Genetically modified acetogenic cell

Abstract

There is provided an acetogenic microbial cell which is capable of producing at least one higher alcohol from a carbon source, wherein the acetogenic microbial cell is genetically modified to comprise an increased expression relative to its wild type cell of at least one enzyme, E.sub.8, a butyryl-CoA:acetate CoA transferase (cat3). There is also provided a method and use of the cell to produce higher alcohols.

Claims

1. An acetogenic microbial cell which is capable of producing at least one higher alcohol from a carbon source, wherein the acetogenic microbial cell is genetically modified to comprise at least one enzyme, E.sub.8, a butyryl-CoA: acetate CoA transferase (cat3), wherein said E.sub.8 comprises an amino acid sequence having at least 100% sequence identity with the amino acid sequence of SEQ ID NO:1 of an enzyme obtained from Clostridium kluyveri, and wherein the higher alcohol comprises the structure of formula I below and has 4 to 10 carbon atoms: ##STR00002## wherein the acetogenic microbial cell is Clostridium ljungdahlii or Clostridium autothenogenum.

2. The acetogenic microbial cell of claim 1, wherein the cell is genetically modified to comprise an increased expression relative to its wild type cell of at least one further enzyme selected from the group consisting of E.sub.1, E.sub.3 to E.sub.7 and E.sub.12, wherein E.sub.1 is an alcohol dehydrogenase (adh) comprising at least a 90% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 19 and comprises alcohol dehydrogenase activity, E.sub.3 is an acetoacetyl-CoA thiolase (thl) comprising at least a 90% sequence identity with SEQ ID NO: 2 and comprises acetoacetyl-CoA thiolase activity, E.sub.4 is a 3-hydroxybutyryl-CoA dehydrogenase (hbd) comprising at least a 90% sequence identity with SEQ ID NO: 3 and comprises 3-hydroxybutyryl-CoA dehydrogenase activity, E.sub.5 is a 3-hydroxybutyryl-CoA dehydratase (crt) comprising at least a 90% sequence identity with SEQ ID NO: 4 and comprises 3-hydroxybutyryl-CoA dehydratase activity, E.sub.6 is a butyryl-CoA dehydrogenase (bcd) comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-7 and comprises butyryl-CoA dehydrogenase activity, E.sub.7 is an electron transfer flavoprotein subunit (etf) comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-13 and comprises electron transfer flavoprotein subunit activity, and E.sub.12 is a trans-2-enoyl-CoA reductase or crotonyl-CoA reductase comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-17 and comprises trans-2-enoyl-CoA reductase or crotonyl-CoA reductase activity.

3. The acetogenic microbial cell of claim 1, wherein the increased expression of the E.sub.8 increases due to an increase in the copy number of a gene for E.sub.8.

4. The acetogenic microbial cell of claim 2, wherein: E.sub.1 comprises at least a 95% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 19 and comprises alcohol dehydrogenase activity; E.sub.3 comprises at least a 95% sequence identity with SEQ ID NO: 2 and comprises acetoacetyl-CoA thiolase activity; E.sub.4 comprises at least a 95% sequence identity with SEQ ID NO: 3 and comprises 3-hydroxybutyryl-CoA dehydrogenase activity; E.sub.5 comprises at least a 95% sequence identity with SEQ ID NO: 4 and comprises 3-hydroxybutyryl-CoA dehydratase activity; E.sub.6 comprises at least a 95% sequence identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5-7 and comprises butyryl-CoA dehydrogenase activity; E.sub.7 comprises at least a 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-13 and comprises electron transfer flavoprotein subunit activity; E.sub.12 comprises at least a 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 15-17 and comprises trans-2-enoyl-CoA reductase or crotonyl-CoA reductase activity.

5. The acetogenic microbial cell of claim 1, wherein the higher alcohol is selected from the group consisting of 1-butanol, 2-methyl-1-butanol, isobutanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol, 1-pentanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-methyl-1-hexanol, 6-methyl-1-heptanol and combinations thereof.

6. A method of producing at least one higher alcohol, the method comprising contacting the acetogenic microbial cell of claim 1 with a medium comprising a carbon source.

7. The method of claim 6, wherein the carbon source comprises CO and/or CO.sub.2.

8. The method of claim 6, wherein the microbial cell is genetically modified to comprise an increased expression relative to its wild type cell of at least one further enzyme selected from the group consisting of E.sub.1, E.sub.3 to E.sub.7 and E.sub.12, wherein E.sub.1 is an alcohol dehydrogenase (adh) comprising at least a 90% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 19, E.sub.3 is an acetoacetyl-CoA thiolase (thl) comprising at least a 90% sequence identity with SEQ ID NO: 2, E.sub.4 is a 3-hydroxybutyryl-CoA dehydrogenase (hbd) comprising at least a 90% sequence identity with SEQ ID NO: 3, E.sub.5 is a 3-hydroxybutyryl-CoA dehydratase (crt) comprising at least a 90% sequence identity with SEQ ID NO: 4, E.sub.6 is a butyryl-CoA dehydrogenase (bcd) comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-7, E.sub.7 is an electron transfer flavoprotein subunit (etf) comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-13, and E.sub.12 is a trans-2-enoyl-CoA reductase or crotonyl-CoA reductase comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-17.

9. The method of claim 8, wherein the acetogenic microbial cell is genetically modified to comprise an increased expression relative to its wild type cell of E.sub.3, E.sub.4 and E.sub.12.

10. The method of claim 6, wherein the higher alcohol is selected from the group consisting of 1-butanol, 2-methyl-1-butanol, isobutanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol, 1-pentanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-methyl-1-hexanol, 6-methyl-1-heptanol and combinations thereof.

11. The method of claim 6, wherein increased expression of E.sub.8 is due to expression of a heterologous E.sub.8 enzyme, an increase in the copy number of a gene for E.sub.8, the use of a heterologous promoter, or combinations thereof.

12. An acetogenic microbial cell which is capable of producing at least one higher alcohol from a carbon source, wherein: the acetogenic microbial cell is genetically modified to comprise increased butyryl-CoA: acetate CoA transferase (cat3 activity) relative to the acetogenic microbial cell before being genetically modified, said increased activity being at least partially due to the enzyme E.sub.8, wherein said E.sub.8 comprises an amino acid sequence having at least 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 of an enzyme of obtained from Clostridium kluyveri; the higher alcohol comprises the structure of formula I below and has 4 to 10 carbon atoms: ##STR00003## wherein R=H or CH.sub.3, and n=1-6; and wherein the acetogenic microbial cell is Clostridium ljungdahlii or Clostridium autothenogenum.

13. The acetogenic microbial cell of claim 12, wherein the cell is genetically modified to comprise increased expression relative to its wild type cell of at least one enzyme selected from the group consisting of E.sub.1, E.sub.3 to E.sub.7 and E.sub.12, wherein E.sub.1 is an alcohol dehydrogenase (adh) comprising at least a 90% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 19 and comprises alcohol dehydrogenase activity, E.sub.3 is an acetoacetyl-CoA thiolase (thl) comprising at least a 90% sequence identity with SEQ ID NO: 2 and comprises acetoacetyl-CoA thiolase activity, E.sub.4 is a 3-hydroxybutyryl-CoA dehydrogenase (hbd) comprising at least a 90% sequence identity with SEQ ID NO: 3 and comprises 3-hydroxybutyryl-CoA dehydrogenase activity, E.sub.5 is a 3-hydroxybutyryl-CoA dehydratase (crt) comprising at least a 90% sequence identity with SEQ ID NO: 4 and comprises 3-hydroxybutyryl-CoA dehydratase activity, E.sub.6 is a butyryl-CoA dehydrogenase (bcd) comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-7 and comprises butyryl-CoA dehydrogenase activity, E.sub.7 is an electron transfer flavoprotein subunit (etf) comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-13 and comprises electron transfer flavoprotein subunit activity, and E.sub.12 is a trans-2-enoyl-CoA reductase or crotonyl-CoA reductase comprising at least a 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-17 and comprises trans-2-enoyl-CoA reductase or crotonyl-CoA reductase activity.

14. The acetogenic microbial cell of claim 13, wherein: E.sub.1 comprises at least a 95% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 19 and comprises alcohol dehydrogenase activity; E.sub.3 comprises at least a 95% sequence identity with SEQ ID NO: 2 and comprises acetoacetyl-CoA thiolase activity; E.sub.4 comprises at least a 95% sequence identity with SEQ ID NO: 3 and comprises 3-hydroxybutyryl-CoA dehydrogenase activity; E.sub.5 comprises at least a 95% sequence identity with SEQ ID NO: 4 and comprises 3-hydroxybutyryl-CoA dehydratase activity; E.sub.6 comprises at least a 95% sequence identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5-7 and comprises butyryl-CoA dehydrogenase activity; E.sub.7 comprises at least a 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-13 and comprises electron transfer flavoprotein subunit activity; E.sub.12 comprises at least a 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 15-17 and comprises trans-2-enoyl-CoA reductase or crotonyl-CoA reductase activity.

15. The acetogenic microbial cell of claim 14, wherein the higher alcohol is selected from the group consisting of 1-butanol, 2-methyl-1-butanol, isobutanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol, 1-pentanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-methyl-1-hexanol, 6-methyl-1-heptanol and combinations thereof.

16. The acetogenic microbial cell of claim 12, wherein the higher alcohol is selected from the group consisting of 1-butanol, 2-methyl-1-butanol, isobutanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol, 1-pentanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-methyl-1-hexanol, 6-methyl-1-heptanol and combinations thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is the vector pSOS95

EXAMPLES

(2) The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

(3) All the sequences within the examples are of the genes connected together and does not include the actual vector pSOS95 backbone sequence.

Example 1

(4) Generation of a genetically modified acetogenic bacteria for the formation of Butanol

(5) Vectors pATh-LEM-04 and pATh-LEM-14

(6) The genes Thiolase from C. acetobutylicum ATTC 824 (thl_Ca) (SEQ ID NO:28), hydroxybutyryl-CoA dehydrogenase from C. kluyveri (hbdl_Ck) (SEQ ID NO: 29), crotonase from C. klyuveri (crtl_Ck) (SEQ ID NO:30) and butyryl-CoA dehydrogenase form C. kluyveri (bcd1_Ck) (SEQ ID NO:31) are amplified from the corresponding genome and were inserted into the vector pEmpty by using KasI and BamHI. This plasmid (pEmpty) was based on the plasmid backbone pSOS95 (FIG. 1). To use pSOS95, it was digested with BamHI and KasI. This removed the operon ctfA-ctfB-adc, but leaves the thl promoter and the rho-independent terminator of adc. The newly generated vector, which bore the named genes, was called pATh-LEM-02 (SEQ ID NO:51 refers to the sequences of the genes connected together in pATh-LEM-02 without the sequence of the actual vector).

(7) In a second cloning step, the vector pATh-LEM-02 was digested with EcoRI and KasI and the CoA-Transferase from C. kluyveri (cat3_Ck) (SEQ ID NO:26) was amplified from genomic DNA and integrated into the vector. The newly designed vector was named pATh-LEM-04. To create the vector pATh-LEM-14, the vector pATH-LEM-04 was digested with KasI and BspEI. The genes etfBA were amplified from genomic DNA of Clostridium kluyveri by using the oligonucleotides of SEQ ID NOs: 46 and 47.

(8) A fragment of cat3 was amplified from pATh-LEM-04 by using the oligonucleotides of SEQ ID NOs: 48 and 49. The resultant fragment has sequence of SEQ ID NO:52. The two fragments of cat3 and etfBA were then fused using PCR with primers of SEQ ID NO: 50 and 49. This fusion insert of cat3 and etfBA was then added to the KasI and BspEI opened vector pATH-LEM-04. The resultant vector was called pATH-LEM-14 (SEQ ID NO:20 is the sequence of the target genes fused together that can be easily inserted into the vector).

(9) Vectors pATh-Syn4-03 and pATh-LEM-23

(10) To generate a vector named pATh-Syn4-03 a cassette with SEQ ID NO:53 was first formed. This cassette comprised the genes: Thiolase from C. acetobutylicum ATTC 824 (thl_Ca) (SEQ ID NO:28), hydroxybutyryl-CoA dehydrogenase from C. kluyveri (hbdl_Ck) (SEQ ID NO: 29), and crotonase from C. klyuveri (crtl_Ck) (SEQ ID NO:30). The cassette with SEQ ID NO:53 was then inserted into the vector pEmpty by using KasI and BamHI.

(11) This plasmid (pEmpty) was based on the plasmid backbone pSOS95 (FIG. 1). To use pSOS95, it was digested with BamHI and KasI. This removes the operon ctfA-cfB-adc, but leaves the thl promoter and the rho-independent terminator of adc. In a second step, the thl promoter was removed from the vector by digesting it with SbfI and BamHI. The pta promoter fragment (SEQ ID NO: 25 (Ueki et al. (2014) mBio. 585): 1636-14) was synthesized and was ligated to the BamHI/SbfI digested vector. The newly generated vector, which bears the named genes and the pta promoter, was called pATh-Syn4-14.

(12) The vector pATh-Syn4-14 was opened with KasI and EcoRI and ligated with SEQ ID NO:54 which was synthesized from CoA-Transferase from C. kluyveri. The generated vector was named pATh-LEM-23 (SEQ ID NO:21)

(13) Vectors pATh-LEM-15, pATh-LEM-16, pATh-LEM-24, pATh-LEM-25, pATh-LEM-26

(14) A cassette containing Thiolase from C. acetobutylicum ATTC 824 (thl_Ca) (SEQ ID NO:29), hydroxybutyryl-CoA dehydrogenase from C. kluyveri (hbdl_Ck) (SEQ ID NO: 29), and crotonase from C. klyuveri (crtl_Ck) (SEQ ID NO:30) were synthesized and were inserted into the vector pEmpty by using KasI and BamHI. This plasmid (pEmpty) is based on the plasmid backbone pSOS95 (FIG. 1). To use pSOS95, it was digested with BamHI and KasI. This removes the operon ctfA-ctfB-adc, but leaves the thl promoter and the rho-independent terminator of adc. The newly generated vector, which bears the named genes, was called pATh-Syn4-03.

(15) The vector pATh-Syn4-03 was opened with KasI and a cassette containing butyrate-dehydrogenase from C. acetobutylicum (bcd_Ca) (SEQ ID NO:34), electron-transfer protein from C. acetobutylicum (etfBA_Ca) (SEQ ID NOs: 35 and 36) and CoA-transferase from C. kluyveri (cat3_Ck) (SEQ ID NO:26) was ligated by in vitro cloning. The newly constructed vector is named pATh-LEM-15 (SEQ ID NO:55).

(16) The vector pATh-Syn4-03 was opened with KasI/EcoRI and ligated with a cassette (SEQ ID NO:56 without the full sequence of the vector containing butyrate-dehydrogenase from C. kluyveri (bcd1_Ck) (SEQ ID NO:5), electron-transfer protein from C. klyuveri (etfBA1_Ck) (SEQ ID NOs:8 and 9) and CoA-transferase from C. kluyveri (cat3_Ck) (SEQ ID NO:1). The newly constructed vector is named pATh-LEM-16.

(17) The vector pATh-Syn4-03 was opened with KasI and EcoRI. A DNA fragment of CoA-Transferase from C. kluyveri (SEQ ID NO:57) was synthesized and ligated to the prepared vector. The generated vector was named pATh-LEM-24 (SEQ ID NO:22).

(18) To generate the vector pATh-LEM-25, the plasmid pATh-Syn4-24 was opened with AsiSI and EcoRI. A DNA fragment containing the Butanol dehydrogenase B from C. acetobutylicum (bdhB_Ca) (SEQ ID NO:44) was synthesized and ligated to the prepared vector. The generated vector was named pATh-LEM-25 (SEQ ID NO:23).

(19) To generate the vector pATh-LEM-26, the plasmid pATh-Syn4-25 (SEQ ID NO:23) was opened with AsiSI and AscI. The Butanol dehydrogenase from E. coli codon optimized for C. ljungdahlii (YghD_E(coCl)) (SEQ ID NO:58) was amplified, fused with a ribosome binding site and ligated to the prepared vector. The generated vector was named pATh-LEM-26 (SEQ ID NO:24).

(20) Vectors pATh-LEM-17, pATh-LEM-18, pATh-LEM-19, pATh-LEM-20, pATh-LEM-21

(21) The vector pATh-LEM-16 was opened with KasI and NotI. A DNA fragment of SEQ ID NO: 59 containing butyrate-dehydrogenase 2 from C. kluyveri (bcd2_Ck) (SEQ ID NO:37) and electron-transfer protein 2 from C. klyuveri (etfBA2_Ck) (SEQ ID NOs: 39 and 38) was ligated. The newly constructed vector was named pATh-LEM-17.

(22) To create pATh-LEM-18 the vector pATh-LEM-16 was opened with KasI and NotI. The DNA fragment containing the codon optimized trans-2-enoyl-CoA reductase from Treponema denticola (TER_Td(coCl)) (SEQ ID NO:41) was ligated. The newly constructed vector is named pATh-LEM-18.

(23) To create pATh-LEM-19 the vector pATh-LEM-16 was opened with Not and AarI. The DNA fragment containing the codon optimized trans-2-enoyl-CoA reductase from Euglena gracilis (TER_Eg(coCl)) (SEQ ID NO:40) was ligated. The newly constructed vector was named pATh-LEM-19.

(24) The vector pATh-LEM-16 was opened with AarI and NotI. The DNA fragment containing the codon optimized trans-2-enoyl-CoA reductase from Caenorhabditis elegans (TER_Ce(coCl)) (SEQ ID NO:42) was ligated. The newly constructed vector was named pATh-LEM-20.

(25) The vector pATh-LEM-16 was opened with FseI and NotI. The synthetic DNA fragment containing the codon optimized crotonyl-CoA reductase from Streptomyces collinus (Ccr_Sc(coCl)) (SEQ ID NO:43) was ligated. The newly constructed vector was named pATh-LEM-21.

(26) Vector pATh-LEM-22

(27) A DNA fragment (SEQ ID NO: 60) containing the butyryl-CoA dehydrogenase from C. klyveri (bcd1_Ck) (SEQ ID NO:31), electron-transfer protein from C. kluyveri (etfBA1_Ck) (SEQ ID NOs:32 and 33), the CoA-transferase from C. kluyveri (cat3_Ck) (SEQ ID NO:26) and transcriptional elements (pta-Promoter and a Terminator). The parental vector pATh-Syn4-03 was opened with EcoRI/XhoI and the DNA fragment (SEQ ID NO: 60) ligated in to produce the vector pATh-LEM-22.

(28) Transformation of Acetogens:

(29) The transformation of C. ljungdahlii DSMZ 13528 and C. autoethanogenum DSMZ 10061 was done as disclosed in Leang et al. (2013) Applied and Environmental Microbiology 79(4): 1102-1109.

Example 2

(30) Fermentation of Genetically Modified Strains on Mixtures of H.sub.2, CO.sub.2 and CO Showing Acid and Higher Alcohol Formation.

(31) For cell culture of

(32) C. ljungdahlii pATh-LEM-04

(33) C. ljungdahlii pATh-LEM-14

(34) C. ljungdahlii pATh-LEM-15

(35) C. ljungdahlii pATh-LEM-16

(36) C. ljungdahlii pATh-LEM-17

(37) C. ljungdahlii pATh-LEM-18

(38) C. ljungdahlii pATh-LEM-19

(39) C. ljungdahlii pATh-LEM-20

(40) C. ljungdahlii pATh-LEM-21

(41) C. ljungdahlii pATh-LEM-22

(42) C. ljungdahlii pATh-LEM-23

(43) C. ljungdahlii pATh-LEM-24

(44) C. ljungdahlii pATh-LEM-25

(45) C. ljungdahlii pATh-LEM-26

(46) C. ljungdahlii pEmpty

(47) C. autoethanogenum pATh-LEM-04

(48) C. autoethanogenum pATh-LEM-14

(49) C. autoethanogenum pATh-LEM-15

(50) C. autoethanogenum pATh-LEM-16

(51) C. autoethanogenum pATh-LEM-17

(52) C. autoethanogenum pATh-LEM-18

(53) C. autoethanogenum pATh-LEM-19

(54) C. autoethanogenum pATh-LEM-20

(55) C. autoethanogenum pATh-LEM-21

(56) C. autoethanogenum pATh-LEM-22

(57) C. autoethanogenum pATh-LEM-23

(58) C. autoethanogenum pATh-LEM-24

(59) C. autoethanogenum pATh-LEM-25

(60) C. autoethanogenum pATh-LEM-26

(61) C. autoethanogenum pEmpty

(62) 5 mL of the culture will be anaerobically grown in 500 ml of LM33-medium with 100 mg/L of erythromycin.

(63) LM 33 media was prepared at pH 5.5 as follows in tables 1-3. All ingredients with the exception of cysteine HCL were mixed in dH.sub.2O to a total volume of 1 L. This solution was made anaerobic by heating to boiling point and allowing it to cool to room temperature under a constant flow of N.sub.2 gas. Once cool, the cysteine HCL (0.5 g/L) was added and the pH of the solution adjusted to 5.5; anaerobicity was maintained throughout the experiments.

(64) TABLE-US-00001 TABLE 1 Media component (LM-33) used in Example 1 Media component concentration MgCl.sub.2 × 6H.sub.2O 0.5 g/L NaCl 0.2 g/L CaCl.sub.2 × 2H.sub.2O 0.135 g/L NaH.sub.2PO.sub.4 × 2H.sub.2O 2.65 g/L KCl 0.5 g/L NH.sub.4Cl 2.5 g/L MES 20.0 g/L LS06-trace element solution 10 mL/L LS03-vitamin solution 10 mL/L FeCl.sub.3-Solution 2 mL/L

(65) TABLE-US-00002 TABLE 2 LS06-trace element solution components concentration Nitriloacetic acid 1.5 g/L MgSO.sub.4 × 7H.sub.2O 3 g/L MnSO.sub.4 × H.sub.2O 0.5 g/L NaCl 1 g/L FeSO.sub.4 × 7H.sub.2O 0.1 g/L Fe(SO.sub.4).sub.2(NH4).sub.2 × 6H.sub.2O 0.8 g/L CoCl.sub.2 × 6H.sub.2O 0.2 g/L ZnSO.sub.4 × 7H.sub.2O 0.2 g/L CuCl.sub.2 × 2H.sub.2O 0.02 g/L KAl(SO.sub.4).sub.2 × 12H.sub.2O 0.02 g/L H.sub.3BO.sub.3 0.3 g/L Na.sub.2MoO.sub.4 × 2H.sub.2O 0.03 g/L Na.sub.2SeO.sub.3 0.02 g/L NiCl.sub.2 × 6H.sub.2O 0.02 g/L Na.sub.2WO.sub.4 × 6H.sub.2O 0.02 g/L

(66) TABLE-US-00003 TABLE 3 LS03-vitamin-solution component concentration Biotin 20 mg/L Folic Acid 20 mg/L Pyridoxine HCl 10 mg/L Thiamin HCl 50 mg/L Riboflavin 50 mg/L Nicotinic Acid 50 mg/L CalciumD-(+)- 50 mg/L pantothenate Vitamin B12 50 mg/L p-Aminobenzoic acid 50 mg/L Lipoic Acid 50 mg/L

(67) Cultivation is carried out in duplicate into 1 L glass bottles with a premixed gas mixture composed of around H.sub.2, CO.sub.2 and CO in an open water bath shaker at 37° C., 150 rpm and aeration of 3 L/h for 70 h. The gas will enter the medium through a filter with a pore size of 10 microns, which will mount in the middle of the reactor, at a gassing tube. When sampling each 5 ml sample will be removed for determination of OD600 nm, pH and the product range. The determination of the product concentration will be performed by semi-quantitative 1 H-NMR spectroscopy. As an internal quantification standard sodium trimethylsilylpropionate will be used. In contrast to the negative controls C. ljungdalii pEmpty and C. autoethanogenum pEmpty the modified strains will produce butyrate, butanol, hexanoate, hexanol, octanoate and octanol.

Example 3

(68) Materials and Methods

(69) In the following examples, genetically modified Clostridium ljungdahlii or Clostridium autoethanogenum were cultivated in order to produce butanol and/or the precursors 3-hydroxybutyrate and/or butyrate. A complex medium with 5 g/L fructose was used, consisting of 1 g/L NH.sub.4Cl, 0.1 g/L KCl, 0.2 g/L MgSO.sub.4×7 H.sub.2O, 0.8 g/L NaCl, 0.1 g/L KH.sub.2PO.sub.4, 20 mg/L CaCl.sub.2×2 H.sub.2O, 20 g/L MES, 1 g/L yeast extract, 0.4 g/L L-cysteine-HCl, 0.4 g/L Na.sub.2S×9 H.sub.2O, 20 mg/L nitrilotriacetic acid, 10 mg/L MnSO.sub.4×H.sub.2O, 8 mg/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.2×6 H.sub.2O, 2 mg/L CoCl.sub.2×6 H.sub.2O, 2 mg/L ZnSO.sub.4×7 H.sub.2O, 0.2 mg/L CuCl.sub.2×2 H.sub.2O, 0.2 mg/L Na.sub.2MoO.sub.4×2 H.sub.2O, 0.2 mg/L NiCl.sub.2×6 H.sub.2O, 0.2 mg/L Na.sub.2SeO.sub.4, 0.2 mg/L Na.sub.2WO.sub.4×2 H.sub.2O, 20 μg/L biotin, 20 μg/L folic acid, 100 μg/L pyridoxine-HCl, 50 μg/L thiamine-HCl×H.sub.2O, 50 μg/L riboflavin, 50 μg/L nicotinic acid, 50 μg/L Ca-pantothenoic acid, 1 μg/L vitamin B12, 50 μg/L p-aminobenzoic acid, 50 μg/L lipoic acid.

(70) The heterotrophic cultivations were performed in 50 mL medium in a 250 mL serum bottle. The serum bottle was continuously shaken in an open water bath Innova 3100 from New Brunswick Scientific at 37° C. and a shaking rate of 150 min.sup.−1.

(71) The experiments were inoculated with 5 mL cell suspension grown in Hungate tubes in above described medium. During the experiment samples of 5 mL were taken for the determination of OD.sub.600, pH and product concentrations. The latter were determined by quantitative .sup.1H-NMR-spectroscopy.

(72) Results and Discussion:

Example 3a

(73) Cultivation of Genetically Modified Clostridium ljungdahlii pATh-LEM-14

(74) Genetically modified C. ljungdahlii pATh-LEM-14 as shown in Examples 1 and 2, was heterotrophically cultivated under above described conditions.

(75) After inoculation, cells grew up to a maximal optical density of 1.82 after 56.6 hours. Besides the natural products acetate and ethanol a maximal butanol concentration of 59 mg/L was measured after 56.6 h. Butyrate was produced up to a concentration of 200 mg/L. The results are shown in Table 4.

(76) TABLE-US-00004 TABLE 4 Results of C. ljungdahlii pATh-LEM-14 fermentation NMR-analytics 3-Hydroxy- Process Acetate, Ethanol, butyrate, n-Butanol, Butyrate, time, h pH OD.sub.600 mg/L mg/L mg/L mg/L mg/L 0.0 5.96 0.14 160 17 n.d. n.d. 41 56.6 5.01 1.82 2650 500 n.d. 59 190 117.7 5.03 1.21 2700 490 n.d. 59 200 (n.d. = not detected)

Example 3b

(77) Cultivation of Genetically Modified Clostridium ljungdahlii pATh-LEM-23

(78) Genetically modified C. ljungdahlii pATh-LEM-23 was heterotrophically cultivated under above described conditions. After inoculation, cells grew up to a maximal optical density of 1.21 after 113.6 hours. Besides the natural products acetate and ethanol a maximal butanol concentration of 8 mg/L was measured after 113.6 h. 3-hydroxybutyrate and butyrate were produced up to concentrations of 230 mg/L and 15 mg/L respectively. The results are shown in Table 6.

(79) TABLE-US-00005 TABLE 5 Results of C. ljungdahlii pATh-LEM-23 fermentation NMR-analytics 3-Hydroxy- Process Acetate, Ethanol, butyrate, n-Butanol, Butyrate, time, h pH OD.sub.600 mg/L mg/L mg/L mg/L mg/L 0.0 5.91 0.14 210 25 25 n.d. n.d. 113.6 4.99 1.21 2950 520 230 8 15 (n.d. = not detected)

Example 3c

(80) Cultivation of Genetically Modified Clostridium ljungdahlii pATh-LEM-24

(81) Genetically modified C. ljungdahlii pATh-LEM-24 was heterotrophically cultivated under above described conditions. After inoculation, cells grew up to a maximal optical density of 1.92 after 113.6 hours. Besides the natural products acetate and ethanol a maximal butanol concentration of 7 mg/L was measured after 113.6 h. 3-hydroxybutyrate and butyrate were produced up to concentrations of 170 mg/L and 12 mg/L respectively. The results are shown in Table 7.

(82) TABLE-US-00006 TABLE 6 Results of C. ljungdahlii pATh-LEM-24 fermentation NMR-analytics 3-Hydroxy- Process Acetate, Ethanol, butyrate, n-Butanol, Butyrate, time, h pH OD.sub.600 mg/L mg/L mg/L mg/L mg/L 0.0 5.94 0.06 91 15 17 n.d. n.d. 113.6 4.95 1.92 3000 580 170 7 12 (n.d. = not detected)

Example 3d

(83) Cultivation of Genetically Modified Clostridium ljungdahlii pATh-LEM-25

(84) Genetically modified C. ljungdahlii pATh-LEM-25 was heterotrophically cultivated under above described conditions. After inoculation, cells grew up to a maximal optical density of 1.52 after 117.4 hours. Besides the natural products acetate and ethanol no butanol was detected. Butyrate had a peak of 13 mg/L after 51.1 hours, but was consumed again thereafter. The precursor 3-hydroxybutyrate was produced up to a concentration of 73 mg/L. The results are shown in Table 8.

(85) TABLE-US-00007 TABLE 7 Results of C. ljungdahlii pATh-LEM-25 fermentation NMR-analytics 3-Hydroxy- Process Acetate, Ethanol, butyrate, n-Butanol, Butyrate, time, h pH OD.sub.600 mg/L mg/L mg/L mg/L mg/L 0.0 6.01 0.07 88 19 17 n.d. n.d. 51.1 5.82 0.61 730 320 55 n.d. 13 117.4 5.04 1.52 2800 640 73 n.d. n.d. (n.d. = not detected)

Example 3e

(86) Cultivation of Genetically Modified Clostridium autoethanogenum pATh-LEM-23

(87) In this example, genetically modified C. autoethanogenum pATh-LEM-23 was heterotrophically cultivated under above described conditions.

(88) After inoculation, cells grew to a maximal optical density of 0.98 after 117.4 hours. Besides the natural products acetate and ethanol no butanol was detected. The precursor butyrate had a peak of 6 mg/L after 51.1 hours, but was consumed again thereafter. The precursor 3-hydroxybutyrate was produced up to a concentration of 140 mg/L. The results are shown in Table 9.

(89) TABLE-US-00008 TABLE 8 Results of C. autoethanogenum pATh-LEM-23 fermentation NMR-analytics 3-Hydroxy- Process Acetate, Ethanol, butyrate, n-Butanol, Butyrate, time, h pH OD.sub.600 mg/L mg/L mg/L mg/L mg/L 0.0 5.98 0.08 120 31 12 n.d. n.d. 51.1 5.93 0.27 350 180 16 n.d. 6 117.4 5.26 0.98 2200 760 140 n.d. n.d. (n.d. = not detected)

Example 3f

(90) Cultivation of Wildtype Clostridium ljungdahlii DSM 13528 (Wildtype)

(91) The wildtype of C. ljungdahlii (DSM 13528) was heterotrophically cultivated under above described conditions. After inoculation, cells began to grow up to a maximal optical density of 1.20 after 68.5 hours. Only the natural products acetate and ethanol were measured after 68.5 h to maximal concentrations of 1197 mg/L and ethanol respectively.

(92) TABLE-US-00009 TABLE 9 Results of C. ljungdahlii wt fermentation NMR-analytics 3-Hydroxy- Process Acetate, Ethanol, butyrate, n-Butanol, Butyrate, time, h pH OD.sub.600 mg/L mg/L mg/L mg/L mg/L 0.0 6.00 0.11 156 15 n.d. n.d. n.d. 68.5 5.61 1.20 1197 402 n.d. n.d. n.d. (n.d. = not detected)