RECOMBINANT MICROORGANISM FOR IMPROVED PRODUCTION OF ALANINE

Abstract

The present invention relates to a recombinant nucleic acid molecule, a recombinant microorganism, to a method for producing alanine and to the use of the recombinant nucleic acid molecule or the recombinant microorganism for the fermentative production of alanine.

Claims

1. A recombinant microorganism comprising a reduced, repressed or deleted activity and/or expression of an asd gene or a gdhA gene.

2. The recombinant microorganism of claim 1 further comprising at least one of the features selected from the group of (a) an introduced, increased or enhanced activity and/or expression of an alaD gene encoding an alanine dehydrogenase, (b) a reduced, repressed or deleted activity and/or expression of a pflB gene encoding a pyruvate formate lyase I, (c) a reduced, repressed or deleted activity and/or expression of an adhE gene encoding a bifunctional acetaldehyde-CoA dehydrogenase/iron-dependent alcohol dehydrogenase/pyruvate-formate lyase deactivase, (d) a reduced, repressed or deleted activity and/or expression of a ldhA gene encoding a NAO-dependent fermentative D-lactate dehydrogenase, (e) a reduced, repressed or deleted activity and/or expression of a pta gene encoding a phosphate acetyltransferase and/or a reduced, repressed or deleted activity and/or expression of an ackA gene encoding an acetate kinase A and propionate kinase 2, (f) a reduced, repressed or deleted activity and/or expression of a frdA gene encoding a fumarate reductase, and/or (g) a reduced, repressed or deleted activity and/or expression of a dadX gene encoding an alanine racemase, wherein the reduction, repression, deletion, introduction, increase or enhancement of the activity and/or expression of a gene is determined compared to a respective reference microorganism.

3-8. (canceled)

9. The recombinant microorganism of claim 1, further comprising at least one feature selected from the group of: (a) a reduced, repressed or deleted activity and/or expression of a brnQ gene encoding a brnQ protein having a branched chain amino acid transporter activity, (b) a reduced, repressed or deleted activity and/or expression of a gcvB gene encoding a non-protein encoding RNA, (c) an increased and/or enhanced activity and/or expression of a zipA gene encoding cell division protein involved in Z ring assembly, (d) an increased and/or enhanced activity and/or expression of a lpd gene encoding a lipoamide dehydrogenase, a changed activity of a lpxD gene encoding encoding an UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase protein, an increased and/or enhanced activity and/or expression of a gcvA gene encoding a DNA-binding protein, and/or an increased and/or enhanced activity and/or expression of a ygaW gene encoding an alanine transporter.

10-15. (canceled)

16. The recombinant microorganism of claim 1, wherein the asd gene or the gdhA gene is selected from the group of (i) a nucleic acid molecule comprising a sequence of SEQ ID NO: 25 or SEQ ID NO: 37, or (ii) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 25 or SEQ ID NO: 37, or (iii) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 25 or SEQ ID NO: 37 under stringent conditions, or (iv) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 26 or SEQ ID NO: 38, or (v) a nucleic acid molecule encoding a polypeptide having at least 60% homology to the polypeptide of SEQ ID NO: 26 or SEQ ID NO: 38.

17. The recombinant microorganism of claim 2, wherein the alaD gene is selected from the group consisting of (AA) a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, or (BB) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 1, or (CC) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 1 under stringent conditions, or (DD) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 2, or (EE) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 2.

18. The recombinant microorganism of claim 2, wherein the pflB gene is selected from the group consisting of (A) a nucleic acid molecule comprising a sequence of SEQ ID NO: 5, or (B) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 5, or (C) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 5 under stringent conditions, or (D) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 6, or (E) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 6.

19. The recombinant microorganism of claim 2, wherein the adhE gene is selected from the group consisting of (F) a nucleic acid molecule comprising a sequence of SEQ ID NO: 7, or (G) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 7, or (H) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 7 under stringent conditions, or (I) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 8, or (J) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 8.

20. The recombinant microorganism of claim 2, wherein the ldhA gene is selected from the group consisting of (K) a nucleic acid molecule comprising a sequence of SEQ ID NO: 9, or (L) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 9, or (M) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 9 under stringent conditions, or (N) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 10, or (O) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 10.

21. The recombinant microorganism of claim 2, wherein the pta gene is selected from the group consisting of (P1) a nucleic acid molecule comprising a sequence of SEQ ID NO: 11, or (Q1) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 11, or (R1) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 11 under stringent conditions, or (S1) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 12, or (T1) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 12 and wherein the ackA gene is selected from the group consisting of (P2) a nucleic acid molecule comprising a sequence of SEQ ID NO: 32, or (Q2) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 32, or (R2) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 32 under stringent conditions, or (S2) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 33, or (T2) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 33.

22. The recombinant microorganism of claim 2, wherein the frdA gene is selected from the group consisting of (U) a nucleic acid molecule comprising a sequence of SEQ ID NO: 13, or (V) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 13, or (W) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 13 under stringent conditions, or (X) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 14, or (Y) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 14.

23. The recombinant microorganism of claim 2, wherein the dadX gene is selected from the group consisting of (Z) a nucleic acid molecule comprising a sequence of SEQ ID NO: 15, or (FF) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 15, or (GG) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 15 under stringent conditions, or (HH) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 16, or (II) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 16.

24. The recombinant microorganism of claim 9, wherein the ygaW gene is selected from the group consisting of (JJ) a nucleic acid molecule comprising a sequence of SEQ ID NO: 17, or (KK) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 17, or (LL) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 17 under stringent conditions, or (MM) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 18, or (NN) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 18.

25. The recombinant microorganism of claim 9, wherein the zipA gene is selected from the group consisting of (OO) a nucleic acid molecule comprising a sequence of SEQ ID NO: 19, or (PP) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 19, or (QQ) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 19 under stringent conditions, or (RR) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 20, or (SS) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 20.

26. The recombinant microorganism of claim 9, wherein the lpd gene is selected from the group consisting of (TT) a nucleic acid molecule comprising a sequence of SEQ ID NO: 21, or (UU) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 21, or (VV) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 21 under stringent conditions, or (WW) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 22, or (XX) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 22.

27. The recombinant microorganism of claim 9, wherein the brnQ gene is selected from the group consisting of (YY) a nucleic acid molecule comprising a sequence of SEQ ID NO: 23, or (ZZ) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 23, or (AAA) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 23 under stringent conditions, or (BBB) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 24, or (CCC) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 24.

28. The recombinant microorganism of claim 9, wherein the lpxD gene is selected from the group consisting of (DDD) a nucleic acid molecule comprising a sequence of SEQ ID NO: 27, or (EEE) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 28.

29. The recombinant microorganism of claim 9, wherein the gcvA gene is selected from the group consisting of (FFF) a nucleic acid molecule comprising a sequence of SEQ ID NO: 29, or (GGG) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 29, or (HHH) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 29 under stringent conditions, or (III) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 30, or (JJJ) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 30.

30. The recombinant microorganism of claim 9, wherein the gcvB gene is selected from the group consisting of (KKK) a nucleic acid molecule comprising a sequence of SEQ ID NO: 31, or (LLL) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 31, or (MMM) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 31 under stringent conditions.

31. The recombinant microorganism of claim 1, wherein the microorganism is selected from a genus of the group consisting of Corynebacterium, Bacillus, Erwinia, Escherichia, Pantoea, Streptomyces, Zymomonas, Rhodococcus, Saccharomyces, Candida or Pichia.

32-36. (canceled)

37. A method of producing alanine comprising culturing one or more recombinant microorganism according to claim 1 under conditions that allow for the production of alanine

38-43. (canceled)

44. The method of claim 37, comprising I) growing the one or more microorganism in a fermenter to obtain a fermentation broth, and II) recovering alanine from the fermentation broth.

Description

[0261] The term expression or gene expression means the transcription of a specific gene(s) or specific genetic vector construct. The term expression or gene expression in particular means the transcription of gene(s) or genetic vector construct into mRNA. The process includes transcription of DNA and may include processing of the resulting RNA-product. The term expression or gene expression may also include the translation of the mRNA and therewith the synthesis of the encoded protein, i.e. protein expression.

[0262] FIG. 1

[0263] Alanine formation in a batch fermentation of E. coli QZ20 with empty plasmid control and F coliQZ20/pACYC-asd in 500 mL AM 1 medium with 120 g/L glucose. The fermentation was controlled at 37 C, 400 rpm, at pH 6.8 with 5 N NH.sub.4OH without aeration.

[0264] FIG. 2

[0265] Alanine formation in a batch fermentation of F col i QZ20 with empty plasmid control and F coliQZ20/pACYC-gdhA in 500 mL AM 1 medium with 120 g/L glucose. The fermentation was controlled at 37 C, 400 rpm, at pH 6.8 with 5 N NH.sub.4OH without aeration.

EXAMPLES

[0266] Chemicals and Common Methods

[0267] Unless indicated otherwise, cloning procedures carried out for the purposes of the present invention including restriction digest, agarose gel electrophoresis, purification of nucleic acids, ligation of nucleic acids, transformation, selection and cultivation of bacterial cells are performed as described (Sambrook et al., 1989). Sequence analyses of recombinant DNA are performed with a laser fluorescence DNA sequencer (Applied Biosystems, Foster City, Calif., USA) using the Sanger technology (Sanger et al., 1977).

[0268] Unless described otherwise, chemicals and reagents are obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, USA), from Promega (Madison, Wis., USA), Duchefa (Haarlem, The Netherlands) or Invitrogen (Carlsbad, Calif., USA). Restriction endonucleases are from New England Biolabs (Ipswich, Mass., USA) or Roche Diagnostics GmbH (Penzberg, Germany). Oligonucleotides are synthesized by Eurofins MWG Operon (Ebersberg, Germany).

Example 1

[0269] E. coli W (LU17032) was engineered for L-alanine production by inactivation of the ackA, adhE, frdABCD and pflB ORFs and replacement of the IdhA ORF by a codon-optimized variant of the alaD gene (alaD-gstear).

[0270] The ackA, adhE, frdABCD and pflB ORFs were inactivated by insertion of an FRT-flanked kanamycin resistance cassette, followed by removal of the antibiotic resistance cassette by FLP recombination.

[0271] The IdhA gene was replaced by alaD-gstear and a downstream FRT-flanked zeocin resistance cassette, which was finally removed by FLP recombination.

[0272] The procedure has been described previously for example in WO2015/044818 which is hereby incorporated by reference.

[0273] Disruption of ackA has been described previously for example in Causey et al (2004), PNAS, 101 (8) pages 2235-2240.

[0274] Further, in said strain comprising an inactivated ackA, adhE, frdABCD, pflB and IdhA and an introduced alaD activity, the activity of the genes lpd, zipA and ygaW have been increased as described previously in WO2015/044818 and the activity of the alaD has been further enhanced by mutating the promoter controlling said gene as described previously in WO2015/044818.

[0275] Bacterial Culture

[0276] E. coli W (LU17032) was cultured in Luria-Bertani (LB) liquid medium or on Luria-Bertani solid medium. Occasionally, clones were passaged over M9 minimal agar containing 10 mM Sucrose to confirm W strain identity. Antibiotics were added to the liquid and solid media as appropriate, to final concentrations of 15 g/ml (kanamycin, chloramphenicol), 25 g/ml (zeocin) or 3 g/ml (tetracyclin).

[0277] Red/ET Recombination

[0278] Red/ET recombination was performed using standard protocols of Gene Bridges GmbH (www.genebridges.com). Briefly, Red/ET-proficient E. coli W was aerobically grown at 30 C. to an OD600 nm of 0.3. Expression of red genes was induced by adding 50 I of 10% (w/v) L-arabinose, followed by a temperature increase to 37 C. Arabinose was omitted from uninduced control cultures. After 35 min of incubation at 37 C. the cells were washed twice with ice cold 10% (v/v) glycerol and electroporated with 500 ng of PCR product at 1.35 kV, 10 F, 6000. The cells were then resuspended in 1 ml ice-cold LB medium and aerobically grown at 37 C. for approximately 1.5 h. Cultures were then plated on LB agar containing 15 g/ml kanamycin (knockouts) or 25 g/ml zeocin (knockin).

[0279] FLP Recombination

[0280] Flanking FRT sites allowed removal of antibiotic resistance markers by FLP recombination following modification of the E. coli chromosome. FLP recombination leaves a single FRT site (34 bp) as well as short flanking sequences (approx. 20 bp each) which are used as primer binding sites in the amplification of the cassettes.

[0281] To perform FLP recombination, plasmid 708-FLPe (Tab. 1) encoding FLP recombinase was introduced into the Red/ET recombinants by electroporation. KanR CmR or ZeoR CmR transformants were used to inoculate 0.2 ml LB cultures, which were incubated at 30 C. for 3 h. FLP activity was then induced by a temperature shift to 37 C., followed by a three-hour incubation at 37 C. Single colonies obtained from these cultures were subsequently screened for a CmS and KanS or ZeoS phenotype.

[0282] DNA Preparation and Analysis

[0283] E. coli genomic DNA (gDNA) was isolated from overnight cultures with the Gentra Puregene Yeast/Bact. Kit B (Qiagen, Hilden, Germany). PCR products harbouring knockout or knockin cassettes were amplified from template plasmids with PRECISOR high-fidelity DNA polymerase (BioCat, Heidelberg) and analytical PCR reactions were performed with the PCR Extender System (5PRIME GmbH, Hamburg, Germany), according to the manufacturer's recommendations. PCR amplicons were purified using the GeneJET PCR Purification Kit or the GeneJET Gel Extraction Kit (Fermentas, St. Leon-Rot, Germany) and sequencing was performed by GATC BioTech (Konstanz, Germany) or BioSpring (Frankfurt am Main, Germany).

TABLE-US-00001 TABLE 1 Plasmids and primers Relevant characteristics/oligo sequences plasmids (5 .fwdarw. 3) Source pRed/ET red expression plasmid, Gene pSC101-based, Tc.sup.R Bridges 708-FLPe FLP recombinase expression plasmid, Gene pSC101-based, Cm.sup.R Bridges primers (BioSpring) SEQ ID NO pACYC-asd-F 35 pACYC-asd-R 36 pACYC-gdhA-F 40 pACYC-gdhA-R 41

Example 2 HPLC Detection and Quantification of Alanine

[0284] The following HPLC method for the alanine detection in the cell culture media was used:

[0285] Column: Aminex HPX-87C column (Bio-Rad), 3007.8 mm, i.d. particle size 9 m

[0286] Mobile phase: Ca(NO3)2 at 0.1 mol/L 90%, Acetonitrile 10%

[0287] Flow rate: 0.6 mL/min

[0288] Column temperature: 60 C.

[0289] Detection: Refractive index detector

[0290] Under above method, major estimated components in the cell culture sample matrix can be well separated from alanine, without interfering alanine's quantitation.

[0291] The amount of the alanine in the sample was determined by external standard calibration method. Standard samples containing alanine from 0.5 to 10.0 g/L were injected and the peak areas were used for calibration. Linear regression coefficient of the calibration curve was 0.9995.

[0292] Samples are injected once at 20 L. Peak areas are used to calculate the amount presenting in the sample by Waters LC Millenium software.

Example 3 HPLC Detection and Quantification of of Glucose, Succinate, Lactate, Formate, Acetate and Ethanol

[0293] HPLC Method Used

[0294] Column: Aminex HPX-87H column (Bio-Rad), 300 x7.8 mm, i.d. particle size 9 m

[0295] Mobile phase: H2SO4 4 mM

[0296] Flow rate: 0.4 mL/min

[0297] Column temperature: 45 C.

[0298] Detection: Refractive index detector

[0299] The amount of the analytes was determined by external standard calibration method. Standard samples containing glucose from 0.1 to 38.0 g/L, succinate, lactate, formate, acetate and ethanol from 0.05 to 10.0 g/L were injected and the peak areas were used for calibration. Linear regression coefficients for all six calibration curves were better than 0.999.

[0300] Samples are injected once at 20 L. Peak areas are used to calculate the amount presenting in the sample by Waters LC Millenium software.

Example 4 Effect of the Increased Expression of the Asd Gene on Alanine Productivity

[0301] An additional copy of the asd gene (SEQ ID NO: 25) was introduced into the pACYC184 plasmid under the control of an IPTG-inducible Ptrc promoter. The vector, designated as pACYC-asd (SEQ ID NO: 34), was constructed via commercial InFusion cloning technology (Clontech, Mountain View, Calif., USA). The pACYC184 vector (NEB) was linearized with HindIII and SalI restriction endonucleases (NEB). The generated vector backbone was purified by agarose gel extraction. The asd gene was PCR amplified from wild-type E. coli W genomic DNA with primers asd-pACYC_F (SEQ ID NO: 35) and asd-pACYC_R (SEQ ID NO: 36). The primers contained additional 15 bp homologous overhangs to the vector backbone and a double-stranded DNA fragment with the Ptrc promoter that was synthesized by IDT. The amplified asd gene, the upstream Ptrc promoter and the pACYC184 vector backbone were cloned together according to the InFusion cloning manual. The resulting pACYC-asd vector was transformed into E. coli via electroporation and selected for on LB chloramphenicol plates. Positive constructs were confirmed by DNA sequencing.

[0302] The effect of asd overexpression on alanine productivity was tested by comparative cultivation of E. coli with an empty control pACYC vector and E. coli harbouring the asd overexpression plasmid pACYC-asd (SEQ ID NO: 34). Precultures were grown in shake flasks with LB medium, 20% filling volume at 37 C and 200 rpm overnight. The fermentation was performed in the DASGIP 1.5 L parallel bioreactor system (Eppendorf) in 500 mL AM 1 medium (2.6 g/L (NH4)2HPO4, 0.87 g/L NH4H2PO4, 0.15 g/L Kill, 0.37 g/L MgSO4.7 H2O, 15 g/L (NH4)2SO4, 1 mM betaine, 1 ml/L trace metal stock solution). The trace metal stock comprised 1.6 g/L FeCL3.6 H2O; 0.2 g/L CoCl2.6 H2O; 0.1 g/L CuCl2.2 H2O; 0.2 g/L ZnCl2; 0.2 g/L NaMoO4.2 H2O; 0.05 g/L H3BO3, 0.1 M HCL. 120 g/L Glucose were used as carbon source in the fermentation medium. 25 ug/mL chloramphenicol were added to stably maintain the plasmid. Expression of the asd gene from the Ptrc promoter was induced with 250 uM isopropyl -D-1-thiogalactopyranoside (IPTG) during the early logarithmic growth phase. Each strain was run in duplicates at 37 C and 400 rpm stirrer speed. 5N NH.sub.4OH was used to control the pH to 6.8 and provide the culture with ammonium as an alanine precursor throughout the fermentation. No air was sparged during the fermentation and the vessel was not pressurized so that after the initial consumption of dissolved oxygen in the medium by the cells the fermentation was run under microaerobic conditions. Samples were taken throughout the fermentation and analyzed by HPLC for alanine and glucose concentrations.

[0303] After 72 h of fermentation time E coliQZ20 in which the asd gene (SEQ ID NO: 25) was overexpressed from the pACYC-asd plasmid (SEQ ID NO: 34) reached a significantly higher L-alanine titer of 66.580.50 g/L compared to the strain harbouring the empty control plasmid with 46.001.85 g/L (FIG. 1).

Example 5 Effect of the Increased Expression of the gdhA Gene on Alanine Productivity

[0304] An additional copy of the gdhA gene (SEQ ID NO: 37) was introduced into the pACYC184 plasmid under the control of an IPTG-inducible Ptrc promoter. The vector, designated as pACYC-gdhA (SEQ ID NO: 39), was constructed via commercial InFusion cloning technology (Clontech, Mountain View, Calif., USA). The pACYC184 vector (NEB) was linearized with HindIII and SalI restriction endonucleases (NEB). The generated vector backbone was purified by agarose gel extraction. The gdhA gene was PCR amplified from wild-type E. coli W genomic DNA with primers gdhA-pACYC_F (SEQ ID NO: 40) and gdhA-pACYC_R (SEQ ID NO: 41). The primers contained additional 15 bp homologous overhangs to the vector backbone and a double-stranded DNA fragment with the Ptrc promoter that was synthesized by IDT. The amplified gdhA gene, the upstream Ptrc promoter and the pACYC184 vector backbone were cloned together according to the InFusion cloning manual. The resulting pACYC-gdhA vector was transformed into E. coli via electroporation and selected for on LB chloramphenicol plates. Positive constructs were confirmed by DNA sequencing.

[0305] The effect of gdhA overexpression on alanine productivity was tested by comparative cultivation of E. coli with an empty control pACYC vector and E. coli harbouring the gdhA overexpression plasmid pACYC-gdhA (SEQ ID NO: 39). Precultures were grown in shake flasks with LB medium, 20% filling volume at 37 C and 200 rpm overnight. The fermentation was performed in the DASGIP 1.5 L parallel bioreactor system (Eppendorf) in 500 mL AM 1 medium (2.6 g/L (NH4)2HPO4, 0.87 g/L NH4H2PO4, 0.15 g/L Kill, 0.37 g/L MgSO4.7 H2O, 15 g/L (NH4)2504, 1 mM betaine, 1 ml/L trace metal stock solution). The trace metal stock comprised 1.6 g/L FeCL3.6 H2O; 0.2 g/L CoCl2.6 H2O; 0.1 g/L CuCl2.2 H2O; 0.2 g/L ZnCl2; 0.2 g/L NaMoO4.2 H2O; 0.05 g/L H3BO3, 0.1 M HCL. 120 g/L Glucose were used as carbon source in the fermentation medium. 25 ug/mL chloramphenicol were added to stably maintain the plasmid. Expression of the gdhA gene from the Ptrc promoter was induced with 250 uM isopropyl -D-1-thiogalactopyranoside (IPTG) during the early logarithmic growth phase. Each strain was run in duplicates at 37 C and 400 rpm stirrer speed. 5N NH.sub.4OH was used to control the pH to 6.8 and provide the culture with ammonium as an alanine precursor throughout the fermentation. No air was sparged during the fermentation and the vessel was not pressurized so that after the initial consumption of dissolved oxygen in the medium by the cells the fermentation was run under microaerobic conditions. Samples were taken throughout the fermentation and analyzed by HPLC for alanine and glucose concentrations.

[0306] After 72 h of fermentation time E. coli QZ20 in which the gdhA gene (SEQ ID NO: 37) was overexpressed from the pACYC-gdhA plasmid (SEQ ID NO: 39) reached a significantly higher L-alanine titer of 62.180.37 g/L compared to the strain harbouring the empty control plasmid with 50.963.41 g/L (FIG. 2).