METHOD AND MICROBES FOR THE PRODUCTION OF CHIRAL COMPOUNDS

Abstract

The present invention provides a Clostridium species comprising a non-native gene capable of expressing (R)-3-hydroxybutyryl-Co A dehydrogenase. Also provided is a method of producing (R)-3-hydroxybutyric acid, or a salt thereof, and/or (R) 1,3-butanediol using such Clostridium.

Claims

1. A method of producing (R)-3-hydroxybutyric acid, and/or a salt thereof, and/or (R)-1,3-butanediol, the method comprising culturing a Clostridium species comprising a non native gene capable of expressing (R)-3-hydroxybutyryl-CoA dehydrogenase.

2. A method as claimed in claim 1 comprising culturing the Clostridium species under anaerobic or microaerophilic conditions.

3. A method as claimed in claim 1, comprising the step of purifying the produced (R)-3-hydroxybutyric acid and/or salt thereof and/or the (R)-1,3-butanediol.

4. A method as claimed in claim 1, wherein the gene is PhaB.

5. A method as claimed in claim 1, wherein: the (R)-3-hydroxybutyric acid isomer form comprises 100% of the 3-hydroxybutyric acid formed; or the (R)-3-hydroxybutyric acid isomer form comprises 90-100% of 3-hydroxybutyric acid formed and (S)-3-hydroxybutyric acid isomer form comprises 0-10% of the 3-hydroxybutyric acid formed.

6. A method as claimed in claim 1 wherein: the 1,3-butanediol formed is 100% in the (R)-1,3-butanediol isomer form; or the 1,3-butanediol formed comprises 90-100% in the (R)-1,3-butanediol isomer form and 0-10% in the (S)-1,3-butanediol isomer form.

7. A method as claimed in claim 1 wherein the Clostridium species also includes a non native gene capable of expressing thioesterase.

8. A method as claimed in claim 7, wherein the gene capable of expressing thioesterase is TesB.

9. A method as claimed in claim 1 wherein the Clostridium species comprises an S stereo specific crotonase.

10. A method as claimed in claim 1 wherein the Clostridium species comprises one or more native non substrate specific dehydrogenase/reductase enzymes able to convert (R)-3-hydroxybutyryl-CoA to (R)-3-hydroxybutyric acid and/or (R)-1, 3-butanediol.

11. A method as claimed in claim 1 wherein the Clostridium species comprises one or more non-native genes capable of expressing a non substrate specific aldehyde dehydrogenase and/or alcohol dehydrogenase able to convert (R)-3-hydroxybutyryl-CoA to (R)-1,3-butanediol.

12. A method as claimed in claim 1 wherein the non native gene capable of expressing (R)-3-hydroxybutyryl-CoA dehydrogenase is integrated into the chromosome of the Clostridium species.

13. A method as claimed in claim 1 wherein the Clostridium species comprises reduced or knocked out expression of a native gene involved in the production of butanol, acetone and ethanol, butyrate, acetate and/or lactate.

14. A method as claimed in claim 13 wherein the Clostridium species comprises reduced or knocked out expression of the native ptb, buk, hbd and/or alcohol/aldehyde dehydrogenases of the Clostridium species.

15. (R)-3-hydroxybutyric acid, and/or a salt thereof, and/or (R)-1,3-butanediol produced by a method of claim 1.

16. A Clostridium species comprising a non native gene capable of expressing (R)-3-hydroxybutyryl-CoA dehydrogenase.

17. A Clostridium species as claimed in claim 16 wherein the non-native gene is PhaB.

18. A Clostridium species as claimed in claim 16 which also includes a non native gene capable of expressing thioesterase.

19. A Clostridium species as claimed in claim 16 comprising an S stereo specific crotonase.

20. A Clostridium species as claimed in claim 16, comprising one or more native non substrate specific dehydrogenase/reductase enzymes able to convert (R)-3-hydroxybutyryl-CoA to (R)-3-hydroxybutyric acid and/or (R)-1, 3-butanediol.

21. A Clostridium species as claimed in claim 16 comprises one or more non-native genes capable of expressing a non substrate specific aldehyde dehydrogenase and/or alcohol dehydrogenase able to convert (R)-3-hydroxybutyryl-CoA to (R)-1,3-butanediol.

22. A Clostridium species as claimed in claim 16, wherein the Clostridium species is C. acetobutylicum, C. butyricum or C. saccharoperbutylacetonicum.

23. A Clostridium species as claimed in claim 16 wherein the non native gene capable of expressing (R)-3-hydroxybutyryl-CoA dehydrogenase is integrated into a chromosome of the Clostridium species.

24. A Clostridium species as claimed in claim 16, wherein the Clostridium species comprises reduced or knocked out expression of a native gene involved in the production of butanol, acetone and ethanol, butyrate, acetate and/or lactate.

25. A Clostridium species as claimed in claim 24, wherein the Clostridium species comprises reduced or knocked out expression of the native ptb, buk, hbd and alcohol/aldehyde dehydrogenases of the Clostridium species.

26. A method of producing the Clostridium as defined in claim 16, comprising incorporating a non native gene capable of expressing (R)-3-hydroxybutyryl-CoA dehydrogenase into the Clostridium species.

27. A method according to claim 26 wherein the non-native gene is integrated into a chromosome of the Clostridium species.

Description

FIGURES

[0081] FIG. 1 (A) shows the native acid and solvent production metabolic pathways in solventogenic Clostridium.

[0082] FIG. 1(B) shows the acid and solvent production metabolic pathways in solventogenic Clostridium after the introduction of a heterologous (R)-3-hydroxybutyryl-CoA dehydrogenase (A) and a heterologous thioesterase (B).

[0083] FIGS. 2A-B shows the codon optimised sequence for: the phaB gene from Cupriavidus necator (A) and the TesB gene from E. coli (B).

[0084] FIGS. 3 A-B details the plasmid maps for pfdx_phaB in pMTL83151 (A), pMTL83251 (B) and pMTL82151 (C).

[0085] FIG. 4 shows the concentration of (R/S)-3-hydroxybutyrate and (R/S)-1,3-butanediol produced in C. acetobutylicum (p83151-pfdx_PhaB).

[0086] FIG. 5 shows the concentration of (R/S)-3-hydroxybutyrate and (R/S)-1,3-butanediol produced in C. acetobutylicum (p83251-pfdx_PhaB).

[0087] FIG. 6 shows the concentration of (R)-3-hydroxybutyrate and (R)-1,3-butanediol produced in C. saccharoperbutylacetonicum using plasmid transformation (p82151-pfdx_PhaB) in CGM (A) and in FMC (B).

[0088] FIG. 7 shows the concentration of (R)-3-hydroxybutyrate produced in C. acetobutylicum (p83251-pfdx_PhaB).

[0089] FIG. 8 shows the concentration of (R/S)-3-hydroxybutyrate produced in C. butyricum (p83151-pfdx_PhaB_TesB) and C. butyricum (p83151-pfdx_PhaB).

[0090] FIGS. 9A-B shows the concentration of (R)-3-hydroxybutyrate (A) and (R)-1,3-butanediol (B) produced in C. saccharoperbutylacetonicum when the gene is integrated into a chromosome of the bacteria.

[0091] FIGS. 10A-B shows the concentration of (R)-3-hydroxybutyrate (A) and (R)-1,3-butanediol (B) produced in C. saccharoperbutylacetonicum when the gene is integrated into a chromosome of the bacteria or introduced by plasmid transformation.

EXAMPLES

Example 1

C. acetobutylicum (PhaB Expression)

1) Gene Synthesis

[0092] The gene Cupriavidus necator PhaB was codon optimised for Clostridia. FIG. 2A shows one example of the codon optimised sequence which was synthesized by Gene Art (Thermo Fisher Scientific).

2) Plasmid Assembly

[0093] PhaB was cloned into plasmid pMTL83151 using restriction sites NdeI and NheI. The C. sporogenes Pfdx promoter was cloned upstream of the gene using Infusion cloning kit yielding plasmid pMTL83151_pfdx_phaB. PfdX-phaB was extracted from pMTL83151_pfdx_phaB using restriction sites NotI and NheI. The extracted fragment was cloned into pMTL83251 using standard cloning methods (FIGS. 3A and 3B).

3) Strain Development

[0094] The designed plasmids were used to transform E. coli TOP10 pAN2 for in vitro methylation using standard transformation protocol. The methylated plasmids were extracted using a commercial kit and used to transform C. acetobutylicum ATCC 824. Briefly, an overnight culture of C. acetobutylicum was used to inoculate 2 YTG. Cells were grown anaerobically at 37 C. to an OD.sub.600 of 0.6-0.8 and were washed with ice cold, anaerobe Electroporation buffer (EPB) (270 mM sucrose, 5 mM sodium phosphate (pH 7.4). The final pellet was re-suspended in a small volume of ice cold, anaerobe EPB and immediately used for transformation. Plasmid DNA (1-2 g) and cells were added to the pre-chilled 0.4 cm gap cuvette. Electroporation was carried out using a BioRad electroporator with following settings: 2.0 kV, 25 F and . Transformed cells were recovered anaerobically at 37 C. for 1-3 h in 2 YTG, pH 5.2 before plated on CGM media containing the required antibiotics (15 g/ml thiamphenicol or 50 g/ml erythromycin) Single colonies were obtained within 24-48 hours. The presence of the plasmid was confirmed using colony PCR and plasmid specific primers. Transformed colonies were picked for each plasmid and stored as 80 C. freezer stock.

[0095] The culture media used:

[0096] Suitable culture media include but is not limited to CBM, CGM and 2 YTG media.

[0097] Exemplified media are:

[0098] CBM containing per 1 L:1 ml FeSO.sub.47H.sub.2O (10 mg/mL), 10 ml MgSO.sub.47H.sub.2O (20 mg/mL), 1 ml MnSO.sub.44H.sub.2O (10 mg/mL), 4 g Casein hydrolysate, 1 ml 4-Aminobenzoic acid (1 mg/ml), 1 ml thiamine-HCL (1 mg/ml), 1.33 l biotin (1.5 mg/ml), 10 ml K.sub.2HPO.sub.4 (50 mg/ml), 10 ml KH.sub.2PO.sub.4 (50 mg/ml), 20 ml CaCO.sub.3 (250 mg/ml), 2.5-5% glucose. For solid media Agar was added 15 g/L and CaCO.sub.3 omitted.

[0099] CGM containing per 1 L:2 g Ammonium Sulphate, 1 g Potassium phosphate dibasic, 0.5 g Potassium phosphate dibasic, 0.2 g Magnesium sulphate heptahydrate, 0.75 ml Iron sulphate heptahydrate (20 g/L), 0.5 ml Calcium Chloride (20 g/L), 0.5 ml Manganese sulphate monohydrate (20 g/L), 0.1 ml Cobalt hydrate ((20 g/L), 0.1 ml Zinc Sulphate (20 g/L), Tryptone 2 g, Yeast extract 1 g, 50 g Glucose, 12 g Agar.

[0100] 2 YTG containing per 1 L; 16 g tryptone, 10 g yeast extract, 5 g NaCl, pH adjusted to 5.2 and sterilised by autoclaving at 121 C. Sterile glucose is added to cool down media at a concentration of 0.5-2%

4) Fermentation Data for C. acetobutylicum

Growth Method

[0101] Transformed strains were re-streaked from 80 C. freezer stocks on CBM or CGM plates containing the appropriate antibiotics (15 g/ml thiamphenicol or 50 g/ml erythromycin) Single colonies were picked and used to inoculate an over-night seed culture (2 YTG, pH 5.2). The seed culture was grown anaerobically at 37 C. for up to 16 h. A 40 ml CGM culture containing 2.5% Glucose was inoculated next day 1:100 using the seed culture. Strains were grown anaerobically at 37 C. Samples for metabolic analysis were taken after 48 hr of growth and analysed for (R/S)-3-hydroxybutyrate (R/S-HB) and (R/S)-1,3-butanediol (R/S-BDO).

Analysis

[0102] Analysis for R/S-3-hydroxybutyrate was carried out using HPLC-MS. The samples were derivatized using DATAN (Diacetyl-tartaric Anhydride) and separation of the S and R form was carried out using a standard non-chiral LC column (Agilent Zorbax Eclipse Plus C18, 2.1150 mm, 1.8 um). Briefly, 10 l supernatant was mixed with 250 l methanol. Samples were dried down at 50 C., followed by the addition of 50 l of freshly prepared DATAN solution (200 g/l DATAN in dichloromethane: acetic acid 4:1 (v/v). Samples were incubated for 120 min at 75 C., followed by evaporation step. Dried down samples were suspend in 500 l water and analysed by LC-MS.

Results

[0103] Expression of phaB leads to the production of (R)-3-hydroxybutyrate and (R)-1,3-butaendiol as shown in FIG. 4 (pMTL83151_pfdx_phaB) and FIG. 5 (pMTL83251_pfdx_phaB). Concentrations of over 250 M of (R)-1,3-butanediol and over 3000 M of (R)-3-hydroxybutyrate were achieved. None or minimal levels of (S)-1,3-butanediol and (S)-3-hydroxybutyrate were detected in the transformed strains.

Example 2

C. saccharoperbutylacetonicum (Plasmid Integration)

1) Gene Synthesis

[0104] The gene Cupriavidus necator PhaB was codon optimised for Clostridia. FIG. 2A shows one example of the codon optimised sequence which was synthesized by Gene Art (Thermo Fisher Scientific).

2) Plasmid Assembly

[0105] PhaB, together with the C. sporogenes Pfdx promoter was cloned into plasmid pMTL82151 using restriction sites NotI and NheI yielding plasmid pMTL82151_pfdx_phaB.

3) Strain Development

[0106] Plasmid pMTL82151_pfdx_phaB was used to transform Clostridium saccharoperbutylacetonicum (Cspa) by standard electroporation methods. Briefly, cells were grown anaerobically at 37 C. to an OD.sub.600 of 0.6-0.8 and were washed with ice cold, anaerobe Electroporation buffer (EPB) (270 mM sucrose, 5 mM sodium phosphate (pH 7.4). The final pellet was re-suspended in a small volume of ice cold, anaerobe EPB and immediately used for transformation. Plasmid DNA (1-2 g) and cells were added to the pre-chilled 0.4 cm gap cuvette. Electroporation was carried out using a BioRad electroporator with following settings: 2.0 kV, 25 F and . Transformed cells were recovered anaerobically at 37 C. in RCM, pH 5.2 before plated on RCM +50 g/ml Chloramphenicol. Single colonies were obtained within 24-48 hours. The presence of the plasmid was confirmed using colony PCR and plasmid specific primers. Transformed colonies were picked for each plasmid and stored as 80 C. freezer stock.

4) Fermentation Data for C. saccharoperbutylacetonicum

Growth Method

[0107] Transformants were grown overnight in seed cultures (growth media: CGM or FMC) at 37 C. A 40 ml CGM culture containing 5% glucose was inoculated the next day to a starting OD of 0.05-0.1. Strains were grown anaerobically at 37 C. Samples for metabolic analysis were taken at regular intervals and analysed for (R/S)-3-hydroxybutyrate (R/S-HB) and (R/S)-1,3-butanediol (R/S-1,3-BDO).

Analysis

[0108] Supernatant samples were analysed using a Aminex Ion-Exclusion Column (HPX-87H, 300 mm 7.8 mm, Bio-Rad) connected to an HPLC. Metabolites were eluted with 5 mM H.sub.2SO.sub.4 at a flow rate of 0.5 ml min

[0109] Chirality analysis of produced 3-hydroxybutyrate and 1,3-butanediol was carried out using HPLC-MS. The samples were derivatized using DATAN (Diacetyl-tartaric Anhydride) and separation of the S and R forms was carried out using a standard non-chiral LC column (Agilent Zorbax Eclipse Plus C18, 2.1150 mm, 1.8 um).

[0110] Briefly, 10 l supernatant was mixed with 250 l methanol. Samples were dried down at 50 C., followed by the addition of 50 l of freshly prepared DATAN solution (200 g/l DATAN in dichloromethane:acetic acid 4:1 (v/v)). Samples were incubated for 120 min at 75 C., followed by evaporation step. Dried down samples were suspend in 500 l water and analysed by LC-MS.

Results

[0111] Expression of phaB in Clostridium saccharoperbutylacetonicum results in the production of (R)-3-hydroxybutyrate and (R)-1,3-butanediol, as shown in FIG. 6.

[0112] Growth media depending, about 5.5-7 mM 3-hydroxybutyrate and 5-6 mM 1,3-butanediol was produced within 72 h. Mass spec analysis confirmed R-chirality of the produced 3-hydroxybutyrate and 1,3-butanediol.

Example 3

C. butyricum

1) Gene Synthesis

[0113] The gene Cupriavidus necator PhaB was codon optimised for Clostridia. FIG. 2A shows one example of the codon optimised sequence which was synthesized by Gene Art (Thermo Fisher Scientific).

2) Plasmid Assembly

[0114] PhaB was cloned into plasmid pMTL83251 under control the C. sporogenes Pfdx promoter using standard cloning techniques yielding plasmid pMTL83251_pfdx_phaB.

3) Strain Development

[0115] Plasmid pMTL83251_pfdx_phaB was conjugated into Clostridium butyricum using E. coli CA434. A standard conjugation protocol was applied. Briefly, overnight cultures of E. coli CA434 carrying plasmid pMTL83251_pfdx_phaB and C. butyricum were used to inoculate 9 ml LB media and RCM respectively. Cultures were grown until OD of 0.5-0.7. 1 ml of E. coli culture was spun down and the pellet mixed with 200 l C. butyricum culture. The cell mix was spotted on a non-selective RCM plate and incubated overnight. The incubated mix was re-suspended into 500 l fresh RCM and plated on selective media containing 10 g/ml erythromycin. Presence of the plasmid within the obtained transconjugants was confirmed by PCR using plasmid specific primers.

4) Fermentation Data for C. butyricum

Growth Method

[0116] RCM containing per 1 L: yeast extract 13 g, Peptone 10 g, soluble starch 1 g, sodium chloride 5. g, sodium acetate 3 g, cysteine hydrochloride 0.5 g, carbohydrate 2%, was used. Calcium carbonate 10 g/L were added to liquid culture for pH regulation. Solid media contained 15 g/L agar.

[0117] Transformants were grown overnight in seed cultures (RCM) at 37 C. 100 ml RC media containing 2% glucose was inoculated to a starting OD of 0.05-0.1. Strains were grown anaerobically at 37 C. in the presence of required antibiotic. Samples for metabolic analysis were taken at regular intervals.

Analysis and Results

[0118] Culture supernatant was analysed for (R)-3-hydroxybutyrate using the 3-hydoxybutyrate assay kit (Sigma Aldrich). The strain expressing phaB produced about 17 mg/L 3-hydroxybutyrate as shown in FIG. 7.

Example 4

C. acetobutylicum

1) Gene Synthesis

[0119] The genes Cupriavidus necator PhaB and E. coli TesB were codon optimised for Clostridia. FIG. 2A shows one example of the codon optimised phaB sequence which was synthesized by Gene Art (Thermo Fisher Scientific). FIG. 2B shows one example of the codon optimised TesB sequence which was synthesized by Gene Art (Thermo Fisher Scientific).

2) Strain Development

[0120] PhaB and TesB were cloned as one operon into pMTL83151 under control of the pfdx promoter using standard cloning techniques. The generated plasmid was used to transform E. coli TOP10 pAN2 for in vitro methylation using standard transformation protocol. The methylated plasmids were extracted using a commercial kit and used to transform C. acetobutylicum ATCC 824. Briefly, an overnight culture of C. acetobutylicum was used to inoculate 2 YTG. Cells were grown anaerobically at 37 C. to an OD.sub.600 of 0.6-0.8 and washed with ice cold, anaerobe Electroporation buffer (EPB) (270 mM sucrose, 5 mM sodium phosphate (pH 7.4). The final pellet was re-suspended in a small volume of ice cold, anaerobe EPB and immediately used for transformation. Plasmid DNA (1-2 g) and cells were added to the pre-chilled 0.4 cm gap cuvette. Electroporation was carried out using a BioRad electroporator with following settings: 2.0 kV, 25 F and . Transformed cells were recovered anaerobically at 37 C. for 1-3 h in 2 YTG, pH 5.2 before plated on CGM media containing 15 g/ml thiamphenicol. Single colonies were obtained within 24-48 hours. The presence of the plasmid was confirmed by colony PCR using plasmid specific primers. Transformed colonies were picked for each plasmid and stored as 80 C. freezer stock.

3) Fermentation Data for C. acetobutylicum
Growth Method Transformants were grown overnight in seed cultures (growth media: CBM) at 37 C. Samples were taken at regular intervals and production of chiral chemicals (R)-1,3-butanediol and (R)-3-hyrdoxybutyrate analysed by HPLC_MS.

Analysis

[0121] Analysis for R/S-3-Hydroxybutyrate was carried out using HPLC-MS. The samples were derivatized using DATAN (Diacetyl-tartaric Anhydride) and separation of the S and R form was carried out using a standard non-chiral LC column (Agilent Zorbax Eclipse Plus C18, 2.1150 mm, 1.8 um). Briefly, 10 l supernatant was mixed with 250 l methanol. Samples were dried down at 50 C., followed by the addition of 50 l of freshly prepared DATAN solution (200 g/l DATAN in dichloromethane: acetic acid 4:1 (v/v). Samples were incubated for 120 min at 75 C., followed by evaporation step. Dried down samples were suspend in 500 l water and analysed by LC-MS.

Results

[0122] Overexpression of phaB in C. acetobutylicum leads to the production of two chiral chemicals (R)-1,3-butanediol and (R)-3-hyrdoxybutyrate as shown in FIG. 8. Addition of TesB increased the titres of (R)-3-hydroxybutyrate by 1.5. TesB is non-chiral specific and can use S/R-3-HB-CoA as substrate resulting in a strain producing (R)-3-hydroxybutyrate and (S)-3-hydroxybutyrate at a ratio of about 10:1.

Example 5

C. saccharoperbutylacetonicum (Genome Integration)

1) Gene Synthesis

[0123] The gene Cupriavidus necator PhaB was codon optimised for Clostridia. FIG. 2 shows one example of the codon optimised sequence which was synthesized by Gene Art (Thermo Fisher Scientific).

2) Strain Development

[0124] PhaB was integrated into the genome of C. saccharoperbutylacetonicum using the published ACE method based on pyrE (for example as described WO2009/101400). Transformants were confirmed using gene specific primers.

3) Fermentation for C. saccharoperbutylacetonicum

Growth Method

[0125] Transformants were grown overnight as provided below using suitable culture media include but are not limited to FMC and CGM. Exemplified media are:

[0126] FMC media containing per 1L: Yeast extract 2.5 g, Tryptone 2.5 g, FeSO.sub.47H.sub.2O 0.025 g (NH4).sub.2SO.sub.4 0.5 g. The pH was checked and adjust before autoclaving to 6.5-7. CaCO.sub.3 5 g-10 g was added to regulate the pH.

[0127] CGM media containing per 1L (pH 6.6): yeast extract 5 g, NaCl 1 g, K.sub.2HPO.sub.4 0.75 g, KH.sub.2PO.sub.4 0.75 g, MgSO.sub.4*7H.sub.2O 0.4 g, FeSO.sub.4*7H.sub.2O 0.01 g, MnSO.sub.4*4H.sub.2O 0.01 g, (NH.sub.4).sub.2SO.sub.4 2 g, asparagine 2 g. Calcium carbonate 5-10 g/L was added to liquid culture for pH regulation

[0128] Samples for metabolic analysis were taken at regular interval and analysed for (R/S)-3-hydroxybutyrate (R/S-HB) and (R/S)-1,3-butanediol (R/S-1,3-BDO).

Analysis

[0129] Supernatant samples were analysed using a Aminex Ion-Exclusion Column (HPX-87H, 300 mm 7.8 mm, Bio-Rad) connected to an HPLC. Metabolites were eluted with 5 mM H.sub.2SO.sub.4 at a flow rate of 0.5 ml min.

[0130] Chirality analysis of produced 3-hydroxybutyrate and 1,3-butanediol was carried out using HPLC-MS. The samples were derivatized using DATAN (Diacetyl-tartaric Anhydride) and separation of the S and R form was carried out using a standard non-chiral LC column (Agilent Zorbax Eclipse Plus C18, 2.1150 mm, 1.8 um). Briefly, 10 l supernatant was mixed with 250 l methanol. Samples were dried down at 50 C., followed by the addition of 50 l of freshly prepared DATAN solution (200 g/I DATAN in dichloromethane:acetic acid 4:1 (v/v). Samples were incubated for 120 min at 75 C., followed by evaporation step. Dried down samples were suspend in 500 l water and analysed by LC-MS.

Results

[0131] Integration of phaB into the genome of C. saccharoperbutylacetonicum leads to the production of two chiral chemicals(R)-1,3-butanediol and (R)-3-hyrdoxybutyrate as shown in FIGS. 9A and 9B. Amounts produced were 0.3 g/L 3-hydroxybutyrate and 3.2 g/L 1,3-butanediol.

Example 6

C. saccharoperbutylacetonicum (Genome Integration vs Replicative Plasmid)

1) Gene Synthesis

[0132] The gene Cupriavidus necator PhaB was codon optimised for Clostridia. FIG. 2 shows one example of the codon optimised sequence which was synthesized by Gene Art (Thermo Fisher Scientific).

2) Strain Development

[0133] In one strain PhaB was integrated into the genome of C. saccharoperbutylacetonicum using the published ACE method based on pyrE (for example as described WO2009101400). In a second strain phaB was expressed on a replicative pMTL82151 plasmid under the control of pfdx promoter. The plasmid was transformed into Cspa using standard electroporation protocol for anaerobic Clostridia.

[0134] The correct genotype of each transformant was confirmed using gene specific primers.

3) Fermentation for C. saccharoperbutylacetonicum

Growth Method

[0135] Transformants were grown overnight as below using suitable culture media include but are not limited to FMC and CGM, as described above.

[0136] Samples for metabolic analysis were taken at regular interval and analysed for (R/S)-3-hydroxybutyrate (R/S-HB) and (R/S)-1,3-butanediol (R/S-1,3-BDO).

Analysis

[0137] Supernatant samples were analysed using a Aminex Ion-Exclusion Column (HPX-87H, 300 mm 7.8 mm, Bio-Rad) connected to an HPLC. Metabolites were eluted with 5 mM H.sub.2SO.sub.4 at a flow rate of 0.5 ml min.

[0138] Chirality analysis of produced 3-hydroxybutyrate and 1,3-butanediol was carried out using HPLC-MS. The samples were derivatized using DATAN (Diacetyl-tartaric Anhydride) and separation of the S and R form was carried out using a standard non-chiral LC column (Agilent Zorbax Eclipse Plus C18, 2.1150 mm, 1.8 um). Briefly, 10 l supernatant was mixed with 250 l methanol. Samples were dried down at 50 C., followed by the addition of 50 l of freshly prepared DATAN solution (200 g/l DATAN in dichloromethane:acetic acid 4:1 (v/v). Samples were incubated for 120 min at 75 C., followed by evaporation step. Dried down samples were suspend in 500 l water and analysed by LC-MS.

Results

[0139] Integration of phaB into the genome of C. saccharoperbutylacetonicum and replicative plasmid expression leads to the production of two chiral chemicals(R)-1,3-butanediol and (R)-3-hyrdoxybutyrate, as shown FIG. 10A and 10B.

[0140] Comparison of plasmid expression versus integration of phaB showed an unexpected result. Integration of phaB into the genome leads to a decrease in (R)-3-hyrdoxybutyrate titres while a 6 increase in (R)-1,3-butanediol production was observed when phaB was integrated into the genome.

[0141] As the plasmid, and subsequently phaB exist in multiple copy numbers, a greater product titre would be expected as seen for (R)-3-hyrdoxybutyrate. However this is not observed for (R)-1,3-butanediol production. Gene integration leads to increased (R)-1,3-butanediol compared to plasmid expression, indicating further regulatory mechanism and possible feedback inhibition by increased enzyme availability within the (R)-1,3-butanediol and (R)-3-hyrdoxybutyrate pathway.