PROCESS OF PRODUCING AN ORGANIC COMPOUND AND AN INTERMEDIARY COMPOUND

Abstract

A process of producing an organic compound and/or an intermediary compound by feeding carbon dioxide to a culture of Cyanobacteria cells and subjecting the culture to light, wherein the cells are capable of expressing a nucleic acid molecule that confers the ability to convert a glycolytic intermediate into said organic/intermediary compound. The expression of the nucleic acid molecule is under the control of a regulatory system which responds to a change in the concentration of a nutrient in the culture.

Claims

1. A process of producing an organic compound and/or an intermediary compound produced in a pathway leading to said organic compound, comprising feeding carbon dioxide to a culture of a Cyanobacteria cell and subjecting said culture to light, wherein said cell is capable of expressing a nucleic acid molecule, wherein the expression of said nucleic acid molecule confers on the cell an ability to convert a glycolytic intermediate into said organic compound and/or into said intermediary compound, and wherein said nucleic acid molecule is under the control of a regulatory system which responds to a change in the concentration of a nutrient in said culture.

2. The process according to claim 1, wherein said enzyme is substantially not sensitive towards oxygen inactivation.

3. The process according to claim 1, wherein the organic product is butanol and the intermediary compound is butyraldehyde, and wherein the nucleic acid molecule codes for at least one enzyme capable of converting pyruvate to butanol selected from the group consisting of: a thiolase, a hydroxybutyrylCoA dehydrogenase, a crotonase, a butyrylCoA dehdyrogenase, a butyraldehyde dehydrogenase, and a butanol dehdyrogenase.

4. The process according to claim 1, wherein the organic product is butanol and the intermediary compound is produced in the pathway leading to butanol, and wherein the nucleic acid molecule codes for at least one enzyme capable of converting pyruvate to butanol selected from the group consisting of: a 2-acetolactate synthetase, an acetolactate decarboxylase, a diacetyl reductase, an acetoin reductase, a glycerol dehydratase, and a 1,3-propanediol dehydrogenase.

5. The process according to claim 1, wherein the organic product is ethanol and the intermediary compound is acetaldehyde, and wherein the nucleic acid molecule codes for at least one enzyme capable of converting pyruvate into ethanol selected from the group consisting of a pdc and an alcohol dehydrogenase.

6. The process according to claim 1, wherein the organic product is propanol and the intermediary compound is acetone, and wherein the nucleic acid molecule codes for at least one enzyme capable of converting pyruvate to propanol selected from the group consisting of: a thiolase, an acetoacetylCoA transferase, an acetoacetylCoA decarboxylase, and a propanol dehydrogenase.

7. The process according to claim 1, wherein the organic product is acetone, and wherein the nucleic acid molecule codes for at least one enzyme capable of converting pyruvate to acetone selected from the group consisting of: a thiolase, an acetoacetylCoA transferase, and an acetoacetylCoA decarboxylase.

8. The process according to claim 1, wherein the organic product is 1,3-propanediol and the intermediary compound is glycerol and/or hydroxypropionaldehyde, and wherein the nucleic acid molecule codes for at least one enzyme capable of converting glyceraldehyde-3-phosphate to propanediol selected from the group consisting of: a glycerol-3-P-dehydrogenase, a glycerol-3-P phosphatase, a glycerol dehydratase, and an oxidoreductase.

9. The process according to claim 1, wherein the organic product is D-lactate, and wherein the nucleic acid molecule codes for at least one enzyme capable of converting pyruvate to D-lactate and comprises a lactate dehydrogenase.

10. The process according to claim 1, wherein the organic product is ethylene, and wherein the nucleic acid molecule code for at least one enzyme capable of converting 2-oxoglutarate into ethylene and succinate.

11. The process according to claim 1, wherein the regulatory system responds to a change in concentration of a nutrient ammonium in said culture, and/or wherein said nucleic acid molecule is integrated into the Cyanobacteria genome, preferably via homologous recombination, and/or wherein the Cyanobacteria cell is derived from a Synechocystis cell, preferably a Synechocystis PCC 6803 cell.

12. The process according to claim 1, wherein the organic product and/or intermediary compound is separated from the culture.

13. A Cyanobacteria capable of expressing a nucleic acid molecule, wherein the expression of said nucleic acid molecule confers on the Cyanobacteria the ability to convert a glycolytic intermediate into an organic compound and/or into an intermediary compound produced in a pathway leading to the organic product, and wherein the nucleic acid molecule is under the control of a regulatory system which responds to a change in the concentration of a nutrient when culturing said Cyanobacteria.

14. The Cyanobacteria according to claim 13, wherein the glycolytic intermediate is glyceraldehyde-3-phosphate or pyruvate, the organic compound is selected from the group consisting of butanol, ethanol, propanol, 1,3-propanediol, acetone, D-lactate and ethylene, the intermediary product is selected from the group consisting of butyraldehyde acetaldehyde, acetone, glycerol and hydroxypropionaldehyde, and the enzyme is selected from the group consisting of a thiolase, a hydroxybutyrylCoA dehydrogenase, a crotonase, a butyrylCoA dehdyrogenase, a butyraldehyde dehydrogenase, a butanol dehdyrogenase2-acetolactate synthetase, an acetolactate decarboxylase, a diacetyl reductase, an acetoin reductase, a glycerol dehydratase, a 1,3-propanediol dehydrogenase, a pdc, an alcohol dehydrogenase, an acetoacetylCoA transferase, an acetoacetylCoA decarboxylase, a propanol dehydrogenase, a glycerol-3-P-dehydrogenase, a glycerol-3-P phosphatase, a glycerol dehydratase, an oxidoreductase, and a lactate dehydrogenase.

15. The Cyanobacteria according to claim 13, wherein the regulatory system responds to a change in concentration of a nutrient ammonium, and/or wherein said nucleic acid molecule is integrated into the Cyanobacteria genome, preferably via homologous recombination, and/or wherein the Cyanobacteria is derived from Synechocystis, preferably Synechocystis PCC 6803.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0171] FIG. 1: The light reaction reproduced from Berg, Tymoczko and Stryer: Biochemistry WH Freeman and Co, New York, 2006.

[0172] FIG. 2: The Calvin Cycle reproduced from Berg, Tymoczko and Stryer: Biochemistry WH Freeman and Co, New York, 1 2006.

[0173] FIG. 3: Construction of a recombinant strain: The cassette is introduced by homologous recombination and positioned downstream the SigE controlled promotor of Synechocystis PCC 6803. The black circle shows the suicide plasmid (e.g. pBluescript) that is not able to replicate in Synechocystis PCC 6803. The cassette(s) of interest (denoted as arrow x in the figure) will be engineered to be flanked by DNA sequences homologous to a non-coding DNA region (shown as a dotted bar in the figure) in the nrt operon. Via a double crossover event, shown in the figure as dashed crosses, the cassettes of interest are integrated into the Synechocystis genome. Alternatively, the construct can be inserted at a neutral docking site.

[0174] FIG. 4: Ammonium controlled production via the NtcA transcriptional regulator. Conditions allowing growth repress production, ammonium depletion promotes production.

[0175] FIG. 5: Alcohol resistance. After five days, 100 l of each culture was diluted and transferred to solid medium prepared from the BG-11 medium. Solid cultures were grown in an incubator at 30 C. under continuous illumination (70 Einstein/m.sup.2/s) using two TL tubes without any addition (control) or with butanol, respectively ethanol added at various concentrations. After a week, single colonies were observed and counted. The amount of colonies was compared to the control sample.

[0176] FIG. 6: A preferred design of expression cassettes is given in FIG. 6.

EXAMPLES

Example 1: Biochemical Background of the Cyanobacteria of the Invention

[0177] The energy, in the form of ATP, as well as the reductive power in the form of NADPH, that are both needed to drive the subsequent highly endergonic dark reactions of photosynthesis, are catalyzed by the two photosystems of oxygenic photosynthesis, PS-II and PS-I, arranged according to the well-known Z-scheme, plus the membrane-bound ATPase (FIG. 1). In phototrophic organisms like Cyanobacteria, CO.sub.2 is fixed in the so-called Calvin cycle. This is a cyclic series of reductive steps that result in the net conversion of CO.sub.2 into C3 compounds, such as glyceraldehyde-3-phosphate, phosphoglycerate and pyruvate (FIG. 2). This pathway is essentially endergonic and in Nature driven by sunlight. It consumes CO.sub.2 and water and yields C.sub.3 compounds (e.g. pyruvate) and oxygen:

CO.sub.2+H.sub.2O+Solar energy.fwdarw.C.sub.3 compounds+O.sub.2(1)

[0178] This reaction cannot proceed spontaneously: It is driven by the consumption of the ATP and NADPH, generated in the light reactions of photosynthesis. Subsequently, the C.sub.3 compounds are used in Nature (i.e. in phototrophic organisms like plants and Cyanobacteria) as the building blocks to make new cells and/or plants. This requires additional amounts of reducing power (as NADPH) and energy conserved during the light capturing reactions (as ATP) and also allows the organisms to proliferate (grow and survive).

[0179] Nature also sustains an entirely different mode of (microbial) life: Numerous bacterial and fungal species are able to conserve sufficient energy (as ATP) to proliferate by fermentation, in which they use so-called substrate level phosphorylation to generate their energy. This respiration-independent mode of energy conservation relies on metabolic pathways that result in redox neutral dissimilation of the energy source. The most abundant pathways have evolved with sugars (e.g. glucose) as energy source and therefore all have glycolysis in common:

Glucose.fwdarw.glyceraldehyde-P.fwdarw.pyruvate+reducing power(2)

Redox neutrality is maintained by the generalized reaction:

pyruvate+reducing power.fwdarw.fermentation products(3)

[0180] Thus, it will contain the functional biochemistry to reduce the above-mentioned intermediates to the end product and will have as its biocatalytic input and output the combination of (1) and (3), respectively:

CO.sub.2+H.sub.2O+Solar energy.fwdarw.organic product+O.sub.2

Example 2: Description of the Expression System Used

Genetic Cassettes

[0181] The identity of an organic product formed (and excreted into the environment) in the process of the invention depends on the species-specific gene cassettes (i.e. nucleic acid molecules represented by nucleotide sequences) that encode the respective biochemical pathway (see table 1). Preferred enzymes encoded by nucleic acid molecules are substantially oxygen insensitive.

TABLE-US-00001 TABLE 1 Examples of preferred donor organisms for the nucleic acid molecules or genes to be introduced into a Cyanobacterium with the pathway they catalyze. For e.g. the production of ethanol and propanediol various alternative donor organisms can be suggested. donor genes pathway Sarcina ventriculi pyruvate decarboxylase, Pyruvate .fwdarw. acetaldehyde Lactobacillus brevis alcohol dehydrogenase Acetaldehyde .fwdarw. ethanol Clostridium thiolase pyruvate .fwdarw. acetoacetylCoA acetobutilicum hydroxybutyrylCoA dehydrogenase crotonase acetoacetylCoA .fwdarw. butyrylCoA butyryl-CoA dehydrogenase butyrylCoA .fwdarw. butyraldehyde Butanol dehydrogenase butyraldehyde .fwdarw. 2-butanol Pseudomonas syringiae ethylene forming enzyme 2-ketoglutarate .fwdarw. ethylene Lactococcus lactis lactate dehydrogenase pyruvate .fwdarw. D-lactate Lactococcus lactis acetolactate synthase pyruvate .fwdarw. 2-acetolactate acetolactate decarboxylase 2-acetolactate .fwdarw. acetoin diacetyl reductase diacetyl .fwdarw. acetoin acetoin reductase acetoin .fwdarw. 2,3 butanediol Klebsiella pneumoniae glycerol dehydratase 2,3 butanediol .fwdarw. 2-butanone 1,3 propanediol dehydrogenase 2-butanone .fwdarw. 2-butanol Clostridium thiolase acetylCoA .fwdarw. acetoacetylCoA acetobutilicum ac.acetylCoA transferase acetoacetylCoA .fwdarw. acetoacetate acetoacetate decarboxylase acetoacetate .fwdarw. acetone Clostridium thiolase acetylCoA .fwdarw. acetoacetylCoA acetobutilicum ac.acetylCoA transferase acetoacetylCoA .fwdarw. acetoacetate acetoacetate decarboxylase acetoacetate .fwdarw. acetone Klebsiella pneumoniae propanol dehydrogenase acetone .fwdarw. propanol Synechocystis PCC glycerol-3-P dehydrogenase GAP .fwdarw. glycerol-P 6083 glycerol-3-P Phosphatase glycerol-P .fwdarw. glycerol K. pneumoniae glycerol dehydratase glycerol .fwdarw. OHpropionaldehyde oxidoreductase OHprop.aldehyde .fwdarw. 1,3-propanediol

Homologous Integration and Ammonium Controlled Expression

[0182] The genes/cassettes, necessary for the different pathways and respective organic products in Synechocystis, are preferably introduced to Synechocystis via chromosomal integration. This will be achieved by homologous recombination which allows to precisely define the chromosomal site of insertion. Appropriate plasmids for this purpose known to be applicable in Synechocystis sp PCC 6830 are pBluescript (Stratagene, USA) or pGEM-T (Promega, USA). A strategy with respect to the construct is exemplified in FIG. 3 but alternative (neutral) docking sites for integration will be considered.

[0183] We will make use of the fact that expression of a number of glycolytic genes of Synechocystis are under control of a group 2 sigma-factor, .sup.E. In turn, expression of the gene encoding this factor, SigE, is switched on by the transcriptional regulator NtcA.sup.1,3. This switch is, amongst other unidentified signals, dependent on the extracellular nitrogen availability via the intracellular a-oxoglutarate/glutamate ratio: under conditions of ammonium depletion of the medium to less than 1 mM.sup.2, NtcA binds to a-oxoglutarate and the resulting NtcA-a-oxoglutarate complex has a high binding affinity for and positive control on the SigE promotor. Thus, a gene cassette under SigE control will be expressed upon ammonium depletion. As a consequence, during ammonium excess conditions, the carbon flux will be directed towards biosynthesis whereas in the stationary phase this flux will be directed to production (see FIG. 4). For Synechococcus the external ammonium treshold for the switch to .sup.E synthesis was found to be submillimolar range.sup.2.

Example 3: Alcohol Resistance

[0184] Synechocystis PCC 6803 strain was grown on BG-11 medium (Stanier R Y, et al. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. (1971) 35:171-205) in an orbital shaker at 30 C. under continuous illumination using two TL tubes, which provided average light intensity of approximately 70 E.Math.m.sup.2.Math.s.sup.1. To quantify the influence of alcohols on the net growth rate, cells were grown without any addition (control) or with butanol, respectively ethanol added at various concentrations.

[0185] After 5 days, 100 l of each culture was appropriately diluted and transferred to solid medium prepared from BG-11 and supplemented with 0.3% sodium thiosulfate, 10 mM N-tris[hydroxymethyl]-2-aminoethanesulfonic acid (TES) pH 8.2, 5 mM glucose and 1.5% bactoagar. Solid cultures were grown in an incubator 30 C. under continuous illumination. After a week, single colonies were observed and counted. The amount of colonies was compared to the control sample.

[0186] From the results shown below in FIG. 5, it is concluded that the net specific growth rate decreases linearly with increasing alcohol concentration and that the growth rate is reduced by 50% at concentrations of approximately 0.17 M butanol respectively 0.29 M ethanol. Therefore, it is to be expected that the two phases production process of the invention is much more efficient than a single phase production process.

TABLE-US-00002 TABLE3 listofallprimersused HOMOLOGYREGION1 SEQIDNO: Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI 37 Reverse AAATCTCGAGACCAGGACATCCGACTTGC XhoI 38 HOMOLOGYREGION2 Forward CACGACTAGTGTGACCGGGTCATTTTTTTGCTATTTATTCC SpeI 39 Reverse AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAG XbaI 40 Product:Lacticacid Forward1dh CAATCTCGAGATGGCTGATAAACAACGTAAG XhoI 41 Reverse1dh CAATGAATTCTTAGTTTTTAACTGCAGAAGCAAATTC EcoRI 42 Product:Ethanol Forwardpdc ATAACTCGAGGACAATAGGTGCTTTAATCAC XhoI 43 Reversepdc CGACGATATCAGGTGTAAAATACCATTTATTAAAATAG EcoRV 44 Forwardadh CATTGATATCATGTCTAACCGTTTGGATGG EcoRV 45 Reverseadh CATACTGCAGCTATTGAGCAGTGTAGCCAC PstI 46 Product:1,3-Propanediol Forwardgpd AAATCTCGAGTCAGTGGAGACAATAGTCG XhoI 47 Reversegpd AAATATCGATATGCGTAATTTCCCAGAAATC ClaI 48 Forwarddhak CATAAAGCTTATGAAATTCTATACTTCAACGACAG HindIII 49 Reversedhak AAATGATATCTTACCAGGCGAAAGCTC EcoRV 50 ForwardG1dehydr AAATATCGATTTATTCAATGGTGTCAGGCTG ClaI 51 ReverseG1dehydr CCAAAAGCTTATGAAAAGATCAAAACGATTTG HindIII 52 Forward GGGTGATATCTTAAGGTAAAGTAAAGTCAACCCAC EcoRV 53 oxidoreductase Reverse AAATGAATTCATGTTAAACGGCCTGAAAC EcoRI 54 oxidoreductase Lacticacid-IIsetofprimers SEQIDNO: Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI 55 HomologyI Reverse GTTGTTTATCAGCCATACCAGGACATCCGACTTG 56 HomologyI Reversefor CTGCGTGCAATCCATCTTGTTCAATCATTTAGTTTTTAACTGCAGAAGCAAATTC 57 ldh Reversefor GCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG 58 KAN Reversefor AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI 59 HomologyII Ethanol-IIsetofprimers Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI 60 HomologyI Reverse GATTAAAGCACCTATTGTCACCAGGACATCCGACTTG 61 HomologyII Reversepdc CTACCTTACCATCCAAACGGTTAGACATAGGTGTAAAATACCATTTATTAAAATAG 62 Reverseadh CAATCCATCTTGTTCAATCATCTATTGAGCAGTGTAGCCACCGTC 63 ReverseKAN GCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG 64 Reverse AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI 65 HomologyII 1,3-Propanediol Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI 66 HomologyI Reverse TATTGTCTCCACTGAACCAGGACATCCGACTTG 67 HomologyI Reversedhg CTGTCGTTGAAGTATAGAATTTCATATGCGTAATTTCCCAGAAATCCAAAATACG 68 Reversedhak GGTTCAGCCTGACACCATTGAATAATTACCAGGCGAAAGCTCCAGTTGGAGC 69 Reverse GTGGTTGACTTTACTTTACCTTAAATGAAAAGATCAAAACGATTTGCAGTACTGG 70 glycerol dehydrts Reverse CAATCCATCTTGTTCAATCATATGTTAAACGGCCTGAAACC 71 oxidored ReverseKAN GCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG 72 Reverse AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI 73 HomologyII Ethylene ForwardEfe TAAAGTCGACAAGGAGACTAGCATGACCAAC SalI 135 ReverseEfe TAAAGAATTCTTAGGAGCCGGTGG EcoRI 94 2-Butanol (Clostridium) forwardthl AAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG 99 reversethl TTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG 100 forward3bdh AAGGAGATTCCAATGAAAAAGGTATGTGTTATAG 101 reverse3bdh TTATTTTGAATAATCGTAGAAACCTTTTCCTG 102 forwardcrt-etf AAGGAGATTCCAATGTCAAAAGAGATTTATGAATCAG 103 reversecrt-etf CTACAATTTTTTTACCAAATTCAAAAACATTCC 104 forwardald AAGGAGATTCCAATGGATTTTAATTTAACAAGAG 105 reverseald TTATCTAAAAATTTTCCTGAAATAACTAATTTTCTGAACTTC 106 forwardbdh AAGGAGATTCCAATGCTAAGTTTTGATTATTCAATAC 107 reversebdh TTAATATGATTTTTTAAATATCTCAAGAAGCATCCTCTG 108 2-Butanol(L.lactisandK.pneumoniae) ForewardL. AAGGAGACTACTATGTCTGAGAAACAATTTGGGGC 109 lactisals ReverseL. TCAGTAAAATTCTTCTGGCAAT 110 lactisals ForewardL. AAGGAGACTACTATGTCAGAAATCACACAACTTTTTCA 111 lactisaldB ReverseL. TCATTCAGCTACATCAATATCTTTTTTCAAAGC 112 lactisaldB ForewardL. AAGGAGACTACTATGTCTAAAGTTGCAGCAGTTACTGG 113 lactisbutA ReverseL. TTAATGAAATTGCATTCCACCATC 114 lactisbutA ForewardL. AAGGAGACTACTGTGGCTTGGTGTGGAATCTGT 115 lactisbutB ReverseL. TTATAGACCTTTTCCAGTTGGTG 116 lactisbutB ForewardK. AAGGAGACTACTATGAAAAGATCAAAACGATTTGCAG 117 pneumoniae dhaB ReverseK. TCAGAATGCCTGGCGGAAAAT 118 pneumoniae dhaB ForewardK. AAGGAGACTACTATGAGCTATCGTATGTTTGATTATCTGG 119 pneumoniae dhaT ReverseK. TCAGAATGCCTGGCGGAAAAT 120 pneumoniae dhaT Acetone Forewardthl AAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG 121 Reversethl TTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG 122 ForewardctfAB AAGGAGGCGGCGATGAACTCTAAAATAATTAG 123 ReversectfAB TTATGCAGGCTCCTTTACTATATAAT 124 Forewardadc AAGGAGGCGGCGATGTTAAAGGATGAAGTA 125 Reverseadc CCCTTACTTAAGATAATCATATATAACTTCAGC 126 Propanol Forewardthl AAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG 127 Reversethl TTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG 128 ForewardctfAB AAGGAGGCGGCGATGAACTCTAAAATAATTAG 129 ReversectfAB TTATGCAGGCTCCTTTACTATATAAT 130 Forewardadc AAGGAGGCGGCGATGTTAAAGGATGAAGTA 131 Reverseadc CCCTTACTTAAGATAATCATATATAACTTCAGC 132 ForewardK. AAGGAGAATTCCAATGCATACCTTTTCTCTGC 133 pneumoniae aad ReverseK. TCATTGCAGGTTCTCCAGCAGTTGC 134 pneumoniae aad

REFERENCES

[0187] .sup.1Aichi, M., Takatani N., Omata T. (2001) Role of NtcB in activation of Nitrate ssimilation Genes in the Cyanobacterium Synechocystis sp. Strain PCC6803. J. Bacteriol. 183, 5840-5847 [0188] .sup.2Gillor, O., Harush, A., Post, A. F., Belkin, S. (2003) A Synechococcus PglnA::luxAB fusion for estimation of nitrogen bioavailability to freshwater cyanobacteria. Appl. Environm. Microbiol. 69, 1465-1474 [0189] .sup.3Osanai, T., Imamura, S., Asayama, M., Shirai, M., Suzuki, I., Murata, N., Tanaka, K, (2006) Nitrogen induction of sugar catabolic gene expression in Synechocystis sp. PCC 6803. DNA Research 13, 185-195