1.58 1.95 16.79 16.46 16.75 8.74 17.54 6 2.78 3.41 3.75 4.33 2.26 1.79 1.65 2.05 20.66 20.70 10.35 21.49 7 3.43 3.91 4.57 4.82 2.34 1.85 1.72 2.15 24.61 24.16 13.10 25.44 8 4.28 5.31 5.44 1.92 1.80 2.26 27.27 15.32 29.39 9 4.98 1.99 31.35 10 5.60 2.08 35.83
TABLE-US-00017 TABLE 10B Compiled from succinic acid production kinetics for each period of 24 hours Day A B C D E A B C D E A B C D E Optical Density Yield (mg * L.sup.1 * Yield (mg * L.sup.1 * H.sup.1 * ( = 600 nm) H.sup.1 * ODu.sup.1) ODu.sup.1 * gMeOH.sup.1) 1 0.54 0.73 0.74 0.86 0.48 2.13 7.35 11.44 22.98 15.33 4.39 6.19 10.28 14.37 17.37 2 2.76 2.05 2.03 2.62 2.64 4.71 5.54 14.68 10.79 17.07 1.06 1.65 3.88 10.18 3.55 3 3.98 2.93 2.37 3.03 3.90 7.66 5.82 8.37 10.20 10.77 2.06 1.75 2.28 8.19 2.73 4 5.92 3.93 3.10 3.94 4.16 5.18 6.19 8.98 8.06 7.07 1.30 1.51 2.13 3.13 1.79 5 6.94 4.93 3.39 4.10 4.64 3.86 6.03 8.44 5.51 6.65 0.93 1.35 2.14 2.44 1.68 6 5.63 3.96 4.32 5.16 4.94 6.94 5.38 4.93 1.17 1.76 3.34 1.25 7 6.15 4.34 4.82 5.67 4.41 4.82 7.10 3.66 1.12 1.39 2.59 0.93 8 4.59 5.24 5.93 3.32 5.85 4.31 1.07 2.63 1.09 9 5.07 5.72 1.40 10 5.46 4.78 1.07 Succinic acid Consumed Malic Consumed concentration (g/L) acid (g/L) Methanol (g/L) 1 0.03 0.13 0.20 0.47 0.18 1.28 1.56 1.36 1.16 1.51 0.49 1.19 1.11 1.60 0.88 2 0.31 0.27 0.72 0.68 1.08 0.40 0.21 0.08 0.13 0.04 4.45 3.37 3.79 1.06 4.81 3 0.73 0.41 0.48 0.74 1.01 0.09 0.14 0.08 0.09 0.18 3.72 3.33 3.67 1.25 3.95 4 0.74 0.58 0.67 0.76 0.71 0.15 0.15 0.07 0.12 0.14 3.97 4.11 4.23 2.57 3.95 5 0.64 0.71 0.69 0.54 0.74 0.12 0.11 0.08 0.08 0.08 4.16 4.46 3.95 2.26 3.95 6 0.67 0.66 0.56 0.61 0.09 0.07 0.07 0.10 4.21 3.95 1.61 3.95 7 0.65 0.50 0.82 0.50 0.08 0.07 0.07 0.10 3.95 3.46 2.74 3.95 8 0.37 0.74 0.61 0.07 0.08 0.11 3.11 2.22 3.95 9 0.70 0.07 4.08 10 0.63 0.09 4.48
Example 9
Genetic Switches
[0224] To eliminate the need of malate addition to produce succinic acid using M. extorquens growth on methanol, the sdhA mutant is complemented by incorporating an sdh operon under the control of a genetic switch, into the background of the sdhA mutant.
[0225] Cumate-dependent genetic switches were first described and successfully used in M. extorquens.sup.50,51. Since cumate is an inexpensive molecule, it is reasonable to consider its use in bioreactors.
[0226] The switches are based on the Pseudomonas putida repressor CymR or on the chimeric transactivator cTA. cTA consists in a fusion between CymR and the activation domain of the VP16 protein (herpes simplex). These two transcriptional regulators bind to specific operator sequences. The presence of cumate prevents the binding of CymR and cTA to the operator sequence, resulting in activation or repression, respectively.
[0227] Similarly, CymR-dependent switches are also used. Cumate is then used at low concentrations to permit temporary complementation for biomass production. Sdh proteins produced from such switches are expected to be exhausted later on during growth and succinic acid would then accumulate.
[0228] As the genetic switches can be activated at any moment during growth, the engineered strains may yield higher biomass resulting in higher succinic acid production. Moreover, it is important to underline that CymR and cTA-dependent switches could theoretically be modulated by the addition of cumate generating opposite regulation effects. Likewise, it would be possible to operate these two kinds of switches together in a single mutant strain, controlling simultaneously the expression of the sdh operon and PHB depolymerisation.
[0229] Results showed that the sdhA mutant carrying a genetic switch capable of controlling expression of the sdh operon can grow without malic acid supplementation in the presence of cumate. Furthermore, its growth was reduced as more cumate was added.
Example 10
Heterologous Genes Expression
[0230] Simultaneous overexpression of pyc and ppc genes was shown to improve aerobic succinate production in some bacteria.sup.54,55. These genes encode pyruvate and phosphoenolpyruvate (PEP) carboxylases, respectively, responsible for the conversion of pyruvate and PEP into OAA. The increase of the OAA pool within M. extorquens cells is expected to provide more carbon input into the EMC, especially if the mdh gene is functionally overexpressed.
[0231] As the pyc gene is missing from the M. extorquens genome, its heterologous expression is therefore needed. For example, the pyc gene from Rhodopseudomonas palustris BisA53 is used. Like M. extorquens, R. palustris is an environmental non-pathogenic bacteria belonging to the rhizobiale group of alphaproteobacteria.sup.56.
[0232] Likewise, heterologous overexpression of an isocitrate lyase (Icl), which catalyzes the formation of glyoxylate and succinate from isocitrate may be performed.sup.57,58. Icl is a key enzyme of the glyoxylate regeneration pathway and is absent from the M. extorquens genome, which uses the EMC. For example, Icl may be used to increase the oxidative flux from citrate within the TCA, which may occur as succinic acid accumulates in a genetic switch complemented sdhA mutant. It could also allow inactivation of the EMC, which theoretically, would result in larger amount of carbon available for succinic acid production.
Example 11
Converting PHB Accumulated Biomass to Succinic Acid
[0233] PhaC mutants were shown to have a growth defect when grown on methanol. However, unidentified suppressor mutations of this specific phenotype also occur at high frequency on methanol.sup.31. Therefore, a shdA phaC double mutant that does not produce PHB, but grows normally on methanol may be obtained.
[0234] Alternatively, in order to make the carbon accumulated in the PHB available for succinic acid synthesis in the sdhA mutant, PHB depolymerases and recycling enzymes are cloned, alone or in combination, under an inducible promoter. PHB depolymerisation may then be induced at any time, for example when a culture reaches mid-stationary phase of growth. AtoCD activity results in the production of succinic acid as by-product during PHB depolymerisation. It may thus be possible to perform a two phase bioprocess in which PHB accumulates in a first phase and succinic acid is produced subsequently.
Example 12
Small RNA Knockdowns
[0235] Since the early 2000's, classes of RNA regulators have been discovered and shown to play a key role in the control of genes through various mechanisms, whether during transcription, translation or even post-translation. An important group of these regulators is composed of so-called small RNAs (sRNA). These genes are transcribed as short (100 bases) RNAs not encoding for any protein. Instead, these sRNAs can bind to target mRNAs through base complementarity, typically in the region of the ribosome binding site. Binding of the sRNA to its target prevents accessibility of the ribosome, therefore repressing translation and, consequently, expression. Close to a hundred sRNAs have been identified in Escherichia coli as well as in other species, especially proteobacteria.sup.59,60.
[0236] Most sRNAs bind the protein Hfq which serves as a facilitator for the interaction between the sRNA and the mRNA to be inhibited, thus allowing more efficient binding and repression. More specifically, Hfq binds a region of the sRNA, while the other part of the sRNA can bind to the target mRNA. It is thus possible to design modified sRNAs capable of repressing any selected target.sup.52. While about a hundred are known in E. coli, a few sRNAs have been found so far in M. extorquens PA1, but there are likely as many as in E. coli.sup.61. Indeed, this specie harbors the hfq gene, indicator of sRNA regulatory pathways.sup.59. Therefore, a sRNA such as MicC should function in M. extorquens as it does in E. coli, provided that it has the appropriate sequence to form base pairs with its target mRNA. For instance, sRNA constructs consist in a promoter, a variable region complementary to the target gene, a MicC sequence and a terminator, for a total of less than 500 bases.
[0237] Results based on GFP expression indicate that a version of the PmxaF promoter consisting of 242 bases upstream of the transcription start site is sufficient to produce a sRNA with almost no extra sequence, for instance, only a single G in 5 of the sRNA target-complementarity-region. The sRNA system in M. extorquens can then be assayed against GFP as a reporter gene. To assess GFP repression, three anti-GFP sRNAs are constructed, these are complementary to positions 19 (relative to the start codon) up to the start codon, positions 11 to +10 and from the start codon up to +20. These sRNAs expressed by the truncated PmxaF target a genome insertion of GFP in M. extorquens, also under the control of PmxaF. Based on the results obtained, a sRNA construct complementary to sdhA is then designed. Succinic acid production using M. extorquens modified with this sRNA is measured as previously described, with and without malic acid supplementation.
[0238] Other sRNA may be designed to target other genes which encode proteins involved in the metabolism of succinic acid or which may divert intermediary metabolites from the main path linking methanol to succinate. For instance, genes involved in the citric acid cycle (e.g. sdhBCD and fumC) as well as other pathways, such as the pentose phosphate pathway (e.g. pgm, pgk, gap . . . ), the PHB pathway, or the formate oxidation pathway. Combinations of sRNAs within the same vector may also be used to increase succinic acid production. For instance, another sRNA may be combined with the sdhA sRNA or may be expressed in an sdhA mutant herein described.
[0239] Alternatively, a modified CRISPR system could also be used in a very similar way. The CRISPR RNAs are bacteria's natural defense mechanisms against bacteriophages, but can be adapted to target a gene and its functionality is irrelevant to the species in which they are used, provided that a modified Cas9 protein is co-expressed.sup.53.
Example 13
Expression of a Heterologous Glyxoxylate Shunt and Ethyl-Malonyl-CoA Pathway Inactivation in a Methylobacterium Extorquens Mutant that Produces Succinic Acid from Methanol
13.1Rationale
[0240] In cells, acetyl-CoA is a major anaplerotic metabolite and assimilation pathways have evolved to maximize its carbon incorporation into the central metabolism. In many organisms, one strategy involves the utilization of both the TCA and the Glyoxylate cycles. Indeed, acetyl-CoA can be condensed with oxaloacetate to produce citrate, thereby beginning the oxidative TCA cycle. Then, instead of being further decarboxylated, the isocitrate produced by the TCA cycle can be taken up by the Glyoxylate cycle to form succinate and glyoxylate. This last step is achieved by the isocitrate lyase enzyme (icl). Next, glyoxylate can be used together with acetyl-CoA to produce malate, making the missing carbon to enter the central metabolism.
[0241] Methylotrophic microorganisms, such as Methylobacterium extorquens, lack the Icl enzyme (the glyoxylate shunt) and use the Ethyl-Malonyl-CoA (EMC) pathway to produce, among other molecules, glyoxylate. This glyoxylate is intended to be used by the Serine Cycle for assimilation of methanol, and not for the synthesis of malate, while methanol can be the sole source of carbon and energy. Acetyl-CoA produced by the serine cycle is used as the primary substrate for the EMC pathway. This pathway involves successive thio-ester-CoA molecule modifications and flows into the TCA cycle by forming succinyl-CoA. In fact, during methylotrophic growth, the TCA cycle works only partially and enzymatic reactions toward malate synthesis complete the EMC pathway. The EMC pathway shares its two first steps with the PHB cycle, i.e. the sequential synthesis of aceto-acetyl-CoA and hydroxybutyryl-CoA (OHB-CoA) from acetyl-CoA, by PhaA (a beta-ketothiolase) and PhaB (an NADPH-linked acetoacetyl-CoA reductase), respectively. The final step of PHB synthesis is performed by the PHB synthase PhaC.
[0242] Accordingly, since EMC requires a lot of carbon and the eventual recombinant glyoxylate shunt would produce succinic acid as well as glyoxylate, overexpression of a heterologous shunt within a metabolically modified M. extorquens that produces succinic acid is herein described. Once the glyoxylate shunt is operational, the inefficient and unnecessary EMC may then be inactivated.
13.2Autosomal Expression of a Heterologous Glyxoxylate Shunt in a M. Extorquens Mutant that Produces Succinic Acid from Methanol
[0243] This example describes the creation of a classic glyoxylate shunt within an isocitrate lyase (icl) negative M. extorquens triple mutant (sdhA gap20 phaC) and the assessment of its functionality. This must be performed prior to EMC pathway inactivation (Example 13.3), as it will replace an essential glyoxylate producing pathway by another. Furthermore, since the first steps of the Citrate cycle are poorly expressed in M. extorquens during growth on methanol, genes leading to isocitrate synthesis (gltA and acnA) will be overexpressed together with the isocitrate lyase shunt (icl gene). These genes encode a citrate synthase and an aconitase, respectively. [0244] AFor the isocitrate lyase shunt, two approaches are assayed: (1) The icl gene from the environmental bacterium Rhodopseudomonas palustris BisA53 is PCR amplified and cloned within the medium copy vector pCHOI2. This plasmid is characterized by a multiple cloning site located just downstream of the strong M. extorquens methanol dehydrogenase promoter (PmxaF). The R. palustris genome is chosen as a template since the codon usage in this microorganism is similar to that of M. extorquens. (2) A custom, completely synthetic icl gene (also driven by PmxaF) is designed based on the amino acid sequence of isocitrate lyase in Escherichia coli K12 and on codon usage in M. extorquens. For gltA and acnA genes, M. extorquens ATCC55366 chromosome may be used as a template for PCR amplifications. Then, gltA and acnA are cloned into pCHOI2 vector. [0245] BEach plasmid insertion, together with PmxaF, is sequenced and transformed into the triple mutant. Expression of icl, gltA, acnA and rpoD (housekeeping) genes are evaluated by semi-quantitative RT-PCR, as described in the table below. This experimental design permits observation of the influence of the overexpressed gene on the expression of selected others. Expression of icl R.p. is compared to that of icl synth. Citrate synthase, aconitase and isocitrate lyase activity is also measured. The triple mutant carrying the empty pCHOI2 vector is used as a control. Experiments are performed using 250 mL baffled shake flasks, in biological triplicates. The sampling is performed during the exponential phase of growth.
TABLE-US-00018 RT-PCR design sdhA gap20::145 phaC derivatives Targeted genes +pCHOI2 (negative or basal rpoD icl R.p. icl synth. expression control) gltA acnA +pCHOI2::icl R.p. rpoD icl R.p. gltA acnA +pCHOI2::icl synthetic rpoD icl synth. gltA acnA +pCHOI2::gltA rpoD gltA acnA +pCHOI2::acnA rpoD gltA acnA
13.3In Chromosome Expression of a Heterologous Glyxoxylate Shunt within an M. Extorquens Mutant that Produces Succinic Acid from Methanol
[0246] This example describes integration of the best (stronger) isocitrate lyase overexpressing system, as determined in Example 13.2, together with gltA and acnA systems, into the chromosome of the triple mutant (sdhA gap20 phaC) and the subsequent characterization. [0247] ATargeted DNA fragments are PCR amplified from pCHOI2 derived vectors obtained in Example 13.2 and integration is performed using suicide vector or Tn7-based system. Selection markers are removed using the Cre-LoxP or the flipase (Flp) system. All integrations are verified by sequencing. [0248] BExpression of icl, gltA, acnA and rpoD (housekeeping) are evaluated by semi-quantitative RT-PCR, as described in the table below. Citrate synthase, aconitase and isocitrate lyase activity are measured. The triple mutant is used as a control. Experiments are performed using 250 mL baffled shake flasks, in biological triplicates. The sampling is performed during the exponential phase of growth.
TABLE-US-00019 RT-PCR design Mutant strains Targeted genes sdhA gap20::145 phaC mutant (basal expression control) rpoD gltA acnA sdhA gap20::145 phaC // ::pmxaF-icl, pmxaF-gltA and rpoD icl R.p. or synth. gltA acnA pmxaF-acnA [0249] CGrowth of the triple mutant, overexpressing or not the glyoxylate shunt, is performed using a 1.5 L DASGIP parallel bioreactor systems equipped with a methanol control system. Two reactors are used for each mutant, and runs are to last 72 h. Expression of icl, gltA and acnA are evaluated by semi-quantitative RT-PCR. Citrate synthase, aconitase and isocitrate lyase activity are measured. Succinic and malic acids are quantified by HPLC. Sampling is done every 24 h.
13.4Inactivation of the EMC Pathway in the M. Extorquens Mutants Expressing the Heterologous Glyoxylate Shunt
[0250] This example describes the interruption of the EMC pathway within the mutant obtained in Example 13.3, and the characterization thereof. [0251] AInactivation of the phaA gene and consequently of the entire EMC pathway is performed using a suicide vector. Selection markers are removed using the Cre-LoxP system. The phaA mutation is verified by sequencing. [0252] BThe EMC-negative mutant is grown using a 1.5 L DASGIP parallel bioreactor system equipped with a methanol control system (3 reactors). For comparative purposes, a fourth reactor is used to grow the EMC positive isogenic mutant. Runs are to last 72 h. Expression of icl, gltA, acnA and rpoD are evaluated by semi-quantitative RT-PCR, as described in the table below. Of note, expression of two genes from the EMC pathway are also be quantified by RT-PCR. Citrate synthase, aconitase and isocitrate lyase activity are measured. Succinic and malic acids are quantified by HPLC. Sampling is done every 24 h.
TABLE-US-00020 RT-PCR design Mutant strains Targeted genes sdhA gap20::145 phaC // ::pmxaF-icl, pmxaF-gltA rpoD, icl R.p. or synth. gltA, acnA, EMC gene 1, EMC and pmxaF-acnA (basal expression control) gene 2 sdhA gap20::145 phaC phaA // ::pmxaF-icl, pmxaF- rpoD icl R.p. or synth. gltA, acnA, EMC gene 1, EMC gltA and pmxaF-acnA gene 2
[0253] Alternatively, since PhaA works upstream of both Gap20 and PhaC in the EMC pathway, inactivating PhaA may be sufficient to inactive both the PHB and EMC pathways, without having to also inactivate Gap20 and PhaC. For example, a sdhA phaA mutant could be created that overexpresses isocitrate lysase, and thus produce a mutant having disrupted PHB and EMC pathways, and an operational glyoxylate shunt.
[0254] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Any publication, document, patent, patent application or publication referred to herein should be construed as incorporated by reference each in their entirety and for all purposes.
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