Means and methods for producing isobutene from acetyl-CoA

Abstract

Described is a recombinant organism or microorganism which is capable of enzymatically converting acetyl-CoA into isobutene, (A) wherein in said organism or microorganism: (i) acetyl-CoA is enzymatically converted into acetoacetyl-CoA, (ii) acetoacetyl-CoA is enzymatically converted into 3-hydroxy-3-methylglutaryl-CoA, (iii) 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted into 3-methylglutaconyl-CoA, (iv) 3-methylglutaconyl-CoA is enzymatically converted into 3-methylcrotonyl-CoA, and (v) wherein said 3-methylcrotonyl-CoA is converted into isobutene by: (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid which is then further enzymatically converted into said isobutene; or (b) enzymatically converting 3-methylcrotonyl-CoA into 3-hydroxy-3-methylbutyryl-CoA which is then further enzymatically converted into 3-hydroxy-3-methylbutyric acid which is then further enzymatically converted into 3-phosphonoxy-3-methylbutyric acid which is then further enzymatically converted into said isobutene; (B) wherein said recombinant organism or microorganism has an increased pool of coenzyme A (CoA) over the organism or microorganism from which it is derived due to: (i) an increased uptake of pantothenate; and/or (ii) an increased conversion of pantothenate into CoA. Moreover, described is the use of such a recombinant organism or microorganism for the production of isobutene. Further, described is a method for the production of isobutene by culturing such a recombinant organism or microorganism in a suitable culture medium under suitable conditions.

Claims

1. A recombinant organism or microorganism which is capable of enzymatically converting acetyl-CoA into isobutene, (A) wherein in said organism or microorganism: (i) acetyl-CoA is enzymatically converted into acetoacetyl-CoA by one of, an acetyl-CoA carboxylase and an acetoacetyl-CoA synthase, or an acetyl-CoA acetyltransferase, optionally wherein said acetyl-CoA acetyltransferase is an acetyl-CoA transferase, (ii) acetoacetyl-CoA is enzymatically converted into 3-hydroxy-3-methylglutaryl-CoA by a 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase), (iii) 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted into 3-methylglutaconyl-CoA by one of a 3-methylglutaconyl-coenzyme A hydratase, a 3-hydroxy-3-methylglutaryl-coenzyme A dehydratase, a 3-hydroxyacyl-CoA dehydratase, or an enoyl-CoA hydratase, (iv) 3-methylglutaconyl-CoA is enzymatically converted into 3-methylcrotonyl-CoA by one of a methylcrotonyl-CoA carboxylase, a geranoyl-CoA carboxylase, a 3-methylglutaconyl-CoA decarboxylase, or a glutaconate CoA-transferase, (v) wherein said 3-methylcrotonyl-CoA is converted into isobutene by enzymatically converting said 3-methylcrotonyl-CoA into 3-methylcrotonic acid by one of a direct conversion by a CoA transferase or a thioester hydrolase, optionally wherein said thioester hydrolase is a 1.4-dihydroxy-2-naphthoyl CoA hydrolase or an acyl-CoA hydrolase, or a two-step conversion comprising conversion of said 3-methylcrotonyl-CoA into 3-methylcrotonyl-phosphate by a phosphate butyryltransferase or a phosphate acetyltransferase followed by conversion of said 3-methylcrotonyl-phosphate into said 3-methylcrotonic acid by a phosphotransferase, optionally wherein said phosphotransferase is a butyrate kinase, a branched-chain-fatty-acid kinase, a propionate kinase, or an acetate kinase, and said 3-methylcrotonic acid is enzymatically converted into said isobutene by a flavin mononucleotide (FMN)-dependent 3-methylcrotonic acid decarboxylase associated with an FMN prenyl transferase, optionally wherein said FMN-dependent 3-methylcrotonic acid decarboxylase is a 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) comprising the amino acid sequence of SEQ ID NO:25 or an UbiD-like decarboxylase comprising the amino acid sequence shown of SEQ ID NO:26 and said FMN prenyl transferase is a prenyl transferase ubiX (3-octaprenyl-4-hydroxybenzoate carboxy-lyase): and (B) wherein said recombinant organism or microorganism recombinantly expresses a pantothenate uptake transporter and has an increased pool of coenzyme A (CoA) over the corresponding organism or microorganism from which it is derived, due to an increased uptake of pantothenate due to a said recombinant expression of a said pantothenate uptake transporter.

2. The recombinant organism or microorganism of claim 1, further wherein said organism or microorganism: a) has phosphoketolase activity; b) (i) has a diminished or inactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivation of the gene(s) encoding phosphofructokinase or by reducing phosphofructokinase activity as compared to a non-modified microorganism, or (ii) does not possess phosphofructokinase activity; and c) (i) has a diminished or inactivated oxidative branch of the pentose phosphate pathway (PPP) by inactivation of the gene(s) encoding glucose-6-phosphate dehydrogenase or by reducing glucose-6-phosphate dehydrogenase activity as compared to a non-modified microorganism, or (ii) does not possessing glucose-6-phosphate dehydrogenase activity.

3. The recombinant organism or microorganism of claim 2, further wherein said organism or microorganism: d) has fructose-1,6-bisphosphate phosphatase activity.

4. The recombinant organism or microorganism of claim 2, wherein the EMPP is further diminished or inactivated by inactivation of the gene(s) encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing glyceraldehyde 3-phosphate dehydrogenase activity as compared to a non-modified microorganism.

5. The recombinant organism or microorganism of claim 2 which has been genetically modified to have an increased phosphoketolase activity over the phosphoketolase activity of the corresponding non-modified organism or microorganism from which it is derived by overexpressing the phosphoketolase.

6. The recombinant organism or microorganism of claim 2, wherein said microorganism is a fungus.

7. The recombinant organism or microorganism of claim 2, wherein said microorganism is a bacterium.

8. The recombinant organism or microorganism of claim 7, wherein the gene(s) encoding the PEP-dependent PTS transporter has/have been inactivated.

Description

(1) FIG. 1: shows artificial pathways for isobutene production from acetyl-CoA via 3-methylcrotonic acid. Moreover, enzymatic recycling of metabolites which may occur during the pathway are shown in steps Xa, Xb, XI and XII.

(2) FIG. 2: Chemical structure of Coenzyme A (CoA).

(3) FIG. 3: shows the main routes of artificial pathway for isobutene production from acetyl-CoA via 3-methylcrotonyl-CoA and the two possible main routes from 3-methylcrotonyl-CoA into isobutene.

(4) FIG. 4: shows the individual steps of the biosynthesis of Coenzyme A (CoA) from pantothenate.

(5) FIG. 5: shows the key enzymes for the conversions from pantothenate to CoA and their respective names in bacteria, yeast and mammals. This Figure is taken from Martinez et al., Biochemical Society Transactions 42(4) (2014) 1112-1117 and supplemented.

(6) FIG. 6: Metabolic pathway for the biosynthesis of isobutene from acetyl-CoA via 3-methycrotonic acid, implemented in Escherichia coli.

(7) FIG. 7: shows the growth of strains E ( CoaA R106A, dotted line) and F (+ CoaA R106A, solid line). Both strains are described in Example 1.

(8) FIG. 8: shows a comparison of IBN volumetric productivities for strains E ( CoaA R106A, dotted line) and F (+ CoaA R106A, solid line). Both strains are described in Example 1. Volumic productivity is the mass of isobutene produced per unit of volume of fermentation broth, and per unit of time.

(9) FIG. 9: shows a comparison of IBN specific productivities for strains E ( CoaA R106A, dotted line) and F (+ CoaA R106A, solid line). Both strains are described in Example 1. Specific productivity is the mass of isobutene produced per unit of biomass and per unit of time. Volumes were not measured during the entire growth phase. Therefore, specific productivities are not represented before t=25 h for strain E and t=39 h for strain F.

(10) FIG. 10: shows a comparison of isobutene production at t=72h for C. ljungdahlii strain SGP244 (MBP-ubiD_HAv6-ubiX-Ec) containing 3-methylcrotonic acid decarboxylase and prenyl transferase (left bar), and C. ljungdahlii strain SGP184 (empty vector) harboring the corresponding empty vector (right bar). Both strains are described in Example 5.

(11) FIG. 11: shows a comparison of isobutene production at t=72h for C. ljungdahlii strain SGP339 (MBP-FDC Ss5v5-ubiX EC) containing the 3-methylcrotonic acid decarboxylase and prenyl transferase (left bar) and C. ljungdahlii strain SGP184 (empty vector) harboring the corresponding empty vector (right bar). Both strains are described in Example 6.

(12) FIG. 12: shows a comparison of isobutene production after 6 days for C. ljungdahlii strain SGP353 containing the isobutene pathway listed in Table F (SGP353 clones 1-5, right), and C. ljungdahlii strain SGP348 harboring the corresponding empty vector (SGP clones 1 & 2, left). Both strains are described in Example 7.

(13) FIG. 13: shows a comparison of the GC-MS signals for the amount of isobutene obtained after 6 days from C. ljungdahlii strain SGP353 containing the isobutene pathway listed in Table F (right panel) and the strain containing the isobutene pathway listed in Table F, and C. ljungdahlii strain SGP348harboring the corresponding empty vector (left panel). Both strains are described in Example 7.

(14) In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

(15) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

EXAMPLES

Example 1: Construction of a New E. coli Chassis for the Production of Isobutene

(16) Like most organisms, E. coli converts glucose into acetyl-CoA. A modified E. coli chassis has previously been described wherein the yield and flux of acetyl-CoA has been increased in order to optimize the yield of acetyl-CoA production (WO2013/007786). Therein, a bacterial chassis, strain A, was constructed with the following genotype:

(17) MG1655 ptsHIzwf_edd_eda pfkA pfkB

(18) Plasmid-based overexpression of a PKT gene from phosphoketolase YP 003354041.1 from Lactococcus lactis in strain A resulted in strain B. This strain B is characterized in that the central carbon metabolism is rewired, wherein a new, phosphoketolase-based carbon catabolic pathway repaces the inactivated Embden-Meyerhoff-Parnas pathway (EMPP), the pentose phosphate pathway (PPP), and the Entner Doudoroff pathway (EDP). Upon introduction of an acetone pathway into strain B, superior acetone yields were observed, as compared with wild type MG1655 strain expressing the same acetone pathway.

(19) In order to construct a strain having a PKT pathway and being capable of growing robustly on sucrose as the carbon source, strain A was further engineered as described below.

(20) A PKT gene was introduced into the chromosome of strain A at the kdgk locus (kdgK:: P1_RBST7_pkt). The resulting strain had the following genotype:

(21) MG1655 ptsHI zwf_edd_eda fkA pfkB kdgK:: P1_RBST7_pkt

(22) This strain was passaged for several months on minimal medium supplemented with glucose as carbon source, while clones or populations were continuously selected for having the highest growth rate, until a doubling time of less than 5 hours was reached.

(23) In order to allow for efficient sucrose consumption, the CscA, CscB and CscK encoding genes were inserted into the chromosome within the zwf locus (zwf::P1_cscA_cscB_cscK_FRT). CscA (Uniprot O86076; NCBI Reference Sequence WP_000194515.1), CscB (Uniprot Q7WZY9; NCBI Reference Sequence WP_001197025.1) and CscK (Uniprot Q7WZY7; NCBI Reference Sequence WP_001274885.1), coding for a sucrose hydrolase, a non-PTS sucrose permease, and a fructokinase, respectively, allow for the uptake of sucrose, its hydrolysis into glucose and fructose, and for the phosphorylation of fructose into fructose-6-phosphate, resulting in the efficient metabolization of fructose (for review, see Biotechnology Advances 32 (2014) 905-919). These modifications resulted in a strain having a PKT pathway, and capable of efficient growth on sucrose.

(24) In order to further improve the sugar consumption, the glk (glucokinase gene, Uniprot P0A6V8; NCBI Reference Sequence: NP_416889.1) gene from E. coli was overexpressed by inserting an additional copy, under the control of the PN25 promoter at the pfkA locus.

(25) The resulting strain is referred to as strain C hereafter.

Example 2: Construction of E. coli Strains for the Production of Isobutene from Acetyl-CoA

(26) This working example shows the production of isobutene by recombinant E. coli strains, expressing the genes: (i) constituting isobutene pathway. (ii) encoding pantothenate kinase, CoaA.

(27) The enzymes used in this study to convert acetyl-CoA into isobutene (IBN) via 3-methylcrotonic acid (FIG. 6) are listed in Table A. The source of the pantothenate kinase was the coaA gene from E. coli MG1655 strain (Uniprot Accession Number: P0A6I3).

(28) TABLE-US-00001 TABLE A Uniprot Accession Step Enzyme Gene NCBI reference number I Acetyl-CoA transferase from thIA WP_010966157.1 P45359 Clostridium acetobulyticum II Hydroxymethylglutaryl-CoA mvaS WP_002357756.1 Q9FD71 synthase from Enterococcus faecalis III Enoyl-CoA hydratase/isomerase PputUW4_01474 WP_015094072.1 K9NHK2 from Pseudomonas sp. UW4 (ECH) IV Glutaconate CoA-transferase from MXAN_4264 WP_011554268.1 Q1D4I3 Myxococcus xanthus (AibA/B) MXAN_4265 WP_011554267.1 Q1D4I4 V 1,4-Dihydroxy-2-naphtoyl-CoA Ydil, menI NP_416201.1 P77781 hydrolase from Escherichia coli VI Variant of UbiD-like GZL_07100 A0A0A8EV26 decarboxylase decarboxylase from Streptomyces sp.769 (UbiD) (A241D-G402A-S403C-C404L- P406A-L448W) Flavin prenyl transferase from ubiX WP_000825700.1 P0AG03 Escherichia coli (UbiX)

(29) Expression of Isobutene Biosynthetic Pathway in E. coli

(30) Strain C as described in Example 1 was used as a host microorganism.

(31) All the listed genes were codon optimized for the expression in E. coli and synthesized either by GeneArt (Thermofisher) or by Twist Bioscience, with the exception of the genes YdiI, UbiX and CoaA. The last ones were directly amplified from the genomic DNA of E. coli MG1655. The mutant of the CoaA (R106A) was then constructed by site-directed mutagenesis. The genes thIA and AibA/B were integrated in the bacterial chromosome into the ssrS and mgsA locus, respectively, resulting in strain D.

(32) An expression vector containing the origin of replication pSC and a tetracycline resistance marker was used for the expression of the genes MvaS, ECH, YdiI and UbiD. The constructed vector was named pGB12762.

(33) The modified version of pUC18 (New England Biolabs) containing a modified Multiple Cloning Site (pUC18 MCS) (WO 2013/007786) and an ampicilline resistance gene was used for the overexpression of the UbiX and YdiI genes (plasmid pGB6546) or for a combination of the UbiX, YdiI and CoaA (R106A) genes (plasmid pGB13095).

(34) The different combinations of the plasmids described above were transformed by electroporation into strain D. The strains produced in this way, strains E and F, are summarized in Table B.

(35) TABLE-US-00002 TABLE B Strain Vectors Strain E, expressing the pGB12762 + pGB6546 whole IBN metabolic pathway, without overexpression of CoaA (R106A) on plasmid Strain F, expressing the pGB12762 + pGB13095 whole IBN metabolic pathway + CoaA (R106A)

Example 3: Growth of E. coli Strains and Production of Isobutene from Acetyl-CoA

(36) Pre-Culture Conditions

(37) The transformed cells were then plated on LB plates supplied with ampicillin (100 g/ml) and tetracycline (10 g/ml). Plates were incubated for 2 days at 30 C. Isolated colonies were used to inoculate LB medium supplemented with ampicillin, tetracycline and 50 mM glutamate. These pre-cultures were grown at 30 C. to reach an optical density of 0.6.

(38) Growth Conditions

(39) The fermentation was performed in a 1 L bioreactor with pH and temperature control (Multifors 2, Infors HT). Cells of the pre-cultures were used to inoculate 500 ml of the fermentation medium (Table C), complemented with ampicillin (100 g/ml), tetracycline (10 g/ml), thiamine (0.6 mM), glutamate (50 mM), pantothenate (5 mM), sucrose (1 g/l) and glycerol (5 g/1), to achieve an initial optical density (OD.sub.600) of 0.05. During the growth phase temperature (T=32 C.), pH=6.5, and pO.sub.2=5% were maintained constant. The feed of sucrose was maintained at 0.1 g/g DCW/h. The pulses addition of 5 g/L of yeast extract were done at 16, 24 and 30 h.

(40) At the end of the growth phase (t=40 h) the cell densities were lower with strain E than with strain F, in the range of 13 g/l for strain E, and 15.5 g/l for strain F (FIG. 7)

(41) IBN Production Phase

(42) During this phase temperature, T=34 C., pH 6.5, and pO.sub.2=5% were maintained constant. Sucrose feed was started at 0.30 g sucrose/g DCW/h and then adjusted according to the strain consumption. Glycerol concentration was maintained superior to 2 g/l.

(43) The isobutene (IBN) production of strains E and F was analyzed continuously using a Gas Chromatograph 7890A (Agilent Technology), equipped with a Flame Ionization Detector (FID) to measure IBN. Volatile organic compounds were chromatographically separated on PoraBond Q column (25 m0.25 mm0.35 mm) (Agilent) and IBN was quantified using standard gas (Sigma).

(44) FIG. 8 shows a comparison of the IBN volumic productivities (mass of IBN produced per unit of volume of fermentation broth and per unit of time) for strains E and F during the fermentation run. Volumic productivity (mass of IBN produced per unit of volume of fermentation broth and per unit of time) was significantly higher for strain F than for strain E over the entire production phase, with maximal volumic productivity for strain E being in the range of 66% of maximal volumic productivity achieved by strain F.

(45) In addition, this higher volumic productivity was not due solely to the higher biomass concentration of strain F, but also to a higher production per unit of biomass. As shown in FIG. 9, specific productivity (mass of isobutene produced per unit of biomass and per unit of time) was higher with strain F as compared to reference strain E for most of the production phase (more precisely, from t=42 h through the end of the run). Maximal IBN specific productivity of strain E was in the range of 82% of the maximal specific productivity achieved by strain F.

(46) TABLE-US-00003 TABLE C Fermentation medium composition (derived from ZYM-5052 medium (Studier FW, Prot. Exp. Pur. 41, (2005), 207-234)). Final concentration in Products bioreactor Yeast Extract 5 g/L Tryptone 10 g/L Sodium sulfate, Na.sub.2SO.sub.4 0.71 g/L Ammonium sulfate, (NH.sub.4).sub.2SO.sub.4 1.34 g/L Potassium phosphate monobasic, KH.sub.2PO.sub.4 3.4 g/L Sodium phosphate dibasic, Na.sub.2HPO.sub.4 4.45 g/L Magnesium sulfate, MgSO.sub.4 4 mM 5000X Trace elements solution 1X Antifoam Struktol J 673 A (Struktol) 80 l/L

Example 4: Quantification of Coenzyme A and its Thioester Intermediates

(47) Extraction

(48) 120 L of bacterial cultures is directly sampled from a fermenter and filtered through a membrane filter under vacuum. Immediately after filtration, the filter is put into an aluminum foil and immersed into liquid nitrogen in order to stop all metabolic reactions. Then, the aluminum foil containing the filter is stored at 80 C. until extraction. Intracellular metabolites are extracted (from the membrane filter) with 2 mL of a cold MeOH/H.sub.2O (80/20) mixture during 15 min at 80 C. in a Falcon tube. After this extraction time, the Falcon tube is centrifuged at 9 C. for 20 min. Then, the supernatants are removed and transferred to a new tube. A second extraction of the membrane filter is performed with 1 mL with the cold MeOH/H.sub.2O mixture following the same process as just described (except that both extraction time at 80 C. and centrifugation step are reduced to 5 min). The combined supernatant (ca. 3 mL) is filtered through a 0.2 m syringe filter and 2.4 mL are evaporated to dryness by a Speed-Vac concentrator. Then, the dry extract is re-dissolved with 120 L of ACN/H.sub.2O (50/50, v/v), filtered again through a 0.2 m syringe filter and transferred into a HPLC vial for LC-MS analysis.

(49) LC-MS Analysis

(50) Analysis are carried out on a UHPLC system coupled to a Q-Exactive mass spectrometer (ThermoFisher Scientific, Massachusetts, USA) in negative ionization mode. For the LC part, a BEH amide column (1.7 m, 2.1100 mm, Waters) conditioned at 25 C. is used. The flow rate is set at 0.5 mL/min and 2 L of samples are injected. The mobile phase consists of a binary gradient (A: ammonium formate (10 mM)+0.1% ammonium hydroxide and B: acetonitrile) starting with 95% B during 1.5 min then, decreasing to 55% B until 8.5 min and staying at 55% B during 2 min and then, go back to the initial conditions. The total analysis time is 19 min. At the mass level, analyzes are performed in Full MS+ddMS.sup.2 mode in negative ionization mode. The set of ions between 80<m/z<1200 Da are considered and all ions with an intensity greater than 1e5 are fragmented with a collision energy of 35 eV in order to obtain additional structural information, if needed. Calibration curves of compounds of interest are recorded under the same LC/MS conditions. Data analysis is performed with Xcalibur v3.0.63 software (ThermoFisher Scientific). LC-MS characteristics of compounds of interest are summarized in the Table D.

(51) TABLE-US-00004 TABLE D LC-MS characteristics of Coenzyme A and its thioesters Molecular Molecular weight RT Compound.sup.1 formula (g/mol) (min) m/z observed CoA C.sub.21H.sub.36N.sub.7O.sub.16P.sub.3S 767.535 6.96 766.1096 [M H].sup.; 382.5509 [M H].sup.2+ AcCoA C.sub.23H.sub.38N.sub.7O.sub.17P.sub.3S 809.570 6.83 808.1204 [M H].sup.; 403.5561 [M H].sup.2+ AcAcCoA C.sub.25H.sub.40N.sub.7O.sub.18P.sub.3S 851.607 6.78 850.1310 [M H].sup.; 424.5618 [M H].sup.2+ HMG-CoA C.sub.27H.sub.44N.sub.7O.sub.20P.sub.3S 911.659 7.27 910.1526 [M H].sup.; 454.5722 [M H].sup.2+ MC-CoA C.sub.26H.sub.42N.sub.7O.sub.17P.sub.3S 849.635 6.53 848.1515 [M H].sup.; 423.5719 [M H].sup.2+ .sup.1CoACoenzyme A; AcCoA: Acetyl-Coenzyme A; AcAcCoA: Acetoacetyl-Coenzyme A; HMG-CoA: 3-Hydroxy-3-Methylglutaryl-Coenzyme A; MC-CoA: 3-Methylcrotonyl-Coenzyme A
HPLC Analysis

(52) Two LC methods are available to detect and quantify Coenzyme A derivative compounds.

(53) The first LC method (method 1) is performed by a HPLC 1260 system coupled to a Multiple Wavelength Detector (MWD) (Agilent, Santa Clara, USA). The separation is carried out by a Zorbax Eclipse coupled to a C18 column (3.5 m, 4.6100 mm, Agilent) conditioned at 30 C. The mobile phase consists of an isocratic elution with acetonitrile (5%) and a phosphate buffer (100 mM) at pH5 (95%) during 10 min. The flow rate is set at 1.5 mL/min and 5 L of samples are injected.

(54) The second method (method 2) is performed with a HPLC 1260 system coupled to a Diode Array Detector (DAD) (Agilent, Santa Clara, USA). A ZorbaxsbAq column (5 m, 4.6250 mm, Agilent) conditioned at 30 C. is used to separate metabolites. 5 L of samples is injected. The flow rate is set at 1.5 mL/min. The mobile phase consists of a binary gradient (A: Acetonitrile; and B: H2SO4 8.4 mM) starting with 100% B and then, decreasing to 30% B until 8 min and staying at 30% B during 1 min. During this 1 min at 30% B, the flow rate is increased to 2 mL/min. After 9 min, the binary gradient goes back to the initial conditions (i.e. 100% B and a flow rate at 1.5 mL/min) in 2 min and stays at 100% B during 3 min. The total run time is 14 min.

(55) In both methods, the detection of metabolites of interest is performed at 260 nm and calibrations curves of pure compounds are recorded under the respective LC conditions used. The ChemStation software (Agilent) is used for data analysis.

(56) TABLE-US-00005 TABLE E Retention time of Coenzyme A and its thioesters in HPLC-based quantification method Compound.sup.1 RT (min) Method 1 CoA 1.8 AcCoA 4.6 AcAcCoA 5.0 HMG-CoA 2.6 MC-CoA Method 2 CoA 3.9 AcCoA 4.2 AcAcCoA 4.2 HMG-CoA 4.2 MC-CoA 5.2 .sup.1CoA: Coenzyme A; AcCoA: Acetyl-Coenzyme A; AcAcCoA: Acetoacetyl-Coenzyme A; HMG-CoA: 3-Hydroxy-3-Methylglutaryl-Coenzyme A; MC-CoA: 3-Methylcrotonyl-Coenzyme A

Example 5: Conversion of 3-methylcrotonic Acid into Isobutene by Recombinant C. ljungdahlii Expressing ubiDHav6

(57) This working example shows the production of isobutene from 3-methylcrotonic acid by recombinant C. jungdahlii expressing the genes encoding (i) 3-methylcrotonic acid decarboxylase ubiD HAv6 and (ii) prenyl transferase ubiX. The 3-methylcrotonic acid decarboxylase converts 3-methylcrotonic acid to isobutene and is derived from Hypocrea atroviridis (Trichoderma atroviride) and further engineered for efficient IBN production. The prenyl transferase converts FMN to prenyl-FMN and thus provides the cofactor for the prenate decarboxylase. The source of this enzyme is E. coli MG1655.

(58) Expression of 3-methylcrotonic Acid Decarboxylase and Prenyl Transferase in C. ljungdahlii DSM 13528

(59) The genes were codon optimized for expression in C. jungdahlii and synthesized by Genscript. An expression vector containing the pCB102 replicon for maintenance in C. Ijungdahlii and an erythromycin resistance gene for selection was used for expression of the genes. The expression was driven by the promoter of thl gene of C. acetobutylicum ATCC 824 (in combination with a ribosome binding site from Bacteriophage T7) and terminated by rrnB Terminator from E. coli MG1655.

(60) Culture Conditions

(61) 2YT Medium supplemented with 20 g/L fructose and 5 g/mL clarithromycin was inoculated from cryo cultures of the transformed cells and pressurized with 2 bars of CO.sub.2. The cells were grown until mid-exponential phase. The pH of the cultures was regulated to maintain the pH between 5 and 6. The cultivation was done in serum bottles at 150 rpm and 37 C. Cells were harvested by centrifugation and either stored at 80 C. or directly resuspended at an OD600 of 13 in PETC 1754 medium supplemented with 20 g/L of fructose. The cells were grown in glass vials for 72 h at 150 rpm and 37 C. The amount of IBN was determined by analyzing the Headspace via GC-MS.

(62) The strain containing the 3-methylcrotonic acid decarboxylase and prenyl transferase (SGP244) was compared to a strain harboring the corresponding empty vector (SGP184) (FIG. 10). SGP244 showed a 201-fold increase in isobutene accumulation compared to SGP 184.

Example 6: Conversion of 3-methylcrotonic Acid into Isobutene by Recombinant C. Ijungdahlii Expressing FDCSs5v2

(63) This working example shows the production of isobutene from 3-methylcrotonic acid by recombinant C. jungdahlii expressing the genes encoding (i) 3-methylcrotonic acid decarboxylase FDCSs5v2 and (ii) prenyl transferase ubiX. The 3-methylcrotonic acid decarboxylase converts 3-methylcrotonic acid to isobutene and is derived from Streptomyces sp. and further engineered for efficient IBN production. The prenyl transferase converts FMN to prenyl-FMN and thus provides the cofactor for the prenate decarboxylase. The source of this enzyme is E. coli MG1655.

(64) Expression of 3-methylcrotonic Acid Decarboxylase and Prenyl Transferase in C. ljungdahlii DSM 13528

(65) The genes were codon optimized for expression in C. Ijungdahlii and synthesized by Genscript. An expression vector containing the pCB102 replicon for maintenance in C. ljungdahlii and an erythromycin resistance gene for selection was used for expression of the genes. The expression was driven by the promoter of thl gene of C. acetobutylicum ATCC 824 (in combination with a ribosome binding site from Bacteriophage T7) and terminated by rrnB Terminator from E. coli MG1655.

(66) Culture Conditions

(67) 2YT Medium supplemented with 20 g/L fructose and 5 g/mL clarithromycin was inoculated from cryo cultures of the transformed cells and pressurized with 2 bars of CO.sub.2. The cells were grown until mid-exponential phase. The pH of the cultures was regulated to maintain the pH between 5 and 6. The cultivation was done in serum bottles at 150 rpm and 37 C. Cells were harvested by centrifugation and either stored at 80 C. or directly resuspended at an OD600 of 13 in PETC 1754 medium supplemented with 20 g/L of fructose. The cells were grown in glass vials for 72 h at 150 rpm and 37 C. The amount of IBN was determined by analyzing the Headspace via GC-MS.

(68) The strain containing the 3-methylcrotonic acid decarboxylase and prenyl transferase (SGP 339) was compared to a strain harboring the corresponding empty vector (SGP184) (FIG. 11). SGP339 showed a 2093-fold increase in isobutene accumulation compared to SGP 184.

Example 7: Construction of C. ljungdahlii Strains for the Production of Isobutene from a CO/CO.SUB.2./H.SUB.2 .Gas Mixture Via Acetyl-CoA

(69) This working example shows the production of isobutene from C1 gas mixtures by recombinant C. jungdahlii expressing the genes constituting the isobutene pathway. The enzymes used in this study to convert acetyl-CoA into isobutene (IBN) via 3-methylcrotonic acid (FIG. 6) are listed in Table F.

(70) TABLE-US-00006 TABLE F Uniprot Accession Step Enzyme Gene NCBI reference number I Acetyl-CoA transferase thIA3 EDK35683.1 A5N3I7 from Clostridium kluyverii II Hydroxymethylglutaryl- mvaS WP_002357756.1 Q9FD71 CoA synthase from Enterococcus faecalis III Enoyl-CoA PputUW4_01474 WP_015094072.1 K9NHK2 hydratase/isomerase from Pseudomonas sp. UW4 (ECH) IV Glutaconate CoA- MXAN_4264 WP_011554268.1 Q1D4I3 transferase from MXAN_4265 WP_011554267.1 Q1D4I4 Myxococcus xanthus (AibA/B) V Acyl-CoA thioesterase tesB AAC73555.1 P0AGG2 VI Variant of UbiD-like FDC1 XP_013946967.1 G9NLP8 decarboxylase from Hypocrea atroviridis (Trichoderma atroviride) (UbiD) with N-terminal MBP fusion Flavin prenyl ubiX WP_000825700.1 P0AG03 transferase from Escherichia coli (UbiX)
Expression of Isobutene Biosynthetic Pathway in C. Ijungdahlii DSM 13528

(71) The genes were codon optimized for expression in C. Ijungdahlii and synthesized by Genscript or IDT. An expression vector containing the pCB102 replicon for maintenance in C. Ijungdahlii and an erythromycin resistance gene for selection was used for expression of the genes which were divided into two operons. The expression of both operons was driven by the promoter of thl gene of C. acetobutylicum ATCC 824 and terminated by rrnB Terminator from E. coli MG1655.

(72) Culture Conditions

(73) For the preculture PETC1754 medium supplemented with 20 g/L fructose and 5 g/mL clarithromycin was inoculated from cryo cultures of the transformed cells and pressurized with 2 bars of CO.sub.2. The cells were grown over night in serum bottles at 150 rpm and 37 C. For the main culture PETC1754 medium supplemented with 5 g/mL clarithromycin was inoculated from the preculture and pressurized with 2 bars of a gas mixture containing 55% CO, 25% H2 and 20% CO.sub.2 (all vol %). The pH of the cultures was regulated to maintain the pH between 5 and 6 and the gas was refilled daily to maintain a pressure of 2 bars. The cultivation was done in serum bottles at 150 rpm and 37 C. for 6 days. The amount of IBN was determined by analyzing the Headspace via GC-MS.

(74) The strain containing the isobutene pathway (SGP353) was compared to a strain harboring the corresponding empty vector (SGP348) (FIG. 12). While SGP353 produced a significant amount of isobutene, only traces could be found in SGP348 (FIG. 13). Thus, by introducing the isobutene pathway in C. ljungdahlii an 1971-fold increased signal was obtained for Isobutene compared to a strain without the isobutene pathway.