METABOLIC TRANSISTOR IN BACTERIA

Abstract

The disclosure relates to a metabolic transistor in microbes such as bacteria and yeast where a competitive pathway is introduced to compete with a product pathway for available carbon so as to control the carbon flux in the microbe.

Claims

1) A method of increasing the production of a product in a microbe, said method comprising: a) providing a microbe having a product pathway producing a product, wherein a competitive pathway competes with said product pathway for available carbon, said competitive pathway requiring a cofactor that is not rate limiting and not used in said product pathway; b) adding one or more diverting gene(s) under the control of a promoter to said microbe to directly or indirectly divert said cofactor away from said competitive pathway; and, c) allowing expression of said diverting gene(s) and reducing levels of said cofactor, and thus reducing levels of said competitive pathway; d) thereby increasing said product pathway and said product.

2) The method of claim 1, wherein said cofactor and said one or more diverting genes are selected from the group consisting of: TABLE-US-00017 Cofactor: Diverting gene(s) encoding: ubiquinone geranylgeranyl diphosphate synthase and phytoene synthase and phytoene desaturase ubiquinone geranyl diphosphate:4-hydroxybenzoate geranyltransferase biotin Biotin-CoA ligase or biotin CoA synthetase pantothenate pantothenate hydrolyase thiamine thiamine pyridinylase or aminopyrimidine aminohydrolase Folate or 4-aminobenzoate 1-monooxygenase or 4-aminobenzoate aminobenzoate decarboxylase Heme Siroheme synthase 3-Methyl-2-oxobutanoic 2-oxoisovalerate dehydrogenase or 3-methyl- acid 2-oxobutanoate dehydrogenase.

3) The method of claim 1, wherein said cofactor and said one or more diverting genes and said products are selected from the group consisting of: TABLE-US-00018 Cofactor: Diverting gene(s) encoding: Product: ubiquinone geranylgeranyl diphosphate Fermentative products or synthase and phytoene lactate or acetate ethanol or synthase and phytoene formate, or acetate or desaturase succinate or 2,3 butane diol ubiquinone geranyl diphosphate:4- Fermentative products or hydroxybenzoate lactate or acetate ethanol or geranyltransferase formate, or acetate or succinate or 2,3 butane diol biotin Biotin-CoA ligase or biotin Glutamate or lysine CoA synthetase pantothenate pantothenate hydrolyase Valine thiamine thiamine pyridinylase or Fermentative products or aminopyrimidine lactate or ethanol or formate, aminohydrolase or acetate or succinate or 2,3 butane diol pr Folate or 4- 4-aminobenzoate 1- Serine amino- monooxygenase or benzoate aminobenzoate decarboxylase Heme Siroheme synthase Fermentative products or lactate or acetate ethanol or formate or acetate or succinate or 2,3 butane diol 3-Methyl-2- 2-oxoisovalerate PHE or TYR. oxobutanoic dehydrogenase or 3-methyl- acid 2-oxobutanoate dehydrogenase

4) The method of claim 1, wherein said promoter is a constitutive promoter.

5) The method of claim 1, wherein said promoter is an inducible promoter.

6) A method of controlling aerobic respiration in a microbe in the presence of O.sub.2, said method comprising: a) adding a diverting gene(s) to a microbe to divert substrates away from ubiquinone or thiamine or heme production, wherein said diverting gene(s) is under the control of a promoter; and, b) inducing said promoter, thereby allowing expression of said diverting gene(s), thus reducing ubiquinone levels and reducing aerobic respiration in the presence of 0.sub.2.

7) The method of claim 6, wherein said diverting gene(s) includes the lycopene synthesis pathway.

8) The method of claim 6, wherein said diverting gene(s) include crtE, crtB and crtI.

9) The method of claim 6, wherein said diverting gene encodes geranyl diphosphate:4-hydroxybenzoate geranyltransferase.

10) The method of claim 6, wherein said diverting gene is lePGT-1.

11) The method of claim 6, wherein said diverting gene encodes thiamine pyridinylase or aminopyrimidine aminohydrolase.

12) The method of claim 6, wherein said diverting gene encodes siroheme synthase.

13) A method of producing a product in a microbe, comprising: a) growing a microbe capable of producing a product in cell culture under aerobic conditions; i) said microbe comprising a diverting gene(s) added to said microbe to divert a cofactor away from an electron transport chain, wherein said diverting gene(s) is under the control of an inducible promoter; b) inducing said promoter, thereby allowing expression of said diverting gene(s), thus reducing cofactors levels and reducing aerobic respiration in the presence of O.sub.2 and thereby driving carbons towards production of said product; and, c) isolating said product.

14) The method of claim 13, wherein said cofactor is ubiquinone and said diverting gene(s) is crtE, crtB and crtI or PGT-1.

15) The method of claim 13, wherein said cofactor is heme and said diverting gene(s) encodes siroheme synthase.

16) The method of claim 13, wherein said cofactor is thiamine and said diverting gene(s) encodes thiamine pyridinylase or aminopyrimidine aminohydrolase.

17) The method of claim 13, wherein said microbe is a bacteria.

18) The method of claim 13, wherein said microbe is a yeast.

19) The method of claim 13, wherein said cofactor and said one or more diverting genes are selected from the group consisting of: TABLE-US-00019 Cofactor: Diverting gene(s) encoding: ubiquinone geranylgeranyl diphosphate synthase and phytoene synthase and phytoene desaturase ubiquinone geranyl diphosphate:4-hydroxybenzoate geranyltransferase Heme Siroheme synthase

20) The method of claim 13, wherein said cofactor and said one or more diverting genes are selected from the group consisting of: TABLE-US-00020 Cofactor: Diverting gene(s) encoding: ubiquinone geranylgeranyl diphosphate synthase and phytoene synthase and phytoene desaturase ubiquinone geranyl diphosphate:4-hydroxybenzoate geranyltransferase Heme Siroheme synthase ubiquinone UDP-glucose:4-hydroxybenzoate 4-O-beta-D- glucosyltransferase (EC 2.4.1.194) ubiquinone 4-hydroxybenzoate carboxy-lyase (EC 4.1.1.61) ubiquinone 4-hydroxybenzoate:CoA ligase (EC 6.2.1.27) ubiquinone 5-phospho-alpha-D-ribose-1-diphosphate:4- hydroxybenzoate 5-phospho-beta-D- ribofuranosyltransferase (EC 2.4.2.54) ubiquinone a 4-HB 3-monooxygenase (Xcc0356) ubiquinone chorismate pyruvate-hydrolase (EC 3.3.2.13) ubiquinone chorismate pyruvate-lyase (EC 4.1.3.45) ubiquinone chorismate hydro-lyase (EC 4.2.1.151) ubiquinone UDPglucose:trans-4-hydroxycinnamate 4-O-beta-D- glucosyltransferase (EC 2.4.1.126) ubiquinone phenylacrylic acid decarboxylase (EC 4.1.1.102)

21) A microbial cell engineered to allow fine control over production of a product, wherein a diverting gene(s) has been added to said micobe, wherein said diverting gene(s) allows the diversion of a key intermediate of a competitive pathway, thus allowing fine control of said competitive pathway including said key intermediate, and increased production of a product not requiring said key intermediate.

22) The method of claim 21, wherein said microbe is a bacteria.

23) The method of claim 21, wherein said microbe is a yeast.

Description

DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 Schematic diagram of a metabolic-transistor in bacteria. ETC=electron transfer chain.

[0048] FIG. 2A-B. Schematic diagrams of an introduced Diverting Gene(s) which reduces Cofactor, thus decreasing Competitor and thereby increasing Product. FIG. 2A is a schematic diagram of competitive pathway C which competes with product pathway P for carbon resources. FIG. 2B shows the introduction of Diverting Gene(s), which divert cofactor away from C, thereby reducing flux to C and increasing flux to P.

[0049] FIG. 3. Manipulation of ubiquinone synthesis pathway as a controller of the respiratory chain in bacteria. Strategy one: thick arrow pathway introduced into E. coli cell; strategy two: dash arrow pathway introduced into E. coli cell.

[0050] FIG. 4. Schematic diagram of the plasmid pAC-LYC. lycopene pathway genes including crtE=gene for geranylgeranyl diphosphate synthase; crtB=gene for phytoene synthase; crtI=gene for phytoene desaturase.

[0051] FIG. 5A-B. Schematic diagram of the plasmids employed in this disclosure. FIG. 5A:_pTrc-lePGT; and FIG. 5B: pBlePGT. Abbreviations: Ptrc, trc promoter; Prom araC, araC promoter; AmpR, ampicillin resistant gene; CmR, chloramphenicol resistant gene; Replicate Ori, origin of replication; pACYC184 Ori, replication origin of pACYC184; lacIq, lac operon repressor; rrnBT1,2, 1,2 terminators of rrnB; restriction enzyme sites: NocI, KpnI, HindIll.

[0052] FIG. 6. Coenzyme Q8 concentration (nmol) per mg dry cell weight (DCW) as a function of the inducer, IPTG, concentration (M).

[0053] FIG. 7A-B. Heme diversion experiment, showing colony size on plates with different levels of inducer are shown after 24 or 48 hours. FIG. 7A: Heme diversion experiment results for GNB 11152 (pCA24N); FIG. 7B: Heme diversion experiment results for GNB 11152 (pCA24NcysG). Abbreviation: GNB11152 is BW25113, the Keio background host strain; pCAN24N is the vector; pCAN24NcysG contains the cysG gene under control of a lac regulated promoter. Cell growth is limited when heme is limited by this diverting gene.

[0054] FIG. 8. Heme diversion experiment, showing increase lactate production when heme is diverted.

DETAILED DESCRIPTION

[0055] We propose a new strategy based on network topology and indirect control of competitive pathways by introducing additional nodes where flow through the biosynthetic pathway of interest can be controlled by partitioning at these newly introduced nodes. In other words, we introduce a diverting pathway and use this diversion to negatively control the level of a key participant of a competitive pathway, and thus to slow the flux through a competitive pathway and thereby increasing the flux through the pathway of interest.

[0056] FIG. 2A shows the desired pathway to product P that competes for carbon flow with the competitive pathway C. Adding diverting genes in FIG. 2B will divert cofactor or key intermediate in pathway C, thus slowing it and instead allowing the carbon flux to flow to P. Ideally, the relative amounts of the enzymes and their Km values need to be within an appropriate range relative to the cofactor or key intermediate. If these factors are within the appropriate range, the level of key intermediate can be reduced substantially by the presence of the new diverting reaction(s) and control of the level of key product P can be controlled over a small range.

[0057] In the case of the ETC, slowing the ETC pathway by adding diverting gene(s) to reduce ubiquinone levels allows the carbons to be channeled into fermentative pathways, even though O.sub.2 is present. Thus, we can now take advantage of the high product yields that are available under fermentative conditions, even though O.sub.2 is present.

[0058] There are many circumstances where it would be desired to control the amount of respiration, as shown herein. Too much respiration and oxidation of carbon substrates, while being beneficial for cell energetics and usually for growth rate, lead to loss of carbon that does not go into product (but into CO.sub.2), especially if a reduced product is desired. Many reduced products of commercial interest, such as fuel molecules, are compounds more reduced than glucose, and many chemical intermediates for pharmaceuticals, lactate or monomers for making polymers, fatty acids, etc. also require reduction reactions and similarly need to limit oxidation of the feedstock for a high yield process.

[0059] It is difficult, however, to limit oxygen utilization in large reactorsthat is to try to control the level of oxygen by feeding in say 1% oxygen and having this be constant and well distributed in the tank. A means of limiting the use of oxygen by the cell even when there is excess air or oxygen would thus be very useful.

[0060] We can control the level of expression of the diverting gene(s) by placing it under control of an inducible promoter that we can control by an added inducer compound. We could also use a static or constitutive promoter of the appropriate strength to keep the proportion at a desired level, however, fine tunable promoters are preferred, especially where external control via culture and media conditions is desired.

[0061] Other variations of this general concept could employ (1) adding enzymes that degrade or use up the key intermediate; (2) adding a protein that binds the key intermediate and sequesters it from entering into its normal function; (3) making or enhancing a pathway that consumes an intermediate of the key intermediate, thus competing it away by altering partitioning through the associated network connected to the biosynthetic pathway.

[0062] The advantage of this kind of indirect diversion approach is that it uses very little cell energy and protein quantity to control an important flux in the cell. This is akin to the way a transistor works, where a small current change is used to control a big current flow. The other advantage is that it can be very finely tuned for optimal or desired level performance. Thus, by making small changes in the availability of essential small cofactor or key component for the activity of the enzyme process, a big flow can be affected by subtle changes in the availability of the key intermediate or cofactor.

Ubiquinone Diversion

[0063] To exemplify the inventive concept, we have established a metabolic transistor control strategy on respiratory chain and internal redox levels based on manipulation of the quinone synthesis pathway in E. coli.

[0064] E. coli cells regenerate NAD.sup.+ and generate proton motive force for ATP production through the respiratory chain. Quinones are lipid-soluble molecules that mediate electron transfer between NADH or FADH dehydrogenases and cytochrome oxidases. The major substrate precursor compounds of ubiquinone synthesis are polyprenyl diphosphate and 4-hydroxybenzoic acid (4-HB). The polyprenyl diphosphate is formed by a combination of multiple units of isopentenyl diphosphate (IPP) by polyprenyl diphosphate synthase.

[0065] Based on the quinone synthesis pathway, two strategies of controlling quinone synthesis can be applied in this invention (FIG. 3), where the thick arrow represents the strategy of using lycopene synthesis pathway as the diverting genes, and the dashed arrow represents the strategy of using the 4-hydroxybenzoate geranyltransferase gene (PGT) as a diverting gene. Either added pathway competes away precursors needed for synthesis of ubiquinone, thus reducing ETC by limiting available ubiquinone, which is not normally rate limiting.

[0066] The accession numbers for the various sequences discussed herein are as follows:

TABLE-US-00003 GenBank Accession Strain Gene or Gene ID Protein_ID Erwinia herbicola crtB JX871356 AFX61742.1 Erwinia herbicola crtE JX871355 AFX61741.1 Erwinia herbicola crtI JX871357 AFX61743.1 Lithospermum lepgt-1 AB055078 BAB84122.1 erythrorhizon Eschehchia coli ubiX 948926 AAC75371.1 Eschehchia coli ubiE 949033 AAT48227.1 Eschehchia coli ubiG 946607 AAC75292.1

[0067] One demonstrated example of the inventive technique is using the lycopene synthesis pathway to drain a common substrateisopentenyl diphosphate (IPP) (FIG. 3).

[0068] In previous studies, it was demonstrated that 1 mol of lycopene (C40) needed 8 mol of IPP (C5). The lycopene synthesis pathway thus can be a competing pathway on the common substrate, IPP, of the quinone synthesis pathway. Therefore we transformed the plasmid, which contained the lycopene production operon (crtB, crtE, crtI), into different genetic type E. coli strains to enhance the lycopene production pathway (FIG. 4), thus effectively competing away IPP, as desired based on the amount of promoter induction, in this case with IPTG.

[0069] A second strategy used the reaction of the geranyl diphosphate:4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon (lePGT-1) to divert both isopentenyl diphosphate (IPP) and 4-hydroxybenzoic acid (4-HB) (FIG. 3) away from the production of ubiquinone.

[0070] In a previous study, it was demonstrated that lePGT-1 had strict substrate specificity for geranyl diphosphate to form geranyl-4-HB (G-4HB) and was not involved in ubiquinone biosynthesis. The geranyl diphosphate is synthesized from dimethylallyl diphosphate and isopentenyl diphosphate by geranyl diphosphate synthase. This means that 2 mol of isopentenyl diphosphate can convert to 1 mol of geranyl diphosphate.

[0071] The reaction catalyzed by lePGT-1 also functions as a competing pathway on the substrates (IPP and 4-HB) of the quinone synthesis pathway. We therefore synthesized lePGT-1 and cloned the gene into two plasmids with different copy numbers (pTrc99a and pBAD33) (FIG. 5A, 5B), and transformed these plasmids into different genetic type E. coli strains to control the quinone synthesis pathway. The 925 by DNA fragment encoding the geranyl diphosphate:4-hydroxybenzoate geranyltransferase gene from Lithospermum erythrorhizon (lePGT-1) was synthesized and cloned into the vector pTrc99a and pBAD33.

[0072] The functionalities of each of these strategies to reduce quinone production leading to an increase in lactate accumulation were successfully demonstrated in aerobic shake flask fermentations (see Table 1-12). The experiments are described in further detail below. Lactate is a typical reductive product, which is usually produced under anaerobic conditions, but it is exemplary only, and other products such as formate, acetate, succinate, 1,2 PDO, ethanol, and the like are also being tested. Lactate is, however, an important chemical, which is used in a wide range of food-processing and pharmaceutical industries. It also can use as the monomer of the biodegradable polylactic acid, which was considered as an environmentally friendly plastic.

Lycopene Diverting Genes

[0073] The lycopene synthesis pathway (crtE, crtI, crtB) was introduced in engineered E coli strains to drain isopentenyl diphosphate (IPP) (FIG. 3) and reduce the activity of the respiratory chain. Two different host strains were applied in this experiment, BW25113 and MG1655. The introduction of these three genes is sufficient for E. coli to produce lycopene from an existing supply of IPP.

[0074] These host strains are commonly used for study of metabolic engineering on E. coli. BW25113 (F, (araD-araB)567, lacZ4787(::rrnB-3), .sup., rph-I, (rhaD-rhaB)568, hsdR514) was the original strain for single gene knockout collection, Keio collection. MG1655(F, .sup., ilvG, rfb-50, rph-l) is a wild-type E. coli that is well studied. The use of two strains also shows that the strategy is not host strain dependent.

[0075] We chose different expressed plasmid systems with different copy number and strength of promoter and combined these in some cases with mutations in the ubiquinone pathway. We chose to use the genes in the ubiquinone synthesis pathway, as mutants were not critical for cell growth, but had the effect of reducing the ubiquinone synthesis, thus amplifying the effects of the diversionary pathway. The overexpression plasmid pBAD33 is a low copy number plasmidwe needed to perform this experiment in a background strain with relatively low ubiquinone synthesis in order to clearly demonstrate the effect of the diversionary pathway.

[0076] These experiments demonstrate the objective can be attained in a variety of host strains and that a combination of some mutations in a host gene especially in a redundant pathway can adjust the basal level so the diversion or competition strategy for the intermediate works well over the levels of expression of the diverting pathway we employed.

[0077] The glucose consumption, lactate production and acetate production from aerobic shake flask fermentation experiments of BW25113 (pAC-LYC), BW25113 ubiX, BW25113 ubiX (pAC-LYC), BW25113 ubiE, BW25113 ubiE (pAC-LYC), BW25113 ubiG, and BW25113 ubiG (pAC-LYC) are shown in Table 1.

[0078] The ratio of carbon burnt in each culture was calculated at two time points, one was at the time with highest lactate concentration of the batch process, and the other one was at the end of the batch process. These ratios are shown in Table 2. The results based on MG1655 are shown in Table 3 and 4.

[0079] The highest concentrations of reduced product, lactate, in the mutants of BW25113 ubiX, BW25113 ubiE and BW25113 ubiG reached to 48.68, 50.03 and 54.47 mM, respectively. The lactate was re-consumed in the ubiX mutant strain, while it changed little in the other two mutants. A combination of a ubiX mutation and lycopene synthesis pathway overexpression in BW25113, which limited quinone synthesis, directed the carbon flux to reductive product in full aerobic conditions. The theoretical yield of lactate was achieved in BW25113 ubiX (pAC-LYC) (around 2 mol/mol glucose). In BW25113 ubiE (pAC-LYC) and BW25113 ubiG (pAC-LYC), the lactate yields were 1.67 and 1.75 mol/mol glucose, respectively (Table 1). No carbon was burnt aerobically in BW25113 ubiX (pAC-LYC), BW25113 ubiE (pAC-LYC) and BW25113 ubiG (pAC-LYC) (Table 2), while without the plasmid pAC-LYC, only 38.34, 15.07 and 10.9% of the carbon source was burnt during the aerobic culture of BW25113 ubiX, BW25113 ubiE and BW25113 ubiG, respectively (Table 2).

[0080] In MG1655, a combination of UbiX mutation (3-octaprenyl-4-hydroxybenzoate decarboxylase) and lycopene synthesis pathway overexpression enhanced the accumulation of lactate from 16.57 mM to 40.67 mM (Table 3). The carbon burnt in cultures of MG1655 ubiX (pAC-LYC) at the point of highest lactate concentration was reduced to 11.8%, while in MG1655 (pACYC184) about 37% of carbon was burnt to CO.sub.2 (Table 4).

PGT-1 Diverting Gene

[0081] The reaction catalyzed by the geranyl diphosphate:4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon (lePGT-1) was introduced into the E. coli strains to drain both isopentenyl diphosphate (IPP) and 4-hydroxybenzoic acid (4-HB) (FIG. 3), thus reducing the amount of ubiquinone that can be produced.

[0082] In this first experiment, lePGT-1 was overexpressed under the trc promoter in pTrc99a, in the constructed plasmid named pTrc-lePGT (FIG. 5A). The pTrc-lePGT was transformed into MG1655. The glucose consumption, lactate production and acetate production from aerobic shake flask fermentation experiments of MG1655 (pTrc-lePGT) using different concentrations of IPTG are shown in Table 5. The ratio of carbon burnt by the culture of the strains was calculated at two time points, one was at the time with highest lactate concentration of the batch process, and the other one was at the end of the batch process, were shown in Table 6.

[0083] The phenomena of glucose consumption, lactate and acetate production were similar when 0, 20 and 40 M concentrations of IPTG were added to induce lePTG expression. The metabolic flux of reduced carbon was highly responsive to induction levels. This was shown when a concentration of more than 40 M of IPTG was applied in the fully aerobic condition. In the conditions of 40, 60, 80, 100, 200, 500 and 1000 M of IPTG, the highest concentration of lactate accumulating in the culture increased from around 15 mM to about 66 mM. The concentration of acetate decreased from 130.04 to 63.77 mM. Lactate can be re-consumed when the concentration of IPTG in the culture was below 200 M.

[0084] The carbon burnt was also highly responsive to different induction conditions. From a concentration of 60 to 1000 M of IPTG, the more IPTG added, the less carbon was burnt during the culture process. When 1000 M of IPTG was added in the culture medium, only 10.05% of carbon was burnt to CO.sub.2 (Table 6).

[0085] In addition, preliminary data indicate the coenzyme Q8 concentration also showed a graded response to the inducer IPTG concentration (FIG. 6). Notice that the concentration of coenzyme Q8 (CoQ8) for the wild type strain at 0 M IPTG is at very low level, only about 1.2 nmole/mg DCW. The CoQ8 concentration dropped by an order of magnitude at higher IPTG concentrations (>80 M). The concentration at that low concentration level (10 pmole/mg DCW range) while detectable is below the accuracy of our current experimental protocol. These preliminary data also indicate that further refinement, such as using even more cells, is needed to increase our measuring capability to a higher accuracy at these super low concentrations.

[0086] Note that the CoQ8 concentration dropped significantly at 40 M IPTG when compared with that of 0 M IPTG (FIG. 6), while the amount of lactate accumulation was very similar (Table 5), suggesting that the wild type strain overproduces CoQ8 under normal aerobic conditions to ensure there is a sufficient quantity of CoQ8 for the ETC system. It is also of interest to note that while the concentration of CoQ8 is very low (FIG. 6), its effect on lactate accumulation is unusually large (in mM range per culture volume) a gain of 10,000,000 or 107 when comparing the change in the metabolite being directly controlled by the new node (ubiquinone) to the consequent major pathway (lactate) change.

[0087] In a second experiment, lePGT-1 was overexpressed under the arabinose-responsive ara promoter in pBAD33, in the constructed plasmid named pBlePGT (FIG. 5B). The pBlePGT plasmid was transformed into MG1655 ubiX, MG1655 ubiE and MG1655 ubiG.

[0088] The glucose consumption, lactate production and acetate production from aerobic shake flask fermentation experiments of MG1655 ubiX (pBlePGT), MG1655 ubiE (pBlePGT) and MG1655 ubiG (pBlePGT) using different concentrations of arabinose are shown in Table 7, 9 and 11, respectively. The ratio of carbon burnt in the cultures of the strains was calculated at two time points, one was at the time with highest lactate concentration of the batch process, and the other one was at the end of the batch process. The results are shown in Table 8, 10, and 12, respectively.

[0089] In the experiments of MG1655 ubiX (pBlePGT), with concentrations of 0, 0.5, 1, 2, 5, 10, 12, 16, 20 and 50 mM arabinose were investigated (Table 7). The peak value of lactate accumulation during the culture was slightly enhanced, from 28.81 to 32.51 mM, when the concentration of arabinose increased from 0 to 5 mM. The lactate production changed significantly when more arabinose (more than 5 mM) was added to the medium. When concentrations of 10, 12, 16, 20 and 50 mM arabinose were applied, the peak value of the lactate accumulation reached 43.06, 53.07, 68.6, 95.58 and 94.71 mM, respectively. The accumulated lactate can be consumed by the culture in the cases where concentrations of 0, 0.5, 1, 2, 5, 10 and 12 mM arabinose were used, while it became stable or kept increasing in concentrations of 16, 20 and 50 mM arabinose.

[0090] The carbon burnt was also highly responsive to different induction conditions. When concentrations of 20 or 50 mM arabinose were added in the medium, almost no carbon was burnt to CO.sub.2 (Table 8).

[0091] In the experiments using MG1655 ubiE (pBlePGT), concentrations of 0, 5, 10, 15 and 20 mM arabinose were investigated (Table 9). The UbiE minus strain with pBAD33 accumulated 64.76 mM of lactate. The induction of pBlePGT further enhanced the lactate production. In the conditions of 0, 5, 10, 15 and 20 mM arabinose, the lactate concentration reached 70.85, 75.23, 82.86, 92.61 and 94.83 mM, respectively.

[0092] The concentration of acetate decreased from 55.93 to 14.82 mM. The presence of the ubiE minus mutation in the strain reduced the amount of carbon burnt, to only 16.33%. Further induction of lePGT-1 can reduce the amount of carbon burnt to 0% (Table 10). The lactate yield reached to the anaerobic theoretical yield in the fully aerobic condition with this strain and growth conditions.

[0093] In the using of MG1655 ubiG (pBlePGT), concentrations of 0, 5, 10, 15 and 20 mM arabinose were investigated (Table 11). Cultures of the UbiG minus strain with pBAD33 accumulated 74.66 mM lactate. The induction of pBlePGT further enhanced the lactate production. In the conditions of 0, 5, 10, 15 and 20 mM arabinose induction of the culture, the lactate concentration reached 77.17, 80.6, 88.97, 93.92 and 92.05 mM, respectively. The concentration of acetate decreased from 50.76 to 10 mM.

[0094] In the cultures of UbiG minus strain the amount of carbon burnt, only 3.2%, much lower than the ubiE minus strain. Further induction of lePGT-1 can reduce the amount of carbon burnt to 0%. The lactate yield also reached to the anaerobic theoretical yield in the fully aerobic condition in this strain.

Methods

[0095] Aerobic shake flask experiments were performed at 30 C. with shaking at 250 rpm for 48 h with 1% inoculation in 50 ml LB broth medium supplied with 20 g/1 glucose, 100 mM phosphate and appropriate quantities of kanamycin, chloramphenicol and ampicillin. The initial pH was 7.0. Different concentrations of IPTG and arabinose were added initially in the media to control the expression of the added genes and/or pathways.

TABLE-US-00004 TABLE 1 Metabolite data from aerobic shake flask fermentation experiments of BW25113 (pAC-LYC), BW25113 ubiX, BW25113 ubiX (pAC-LYC), BW25113 ubiE, BW25113 ubiE (pAC-LYC), BW25113 ubiG, and BW25113 ubiG (pAC-LYC). lyco.sup.+: overexpression of CrtIBE from Erwinia herbicola in pACYC184 Time (h) Strain Relevant genotype Substrate and product 0 3 6 12 24 BW25113 (pAC-LYC) lyco.sup.+ Glucose consumption 0.00 0.96 11.15 50.38 63.37 BW25113 ubiX ubiX 0.00 0.81 8.59 46.81 60.95 BW25113 ubiX (pAC-LYC) ubiX, lyco.sup.+ 0.00 0.81 5.15 40.57 47.61 BW25113 ubiE ubiE 0.00 0.50 7.31 40.88 49.60 BW25113 ubiE (pAC-LYC) ubiE, lyco.sup.+ 0.00 0.00 6.93 39.47 47.40 BW25113 ubiG ubiG 0.00 0.058 9.55 41.95 50.37 BW25113 ubiG (pAC-LYC) ubiG, lyco.sup.+ 0.00 0.18 7.44 39.86 47.91 BW25113 (pAC-LYC) lyco.sup.+ Lactate production 0.00 0.081 2.00 27.00 0.0081 BW25113 ubiX ubiX 0.12 0.67 10.39 48.68 0.20 BW25113 ubiX (pAC-LYC) ubiX, lyco.sup.+ 0.00 0.50 11.75 95.69 101.50 BW25113 ubiE ubiE 0.00 2.00 12.42 50.03 46.94 BW25113 ubiE (pAC-LYC) ubiE, lyco.sup.+ 0.00 2.33 15.10 69.83 79.00 BW25113 ubiG ubiG 0.00 2.26 14.30 54.36 54.47 BW25113 ubiG (pAC-LYC) ubiG, lyco.sup.+ 0.00 2.08 15.28 73.52 83.66 BW25113 (pAC-LYC) lyco.sup.+ Acetate production 1.33 3.56 18.52 60.24 116.33 BW25113 ubiX ubiX 0.92 2.35 7.56 39.33 112.45 BW25113 ubiX (pAC-LYC) ubiX, lyco.sup.+ 1.01 2.10 3.57 6.98 17.23 BW25113 ubiE ubiE 0.80 1.16 5.65 34.66 55.95 BW25113 ubiE(pAC-LYC) ubiE, lyco.sup.+ 0.82 1.48 5.72 24.14 34.13 BW25113 ubiG ubiG 0.80 1.77 8.92 34.25 52.94 BW25113 ubiG (pAC-LYC) ubiG, lyco.sup.+ 0.00 1.38 6.17 20.07 27.01

TABLE-US-00005 TABLE 2 The ratio of carbon burnt from aerobic shake flask fermentation experiments of BW25113 (pAC-LYC), BW25113 ubiX, BW25113 ubiX (pAC-LYC), BW25113 ubiE, BW25113 ubiE (pAC-LYC), BW25113 ubiG, and BW25113 ubiG (pAC-LYC). Carbon burned.sup.a at the time Carbon burned point of highest at the time Relevant lactate concen- point of 24 Strain genotype tration (%) hours (%) BW25113 lyco.sup.+ 33.34 38.8 (pAC-LYC) BW25113 ubiX ubiX 20.00 38.34 BW25113 ubiX ubiX, lyco.sup.+ 0.00 0.00 (pAC-LYC) BW25113 ubiE ubiE 10.53 15.07 BW25113 ubiE ubiE, lyco.sup.+ 0.00 0.00 (pAC-LYC) BW25113 ubiG ubiG 10.9 10.9 BW25113 ubiG ubiG, lyco.sup.+ 0.00 0.00 (pAC-LYC) lyco.sup.+: overexpression of CrtIBE from Erwinia herbicola in pACYC184 .sup.aCarbon burnt refers to the difference between glucose consumption and metabolite accumulation, which can be attributed to CO.sub.2 being released from the culture. Reduced product refers to lactate, ethanol, and succinate, which consume NADH during synthesis

TABLE-US-00006 TABLE 3 Metabolite data from aerobic shake flask fermentation experiments of MG1655 (pACYC184), MG1655 (pAC-LYC), MG1655 ubiX (pAC-LYC), and MG1655 ubiX ldhA (pAC-LYC). lyco.sup.+: overexpression of CrtIBE from Erwinia herbicola in pACYC184 Substrate and Time (h) Strain Relevant genotype product 0 3 6 9 12 24 MG1655 (pACYC184) Glucose 0.00 1.38 14.38 32.28 38.00 52.14 MG1655 (pAC-LYC) lyco.sup.+ consumption 0.00 0.52 8.29 30.88 43.75 56.60 MG1655ubiX (pAC-LYC) ubiX, lyco.sup.+ 0.00 0.00 5.42 21.41 32.96 49.18 MG1655ubiX ldhA (pAC-LYC) ubiX, ldhA, lyco.sup.+ 0.00 0.070 0.31 3.67 17.25 53.46 MG1655 (pACYC184) Lactate 0.00 0.29 2.01 16.57 8.00 0.00 MG1655 (pAC-LYC) lyco.sup.+ production 0.00 0.00 0.35 14.98 13.91 0.00 MG1655ubiX (pAC-LYC) ubiX, lyco.sup.+ 0.00 1.10 13.03 34.18 40.67 0.00 MG1655ubiX ldhA (pAC-LYC) ubiX, ldhA, lyco.sup.+ 0.00 0.00 0.00 0.00 0.00 0.00 MG1655 (pACYC184) Acetate 0.00 4.85 20.08 36.16 56.97 96.26 MG1655 (pAC-LYC) lyco.sup.+ production 0.93 3.11 16.67 36.15 56.44 108.23 MG1655ubiX (pAC-LYC) ubiX, lyco.sup.+ 0.00 1.51 4.54 14.16 26.22 92.62 MG1655ubiX ldhA (pAC-LYC) ubiX, ldhA, lyco.sup.+ 0.00 1.51 2.04 5.68 22.06 94.91

TABLE-US-00007 TABLE 4 The ratio of carbon burnt from aerobic shake flask fermentation experiments of MG1655 (pACYC184), MG1655 (pAC-LYC), MG1655ubiX (pAC-LYC), and MG1655ubiXldhA (pAC-LYC) at 24 hours. Carbon burned at the time Carbon burned point of highest at the time Relevant lactate concen- point of 24 Strain genotype tration (%) hours (%) MG1655 37.00 38.46 (pACYC184) MG1655 lyco.sup.+ 36.71 36.26 (pAC-LYC) MG1655ubiX ubiX, lyco.sup.+ 11.80 37.22 (pAC-LYC) MG1655ubiX ubiX, ldhA, lyco.sup.+ 40.82 40.82 ldhA (pAC- LYC) lyco.sup.+: overexpression of CrtIBE from Erwinia herbicola in pACYC184

TABLE-US-00008 TABLE 5 Metabolite data from aerobic shake flask fermentation experiments of MG1655 (pTrc-lePGT) in different concentrations of IPTG. PGT-1.sup.+: overexpression of geranyl diphosphate: 4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pTrc99A. Relevant IPGT Substrate Time (h) Strain genotype (m) and product 0 3 6 9 12 15 24 30 36 48 MG1655 PGT-1.sup.+ 0 Glucose 0.00 0.29 7.15 24.51 34.19 43.82 53.68 58.68 61.86 67.81 (pTrc- 20 consumption 0.00 0.00 6.91 22.86 32.93 41.54 51.43 56.23 59.68 64.56 lePGT) 40 0.00 0.41 6.74 22.64 34.21 41.14 52.20 56.07 60.37 66.00 60 0.00 0.55 3.82 19.37 39.44 45.64 53.71 58.63 63.20 69.28 80 0.00 0.63 3.23 19.38 3.00 46.36 52.94 57.26 62.21 68.54 100 0.00 0.68 3.10 20.18 38.21 46.50 52.63 56.39 60.96 68.26 200 0.00 2.76 5.88 24.76 38.09 45.20 50.32 53.98 57.30 61.02 500 0.00 1.58 5.88 19.91 38.62 44.68 50.79 54.04 56.87 60.56 1000 0.00 1.83 5.53 19.84 38.36 44.24 50.50 53.93 56.54 60.19 MG1655 PGT-1.sup.+ 0 Lactate 0.00 0.074 0.38 6.75 15.06 3.65 0.00 0.00 0.00 0.00 (pTrc- 20 production 0.00 0.00 0.20 5.81 13.58 3.61 0.00 0.00 0.00 0.00 lePGT) 40 0.00 0.00 0.00 5.00 15.09 5.65 0.00 0.00 0.00 0.00 60 0.00 0.00 0.6 6.77 25.78 25.27 1.36 0.00 0.00 0.00 80 0.00 0.00 0.86 10.97 37.79 39.79 17.51 8.71 4.08 4.50 100 0.00 0.00 1.08 14.94 48.39 51.82 35.2 28.54 26.97 30.04 200 0.00 0.00 1.28 19.95 53.33 55.72 55.6 57.46 58.44 64.54 500 0.00 0.00 1.04 20.96 52.94 56.54 57.49 59.16 60.94 66.1 1000 0.00 0.00 0.38 21.01 53.11 58.17 58.24 59.48 60.5 65.77 MG1655 PGT-1.sup.+ 0 Acetate 0.00 2.12 10.27 29.67 45.1 67.12 97.74 107.25 114.31 130.79 (pTrc- 20 production 0.00 2.29 11.05 30.1 46.99 66.00 96.09 105.33 112.15 128.62 lePGT) 40 0.00 0.5 10.4 29.15 46.20 64.43 93.41 104.81 112.55 130.4 60 0.00 2.14 8.64 25.65 39.60 58.79 102.66 113.26 119.83 136.65 80 0.00 2.05 5.39 24.21 37.24 53.45 87.68 105.40 115.20 133.06 100 0.00 1.91 8.01 21.67 33.97 47.22 75.21 88.92 97.09 110.08 200 0.00 2.28 8.01 14.96 28.93 35.65 49.62 55.38 59.25 64.79 500 0.00 0.00 6.99 18.24 27.50 34.90 48.29 53.98 58.01 64.01 1000 0.00 2.04 7.09 18.15 27.21 34.65 47.84 53.24 58.00 63.77

TABLE-US-00009 TABLE 6 The ratio of carbon burnt from aerobic shake flask fermentation experiments of MG1655 (pTrc- lePGT) in different IPTG concentration. Carbon burned.sup.a at the time Carbon burned point of highest at the time Relevant IPGT lactate concen- point of 48 Strain genotype (m) tration (%) hours (%) MG1655 PGT-1.sup.+ 0 33.99 35.70 (pTrc-lePGT) 20 31.81 33.59 40 32.92 34.13 60 33.84 34.25 80 18.66 32.01 100 10.44 24.24 200 11.72 11.72 500 10.19 10.19 1000 10.05 10.05 PGT-1.sup.+: overexpression of geranyl diphosphate:4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pTrc99A. .sup.aCarbon burnt refers to the difference between glucose consumption and metabolite accumulation, which can be attributed to CO.sub.2 released from the culture. Reduced product refers to lactate, ethanol, and succinate, which consume NADH during synthesis.

TABLE-US-00010 TABLE 7 Relevant Arabinose Substrate and Time (h) Strain genotype (mM) product 0 3 6 9 12 15 24 30 36 48 Metabolite data from aerobic shake flask fermentation experiments of MG1655 ubiX (pBlePGT) in different concentrations of arabinose. PGT-1.sup.+: overexpression of geranyl diphosphate: 4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pBAD33 which is under the control of the arabinose inducible promoter. MG1655ubiX, ubiX, 0 Glucose 0.00 3.01 7.84 18.14 30.86 35.69 49.22 49.78 57.01 61.7 (pBlePGT) PGT-1.sup.+ 0.5 consumption 0.00 0.54 6.17 18.23 29.52 33.96 47.74 51.02 53.85 60.6 1 0.00 0.23 6.13 17.27 30.25 34.53 47.25 51.16 55.77 59.5 2 0.00 0.00 3.48 16.82 30.18 34.15 47.00 51.28 54.6 59.89 5 0.00 0.00 2.61 16.9 30.37 34.87 47.00 51.02 55.26 59.81 10 0.00 0.71 4.14 19.24 35.4 41.81 45.73 52.28 57.29 61.51 12 0.00 0.028 4.14 20.44 35.94 41.61 47.89 50.42 54.24 59.51 16 0.00 0.69 3.53 22.39 39.09 42.79 48.47 52 55.35 57.81 20 0.00 0.00 1.95 21.46 36.15 38.65 44.13 45.81 48.03 49.7 50 0.00 0.64 6.4 19.72 35.95 37.41 42.8 43.11 45.16 46.43 MG1655ubiX, ubiX, 0 Lactate 0.00 0.35 7.4 22.14 28.81 19.67 0.00 0.00 0.00 0.00 (pBlePGT) PGT-1.sup.+ 0.5 production 0.00 0.3 7.75 22.14 29.4 18.87 0.00 0.00 0.00 0.00 1 0.00 0.26 7.57 22.08 28.33 21.22 0.00 0.00 0.00 0.00 2 0.00 0.25 7.17 22.19 30.96 21.2 0.00 0.00 0.00 0.00 5 0.00 0.34 7.36 22.9 32.51 22.24 0.00 0.00 0.00 0.00 10 0.00 0.00 6.93 25.81 43.06 42.12 3.69 0.00 0.00 0.00 12 0.00 0.00 5.36 28.4 51.86 53.07 33.018 22.25 16.07 11.97 16 0.00 0.00 5.51 35.83 64.09 66.29 64.45 65.31 66.3 68.6 20 0.00 0.00 7.3 44.79 76.32 79.11 85.22 89.26 91.26 95.58 50 0.00 0.00 7.28 42.65 79.11 83.04 86.34 90.02 91.04 94.71 Metabolite data from aerobic shake flask fermentation experiments of MG1655 ubiX (pBlePGT) in different concentrations of arabinose. PGT-1.sup.+: overexpression of geranyl diphosphate: 4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pBAD33. MG1655ubiX, ubiX, 0 Acetate 0.82 1.61 4.50 14.43 26.80 40.42 86.66 95.95 99.77 116.65 (pBlePGT) PGT-1.sup.+ 0.5 production 0.00 1.40 4.77 14.68 26.92 40.88 87.08 96.63 102.78 118.21 1 0.00 1.58 4.66 14.52 26.98 41.13 87.52 96.99 103.48 119.41 2 0.00 1.47 4.57 14.41 26.88 41.53 87.40 96.85 101.44 119.51 5 0.00 1.44 4.36 14.42 26.65 40.56 87.51 97.12 99.60 119.12 10 0.00 1.53 4.47 13.46 24.37 38.39 80.60 100.02 104.58 124.94 12 0.00 1.03 4.24 13.10 21.85 33.32 59.09 74.51 84.96 104.84 16 0.00 1.27 4.17 11.62 19.14 22.62 36.02 41.92 46.01 52.93 20 0.00 1.54 4.24 9.47 14.30 16.04 20.84 23.53 25.33 28.85 50 0.00 1.50 3.79 6.43 8.71 9.57 10.79 11.95 12.35 13.12

TABLE-US-00011 TABLE 8 The ratio of carbon burnt from aerobic shake flask fermentation experiments of MG1655 ubiX (pBlePGT) in different IPTG concentrations at the time point of highest lactate concentration. Carbon burned.sup.a at the time Carbon burned Arab- point of highest at the time Relevant inose lactate concen- point of 48 Strain genotype (mM) tration (%) hours (%) MG1655ubiX ubiX, 0 24.36 36.99 (pBlePGT) PGT-1.sup.+ 0.5 19.79 34.97 1 23.44 33.10 2 19.01 33.48 5 17.23 33.61 10 16.23 32.29 12 9.54 31.22 16 10.16 10.16 20 0.00 0.00 50 0.00 0.00 PGT-1.sup.+: overexpression of geranyl diphosphate:4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pBAD33. .sup.aCarbon burnt refers to the difference between glucose consumption and metabolite accumulation, which can be attributed to CO2 released from the culture. Reduced product refers to lactate, ethanol, and succinate, which consume NADH during synthesis.

TABLE-US-00012 TABLE 9 Metabolite data from aerobic shake flask fermentation experiments of MG1655 ubiE (pBlePGT) in different concentrations of arabinose. PGT-1.sup.+: overexpression of geranyl diphosphate: 4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pBAD33. Relevant Arabinose Substrate Time (h) Strain genotype (mM) and product 0 6 9 12 15 24 30 36 48 MG1655 ubiE, ubiE Glucose 0.00 1.15 8.05 18.85 37.35 48.51 52.00 57.17 62.38 (pBAD33) consumption MG1655ubiE, ubiE, 0 0.00 2.98 6.72 11.53 28.57 48.20 52.51 57.14 60.22 (pBlePGT) PGT-1.sup.+ 5 0.00 0.85 5.15 9.12 29.32 48.04 52.09 55.83 58.49 10 0.00 1.15 5.07 9.37 31.03 47.73 51.37 53.90 55.05 15 0.00 3.31 5.02 9.81 29.02 44.97 48.18 50.63 52.28 20 0.00 2.77 5.56 8.51 24.85 43.17 46.99 48.75 49.97 MG1655ubiE, ubiE Lactate 0.00 1.07 5.99 23.73 47.84 52.17 54.07 57.51 64.76 (pBAD33) production MG1655ubiE, ubiE, 0 0.00 0.91 3.58 13.87 39.51 57.33 59.09 61.50 70.85 (pBlePGT) PGT-1.sup.+ 5 0.00 0.80 3.41 13.56 41.04 61.14 64.53 67.53 75.23 10 0.00 0.81 3.33 13.75 48.84 71.00 73.45 77.43 82.86 15 0.00 0.70 3.04 12.34 50.67 82.02 84.73 86.78 92.61 20 0.00 0.69 2.88 10.94 41.08 85.19 87.14 88.91 94.83 MG1655ubiE, ubiE Acetate 0.00 1.00 2.34 8.75 20.94 44.03 50.82 54.59 59.44 (pBAD33) production MG1655ubiE, ubiE, 0 0.00 0.78 1.67 4.39 14.39 40.23 45.97 50.72 55.93 (pBlePGT) PGT-1.sup.+ 5 0.00 0.92 1.74 4.17 13.24 36.02 40.69 44.86 48.53 10 0.00 1.00 1.61 3.95 11.42 27.64 30.77 33.14 36.18 15 0.00 1.03 1.59 3.09 6.56 15.41 17.35 18.48 19.75 20 0.00 1.02 1.44 2.59 4.28 11.02 12.45 13.66 14.82

TABLE-US-00013 TABLE 10 The ratio of carbon burnt.sup.a from aerobic shake flask fermentation experiments of MG1655 ubiE (pBlePGT) in different IPTG concentrations at the time point of highest lactate concentration. Carbon burned.sup.a at the time Carbon burned Arab- point of highest at the time Relevant inose lactate concen- point of 48 Strain genotype (mM) tration (%) hours (%) MG1655 ubiE, ubiE 16.33 16.33 (pBAD33) MG1655ubiE, ubiE, 0 10.21 10.21 (pBlePGT) PGT-1.sup.+ 5 8.03 8.03 10 2.83 2.83 15 0.00 0.00 20 0.00 0.00 PGT-1.sup.+: overexpression of geranyl diphosphate:4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pBAD33. .sup.aCarbon burnt refers to the difference between glucose consumption and metabolite accumulation, which can be attributed to CO2 released from the culture. Reduced product refers to lactate, ethanol, and succinate, which consume NADH during synthesis.

TABLE-US-00014 TABLE 11 Metabolite data from aerobic shake flask fermentation experiments of MG1655 ubiG (pBlePGT) in different concentrations of arabinose. PGT-1+: overexpression of geranyl diphosphate: 4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pBAD33. Relevant Arabinose Substrate Time (h) Strain genotype (mM) and product 0 12 15 18 21 24 27 36 48 MG1655ubiG, ubiG Glucose 0.00 9.37 11.77 32.38 39.67 44.87 46.78 50.56 55.96 (pBAD33) consumption MG1655ubiG, ubiG, 0 0.00 7.74 17.56 36.47 42.36 45.46 46.54 51.28 56.51 (pBlePGT) PGT-1.sup.+ 5 0.00 0.00 14.40 35.58 41.46 43.75 44.42 49.78 54.38 10 0.00 0.59 13.79 36.66 40.98 43.19 44.15 47.74 51.05 15 0.00 0.61 7.12 34.08 39.38 41.57 42.54 45.26 48.10 20 0.00 1.77 4.96 25.42 39.67 41.05 41.99 45.06 47.19 MG1655ubiG, ubiG Lactate 0.00 7.38 26.44 51.19 60.03 60.28 61.06 68.11 74.66 (pBAD33) production MG1655ubiG, ubiG, 0 0.00 9.27 33.10 57.32 61.47 61.72 64.16 70.11 77.17 (pBlePGT) PGT-1.sup.+ 5 0.00 8.76 30.53 58.63 63.87 64.81 67.95 73.84 80.60 10 0.00 7.86 31.76 69.91 73.28 75.03 78.18 84.02 88.97 15 0.00 6.23 22.41 69.44 80.09 81.99 83.98 88.83 93.92 20 0.00 4.87 15.94 45.99 78.09 81.94 84.53 87.84 92.05 MG1655ubiG, ubiG Acetate 0.00 2.71 7.41 17.82 29.14 34.36 38.27 45.56 50.52 (pBAD33) production MG1655ubiG, ubiG, 0 0.00 2.78 10.32 21.89 30.85 35.54 39.20 46.23 50.76 (pBlePGT) PGT-1.sup.+ 5 0.00 2.68 7.75 19.60 28.20 32.49 35.38 41.28 45.45 10 0.00 2.35 7.58 15.15 20.56 23.56 25.51 29.50 32.15 15 0.00 1.82 3.23 6.33 8.92 10.31 11.02 12.60 13.82 20 0.00 1.52 2.58 3.68 5.93 7.14 7.87 9.50 10.00

TABLE-US-00015 TABLE 12 The ratio of carbon burnt from aerobic shake flask fermentation experiments of MG1655 ubiG (pBlePGT) at different IPTG concentrations at the time point of highest lactate concentration. Carbon burned.sup.a at the time Carbon burned Arab- point of highest at the time Relevant inose lactate concen- point of 48 Strain genotype (mM) tration (%) hours (%) MG1655 ubiG, ubiG 3.20 3.20 (pBAD33) MG1655ubiG, ubiG, 0 1.78 1.78 (pBlePGT) PGT-1.sup.+ 5 0.00 0.00 10 0.00 0.00 15 0.00 0.00 20 0.00 0.00 PGT-1.sup.+: overexpression of geranyl diphosphate:4-hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon in pBAD33. .sup.aCarbon burnt refers to the difference between glucose consumption and metabolite accumulation, which can be attributed to CO2 released from the culture. Reduced product refers to lactate, ethanol, and succinate, which consumes NADH during synthesis.

Biotin Diversion to Increase Glutamate

[0096] Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is already known that biotin limitation triggers L-glutamate production (Peters-Wendisch , 2012). The inventive method could therefore be used to limit biotin availability in the host and thus increase glutamate production.

[0097] Biotin-CoA ligase aka biotin CoA synthetase [E.C. 6.2.1.11] catalyzes the following reaction:

ATP+biotin+CoA=AMP+diphosphate+biotinyl-CoA

[0098] Cloning and overexpressing biotin coA synthetase (e.g. YP_004681889; YP_841268.1; AEI80656.1; CAJ96538.1) will thus reduce the level of biotin for forming active carboxylating enzymes, thus leading to increased glutamate production.

Folate Diversion to Increase Serine

[0099] In the case of serine production by Corynebacterium glutamicum, the reduction of folate allows higher production of serine (Stolz, 2007). Folate availability can be reduced by diversion of biosynthetic intermediates. For example 4-aminobenzoate is needed in the synthesis of 7,8-dihydropteroatea precursor of folate. This could be depleted by overexpression of 4-aminobenzoate 1-monooxygenase [E. C. 1.14.13.27] or aminobenzoate decarboxylase [E.C. 4.1.1.24] and thus depleting the level of the intermediate 4-aminobenzoate. Either enzyme could be overexpressed and deplete the availability of this precursor and reduce the level of activity of the folate compounds with the consequences of increased serine production.

Pantothenate Diversion to Increase Valine

[0100] In this example the biosynthesis of pantothenate, a precursor of coenzyme A, can be reduced by diversion and this will lead to flow of metabolism to the precursors of valine, resulting in increased valine production. The level of pantothenate can be reduced by overexpression of the enzyme a pantothenate hydrolyase [E.C. 3.5.1.22]. The effects of reduced pantothenate were examined in Blombach (2007), and increased valine was observed.

[0101] There are many other ways to induce a shift away from aerobic respiration towards anaerobic pathways, and thus increase the production of products such as lactate, succinate and the like. In the experiments above, this was demonstrated using lycopene diverting genes as well as the PGT gene to divert precursors away from ubiquinone production, thus slowing the ETC. However, there are other cofactors that can be targeted in the same pathway. The following examples describe some of these additional methods.

Heme Diversion

[0102] Another targetable cofactor in the ETC is the heme normally required to reduce cytochrome oxidase activity and allow aerobic respiration.

[0103] In aerobic respiration, cytochrome proteins play an essential role as terminal oxidases with Cyo and Cyd being dominant at high oxygen and low oxygen levels, respectively (Borisov et al, 2011; Thony-Meyer, 1997). Heme synthesis and incorporation are important in production of functional cytochromes and in efforts to overproduce heme-containing proteins in E. coli where difficulties have often been encountered (Londer, 2011; Tsai et al, 2000; Varnado et al, 2013). The biosynthetic pathway of heme has been determined and enzymes and genes are known.

[0104] Heme deficient mutants do not consume oxygen and form lactate as the main fermentation product from glucose (Schellhorn & Hassan, 1988). At a branch the pathway is connected to the formation of siroheme via CysG, uroporphyrinogen III methylase (Spencer et al, 1993; Warren et al, 1994; Warren et al, 1990). This reaction branches from uroporphyrinogen III to siroheme while that catalyzed by HemE proceeds along the heme biosynthetic pathway via HemeNGH to protoheme IX and on to form heme o and heme d, the prosthetic groups of cytochrome o oxidase and cytochrome d oxidase.

[0105] This arrangement of the metabolic pathway suggests that overexpression of cysG could divert substrate from the pathway and reduce the level of heme for the respiratory cytochrome oxidases and thereby limit respiration. In preliminary experiments to prove this, we have observed a reduction of aerobic growth of cysG overexpressing cells where cysG is overexpressed from plasmid under control of a lac-controlled promoter (see FIG. 7A-B). This is particularly evident in hemN or hemF background strains where heme synthesis is lowered by mutation of one of these redundant pathway genes and smaller colonies were seen on plates where cysG was partially induced. Increased lactate was also observed (see FIG. 8).

Thiamine Diversion

[0106] Another route to achieve divert carbons towards the products of anaerobic metabolism, is to limit the availability of thiamine-needed for aerobic pyruvate dehydrogenase (PDH) formation (Taboada, 2008). This would allow us to make products derived from pyruvate, such as lactate, alanine, butane diol and the like, and acetolactate derived products, such as branched chain amino acids and the like. Precursors of thiamine could be diverted to reduce the availability of subsequent thiamine precursors or by introducing a degradative enzyme of thiamine, such as thiamine pyridinylase [EC:2.5.1.2] or the enzyme aminopyrimidine aminohydrolase [E.C. 3.5.99.2] catalyzing the hydrolysis of thiamine.

Lipoic Acid Diversion

[0107] The reduction of lipoate would also be expected to result in a shift toward more lactate, indicative of a loss in ability to perform aerobic respiration, as has been observed in mutants where the pyruvate dehydrogenase (PDH) is completely knocked out and non-functional.

[0108] Very little lipoic acid is needed by cells. Analysis of lipoate biosynthetic mutants showed that lipB mutants have reduced PDH activity and reduced alpha ketoglutarate dehydrogenase (KGDH) activity and had about a 10 fold reduction in protein bound lipoic acid and free lipoic acid (Reed & Cronan, 1993). The levels of LipA and LipB are below the limits of proteomic analyses (300 molecules/cell) (Hassan & Cronan, 2011; Lu et al, 2007) and PDH complexes can retain full enzymatic activity despite containing E2 proteins that have only partial lipoylation and either lack lipoyl domains or contain some lipoyl domains that cannot be modified (Perham, 1991).

[0109] The PDH activity of the lipB deletion strain ZX221 is about 4% of wild type and it is unable to grow aerobically on glucose minimal medium without supplementation with lipoic acid, octanoic acid or succinate and acetate (Hermes & Cronan, 2009). The overproduction of LplA protein, a protein that can use free octanoic acid as substrate, or high affinity mutants of LplA can allow growth of lipB mutants by using octanoic acid (naturally present in cells at a concentration of 28 micromolar) as a precursor of lipoate (Hermes & Cronan, 2009; Morris et al, 1995).

[0110] If a diverting gene is added to the cell that can utilize the octanoate at an appropriately low level, e.g. the fatty acid degradation system, this would be expected to reduce the level of this needed substrate for LplA and reduce the amount of functional PDH containing its essential lipoate prosthetic group. We could employ the genes of the fatty acid degradation system (e.g. fadD of E. coli expressed at varying levels, or if not sufficient, the more efficient enzymes form Salmonella enterica) (Iram & Cronan, 2006). The Km of E. coli acyl-CoA synthase on octanoic acid is reported between 6 and 40 micromolar, near the estimated concentration of octanoic acid in normal cells, (Kameda & Imai, 1985) and below that of the Km of normal LplA (Hermes & Cronan, 2009).

[0111] The base strain would be ZX221 (Hermes & Cronan, 2009) or equivalent, and the fad system, fadD the fatty acid acyl-CoA synthase, would be cloned and expressed on a low copy number vector to create a diverting pathway to reduce lipase levels. Alternatively, its expression could be altered in the chromosome by using a fadR mutant to deregulate its expression or introducing a modified set of promoters to replace the fadD natural promoter within the chromosome to create a library of strains with different expression levels of fadD to reduce the level of octanoic acid in the cell. Methods for analysis of those low levels of octanoic acid have been described (Hermes & Cronan, 2009).

[0112] It is expected that modifying the level of fadD will divert lipoic acid, thus decreasing the levels of active PDH, and allowing increased production of lactate, and other products produced by fermentation.

Inhibitor Diversion to Increase TYR & PHE

[0113] Another way of affecting metabolism via diversion of a metabolite is through the removal of a feedback process, e.g. reducing the level of a metabolite that has a negative feedback on a pathway via inhibition of an enzyme. Reducing of the feedback is often addressed by isolating or using a feedback-resistant variant of the enzyme. Instead, we suggest reducing the level of the molecule causing the feedback, either through diversion of formation of the feedback metabolite, or reducing its level by metabolism (degradation or conversion to a non-feedback active molecule) of the feedback molecule.

[0114] One example in the formation of aromatic amino acids. Tyrosine and phenylalanine are synthesized by a final enzyme TyrB, which is inhibited by 3-methyl-2-oxobutanoate. The effects of altering the level of 3-methyl-oxobutanoate would be easiest to see in mutants with ilvE and aspC mutations, the other transaminases that can make the PHE and TYR. If valine is degraded in the cell, the compound 3-methyl-2-oxobutanoate is formed. We can further consume the 3-methyl-2-oxobutanoate by activating it to a CoA-derivative using 3-methyl-2-oxobutanoate dehydrogenase [E.C. 1.2.7.7], this would deplete the 3-methyl-2oxobutanoate level and reduce inhibition of the pathway to the aromatic amino acids PHE and TYR, thereby increasing the level of these desired amino acids.

[0115] Another enzyme that could be used to reduce the level of 3-methyl-2-oxobutanoate is Kegg Reaction R01210, 2-oxoisovalerate dehydrogenase, which catalyzes the following:

3-Methyl-2-oxobutanoic acid+CoA+NAD.sup.+<=>2-Methylpropanoyl-CoA+CO.sub.2+NADH+H.sup.

[0116] Overexpression of either gene to make these enzymes should deplete the feedback from the 3-methyl-2-oxobutanoate on the transaminase and thus increase formation of PHE or TYR, as compared to when these enzymes are not expressed in excess.

[0117] Current experiments are underway in yeast (S. cerevisea) to demonstrate the invention will work in these diverse species. Exemplary yeast have been made, but testing is ongoing.

[0118] Other possible nodes and enzymes for ETS limitation through competition include:

TABLE-US-00016 Acting at the 4-hydroxybenzoate node 1. UDP-glucose:4-hydroxybenzoate 4-O-beta-D-glucosyltransferase UDP-glucose + 4-hydroxybenzoate = UDP + 4-(beta-D-glucosyloxy)benzoate EC 2.4.1.194 2. 4-hydroxybenzoate carboxy-lyase 4-Hydroxybenzoate <=> Phenol + CO2 4.1.1.61 3. 4-hydroxybenzoate:CoA ligase ATP + 4-Hydroxybenzoate + CoA <=> AMP + Diphosphate + 4-Hydroxybenzoyl-CoA 6.2.1.27 4. 5-phospho-alpha-D-ribose-1-diphosphate:4-hydroxybenzoate 5-phospho-beta-D- ribofuranosyltransferase 5-Phospho-alpha-D-ribose 1-diphosphate + 4-Hydroxybenzoate <=> 4-(beta-D-Ribofuranosyl)phenol 5-phosphate + CO2 + Diphosphate 2.4.2.54 5. PobA of Xanthanmonas gene Xcc0356 a 4-HB 3-monooxygenase that converts 4-Hydroxybenzoate into PCA, 3,4 dihydroxybenzoate Competition at the chorismate node 1) chorismate pyruvate-hydrolase Chorismate + H2O <=> (4R,5R)-4,5-Dihydroxycyclohexa-1(6),2-diene-1- carboxylate + Pyruvate 3.3.2.13 2) chorismate pyruvate-lyase (3-hydroxybenzoate-forming) Chorismate <=> 3-Hydroxybenzoate + Pyruvate 4.1.3.45 3) chorismate hydro-lyase (3-[(1-carboxyvinyl)oxy]benzoate-forming) Chorismate <=> 3-[(1-Carboxyvinyl)oxy]benzoate + H2O 4.2.1.151 Acting on precursors of 4-HB 1) UDPglucose:trans-4-hydroxycinnamate 4-O-beta-D-glucosyltransferase UDP-glucose + 4-Coumarate <=> UDP + 4-O-beta-D-Glucosyl-4-hydroxycinnamate 2.4.1.126 2) phenylacrylic acid decarboxylase 4-Coumarate <=> 4-Hydroxystyrene + CO2 4.1.1.102

[0119] Other exporting systems that would remove the precursor (e.g., if overexpressed, they would reduce the level of the ubiquinone precursor) include:

[0120] Overexpression of an aromatic carboxylic acid efflux system such as: aaeB (ychP), aaeA (yhcQ), aaeX (yhcR), and aaeR (yhcS) would have the same effect of reducing the availability of 4-hydroxybenzoate for ubiquinone synthesis, Van Dyk, et al., Characterization of the Escherichia coli AaeAB Efflux Pump: a Metabolic Relief Valve? J Bacteriol. 2004 November; 186(21): 7196-7204.

[0121] Similar efflux pumps have also been mentioned in the following:

[0122] 1) Wang, et al., A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity, Sci Rep. 2015; 5: 18456 (2015)

[0123] 2) Zhou, et al., The Rice Bacterial Pathogen Xanthomonas oryzae pv. oryzae Produces 3-Hydroxybenzoic Acid and 4-Hydroxybenzoic Acid via XanB2 for Use in Xanthomonadin, Ubiquinone, and Exopolysaccharide Biosynthesis [Genes Xcc4168-4171 or Xcc1998-Xcc1400], Molecular Plant-Microbe Interactions 26(10): 1239-1248 (2013)

[0124] A 4-HBA efflux pump AaeXAB was characterized in E. coli MG1655 (Van Dyk et al. 2004) (FIG. 8A). X campestris pv. Campestris ATCC33913 contains homologs of aaeX (XCC4168), aaeA (XCC4169), and aaeB (XCC4171), as well as an additional XCC4170 encoding an outer membrane efflux protein (FIG. 8A). Overexpression of the gene cluster of XCC4168 to XCC4171 in PXO99A significantly increased the extracellular level of 3-HBA and 4-HBA

[0125] Each of the following references are incorporated herein in their entireties for all purposes:

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[0153] Van Dyk, et al., Characterization of the Escherichia coli AaeAB Efflux Pump: a Metabolic Relief Valve? J Bacteriol. 2004 November; 186(21): 7196-7204.

[0154] Wang, et al., A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity, Sci Rep. 2015; 5: 18456 (2015)

[0155] Zhou, et al., The Rice Bacterial Pathogen Xanthomonas oryzae pv. oryzae Produces 3-Hydroxybenzoic Acid and 4-Hydroxybenzoic Acid via XanB2 for Use in Xanthomonadin, Ubiquinone, and Exopolysaccharide Biosynthesis [Genes Xcc4168-4171 or Xcc1998-Xcc1400], Molecular Plant-Microbe Interactions 26(10): 1239-1248 (2013)

[0156] ATCC33913

[0157] XCC4168

[0158] XCC4169

[0159] XCC4171

[0160] XCC4170

[0161] XCC4168

[0162] XCC4171

[0163] PXO99A