阅读说明：本技术 用于从乙酰-CoA生产异丁烯的改进手段和方法 (Improved means and methods for production of isobutene from acetyl-CoA ) 是由 R·夏约 M·阿尼斯莫瓦 F·马丁 F·科拉斯 O·马哈茂德 于 2020-03-19 设计创作，主要内容包括：重组生物体或微生物,(A)其中在所述生物体或微生物中：(i)将乙酰-CoA酶促转化成乙酰乙酰-CoA,(ii)将乙酰乙酰-CoA酶促转化成3-羟基-3-甲基戊二酰-CoA,(iii)将3-羟基-3-甲基戊二酰-CoA酶促转化成3-甲基戊烯二酰-CoA,(iv)将3-甲基戊烯二酰-CoA酶促转化成3-甲基巴豆酰-CoA,和(v)其中所述3-甲基巴豆酰-CoA通过以下方式转化成异丁烯：(a)将3-甲基巴豆酰-CoA酶促转化成3-甲基巴豆酸,然后将其进一步酶促转化成所述异丁烯；或(b)将3-甲基巴豆酰-CoA酶促转化成3-羟基-3-甲基丁酰-CoA,然后将其进一步酶促转化成3-羟基-3-甲基丁酸,然后将其进一步酶促转化成3-膦氧基-3-甲基丁酸,然后将其进一步酶促转化成所述异丁烯；(B)其中所述重组生物体或微生物具有增加的超过衍生其的生物体或微生物的辅酶A(CoA)池,这是由于：(i)增加的泛酸摄取；和/或(ii)增加的泛酸向CoA的转化。此外,描述了这种重组生物体或微生物用于生产异丁烯的用途。此外,描述了通过在合适的培养基中在合适的条件下培养这样的重组生物体或微生物来生产异丁烯的方法。(A recombinant organism or microorganism, (a) wherein in said organism or microorganism: (i) enzymatically converting acetyl-CoA to acetoacetyl-CoA, (ii) enzymatically converting acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA, (iii) enzymatically converting 3-hydroxy-3-methylglutaryl-CoA to 3-methylpentadienyl-CoA, (iv) enzymatically converting 3-methylpentadienyl-CoA to 3-methylcrotonyl-CoA, and (v) wherein said 3-methylcrotonyl-CoA is converted to isobutylene 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-phosphinyloxy-3-methylbutyric acid, which is then further enzymatically converted into said isobutene; (B) wherein the 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) increased pantothenate uptake; and/or (ii) increased conversion of pantothenate to CoA. Furthermore, the use of such recombinant organisms or microorganisms for the production of isobutene is described. Furthermore, a method for producing isobutene by culturing such a recombinant organism or microorganism in a suitable medium under suitable conditions is described.)

1. A recombinant organism or microorganism capable of enzymatically converting acetyl-CoA to isobutene,

(A) wherein in the organism or microorganism:

(i) enzymatically converting acetyl-CoA into acetoacetyl-CoA,

(ii) enzymatically converting acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA,

(iii) enzymatically converting 3-hydroxy-3-methylglutaryl-CoA into 3-methylpentadienyl-CoA,

(iv) enzymatically converting 3-methylpentenediacyl-CoA into 3-methylcrotonyl-CoA, and

(v) wherein the 3-methylcrotonyl-CoA is converted to isobutylene 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, then further enzymatically converting it into 3-hydroxy-3-methylbutyric acid, then further enzymatically converting it into 3-phosphinyloxy-3-methylbutyric acid, then further enzymatically converting it into said isobutylene;

(B) wherein the 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) increased pantothenate uptake; and/or

(ii) Increased conversion of pantothenate to CoA.

2. The recombinant organism or microorganism of claim 1, wherein said increased pantothenate uptake of (B) (i) is due to recombinant expression of a pantothenate uptake transporter.

3. The recombinant organism or microorganism of claim 1 or 2, wherein said increased conversion of pantothenate to CoA is due to recombinant expression of at least one of (i) to (v):

(i) an enzyme that catalyzes the enzymatic conversion of pantothenate to 4' -phosphopantothenate, preferably pantothenate kinase;

(ii) an enzyme catalyzing the enzymatic conversion of 4 '-phosphopantothenic acid and L-cysteine to 4' -phosphopanthenyl-L-cysteine, preferably phosphopanthenyl cysteine synthetase;

(iii) an enzyme that catalyzes the enzymatic conversion of 4 '-phosphopantetheine-L-cysteine to pantetheine-4-phosphate, preferably 4' -phosphopantetheine decarboxylase;

(iv) an enzyme catalyzing the enzymatic conversion of pantetheine-4-phosphate to dephosphorylated-CoA, preferably pantetheine-phosphate adenylyltransferase; and

(v) an enzyme catalyzing the enzymatic conversion of dephosphorylated-CoA into CoA, preferably a dephosphorylated-CoA kinase.

4. The recombinant organism or microorganism of any one of claims 1 to 3, wherein said organism or microorganism is further characterized by:

a) having phosphoketolase activity;

b) (ii) having a reduced or inactivated Embden-Meyerhof-Parnas pathway (EMPP) by inactivation of a gene encoding phosphofructokinase or by decreasing phosphofructokinase activity compared to an unmodified microorganism; or

(ii) Has no phosphofructokinase activity

And

c) (ii) (i) has a reduced or inactivated oxidative branch of the Pentose Phosphate Pathway (PPP) by inactivating an enzyme encoding glucose-6-phosphate dehydrogenase or by reducing glucose-6-phosphate dehydrogenase activity compared to a non-modified microorganism; or

(ii) Has no glucose-6-phosphate dehydrogenase activity.

5. The recombinant organism or microorganism of claim 4, further characterized by:

d) has fructose-1, 6-bisphosphate phosphatase.

6. The recombinant organism or microorganism of claim 4 or 5, wherein EMPP is further reduced or inactivated by inactivation of a gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing glyceraldehyde 3-phosphate dehydrogenase activity compared to an unmodified microorganism.

7. The recombinant organism or microorganism of any one of claims 4 to 6 which has been genetically modified to have increased phosphoketolase activity.

8. The recombinant organism or microorganism of any one of claims 4 to 7, wherein said microorganism is a fungus.

9. The recombinant organism or microorganism of any one of claims 4 to 7, wherein said microorganism is a bacterium.

10. The recombinant organism or microorganism of claim 9, wherein the gene encoding the PEP-dependent PTS transporter has been inactivated.

11. Use of a recombinant organism or microorganism as defined in any one of claims 1 to 10 for the production of isobutene.

12. A method for producing isobutene by culturing a recombinant organism or microorganism as defined in any one of claims 1 to 10 in a suitable medium under suitable conditions.

13. A method of producing isobutene from acetyl-CoA in a recombinant organism or microorganism comprising:

(A) (i) enzymatically converting acetyl-CoA to acetoacetyl-CoA,

(ii) enzymatically converting said produced acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA,

(iii) enzymatically converting said produced 3-hydroxy-3-methylglutaryl-CoA into 3-methylpentadienyl-CoA,

(iv) enzymatically converting said produced 3-methylpentenoyl-CoA into 3-methylcrotonyl-CoA, and

(v) wherein the produced 3-methylcrotonyl-CoA is converted to isobutylene 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, then further enzymatically converting it into 3-hydroxy-3-methylbutyric acid, then further enzymatically converting it into 3-phosphinyloxy-3-methylbutyric acid, then further enzymatically converting it into said isobutylene;

(B) wherein the method further comprises:

enzymatically providing acetyl-CoA from CoA,

(C) wherein the method further comprises providing coenzyme a (coa) by culturing a recombinant organism or microorganism in a suitable medium under suitable conditions, wherein the 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) increased pantothenate uptake; and/or

(ii) Increased conversion of pantothenate to CoA.

14. The process of claim 13, wherein said increased pantothenate uptake of (C) (i) is due to recombinant expression of a pantothenate uptake transporter.

15. The process of claim 13 or 14, wherein said increased conversion of pantothenate to CoA is due to recombinant expression of at least one of (i) through (vi):

(i) an enzyme that catalyzes the enzymatic conversion of pantothenate to 4' -phosphopantothenate, preferably pantothenate kinase;

(ii) an enzyme catalyzing the enzymatic conversion of 4 '-phosphopantothenic acid and L-cysteine to 4' -phosphopanthenyl-L-cysteine, preferably phosphopanthenyl cysteine synthetase;

(iii) an enzyme that catalyzes the enzymatic conversion of 4 '-phosphopantetheine-L-cysteine to pantetheine-4-phosphate, preferably 4' -phosphopantetheine decarboxylase;

(iv) an enzyme catalyzing the enzymatic conversion of pantetheine-4-phosphate to dephosphorylated-CoA, preferably pantetheine-phosphate adenylyltransferase; and

(v) an enzyme catalyzing the enzymatic conversion of dephosphorylated-CoA into CoA, preferably a dephosphorylated-CoA kinase.

16. The process of any one of claims 12 to 15, further comprising supplementing the medium with pantothenate.

17. The process of any one of claims 12 to 16, further comprising collecting the isobutylene produced.

The term "inactivated" in the context of the present invention preferably means completely inactivated, i.e. the organism or microorganism does not show phosphofructokinase activity. This means in particular that, irrespective of the growth conditions used, the organism or the microorganism does not exhibit phosphofructokinase activity.

Preferably, "inactivated" means that the gene encoding phosphofructokinase present in the organism or microorganism is genetically modified such that expression of the enzyme is prevented. This can be achieved, for example, by deleting the gene or a part thereof, wherein deletion of the part prevents expression of the enzyme, or by disrupting the gene in the coding region or promoter region, wherein the disruption has the effect of not expressing the protein or of expressing a dysfunctional protein.

In a preferred embodiment, the recombinant organism or microorganism of the invention is a recombinant organism or microorganism having a reduced Embden-Meyerhof-Parnas pathway (EMPP) by reducing phosphofructokinase activity compared to an unmodified organism or microorganism. Preferably, this reduction is achieved by genetic modification of the organism or microorganism. This can be achieved, for example, by random or site-directed mutagenesis of the promoter and/or enzyme and subsequent selection of a promoter and/or enzyme with the desired properties, or by a complementary nucleotide sequence or RNAi effect as described above.

In the context of the present invention, "reduced activity" means that the expression and/or activity of an enzyme, in particular phosphofructokinase, in a genetically modified organism or microorganism is at least 10% lower, preferably at least 20% lower, more preferably at least 30% or 50% lower, even more preferably at least 70% or 80% lower and particularly preferably at least 90% or 100% lower than in the corresponding unmodified organism or microorganism. Methods for measuring the expression level of a given protein in a cell are well known to those skilled in the art. Assays for measuring reduced enzymatic activity of phosphofructokinase are known in the art.

In another embodiment, the organism or microorganism according to the invention is an organism or microorganism having no phosphofructokinase activity. This preferably means that such an organism or microorganism does not naturally have phosphofructokinase activity. This means that such an organism or microorganism does not naturally contain in its genome a nucleotide sequence encoding an enzyme having phosphofructokinase activity. Examples of such microorganisms are Zymomonas mobilis (J.S.Suo et al, nat. Biotechnol.23:63(2005)) and Ralstonia eutropha (C.Fleige et al, appl. Microb. cell Physiol.91:769 (2011)).

The organisms or microorganisms according to the invention are preferably further characterized by having a reduced or inactivated oxidative branch of the Pentose Phosphate Pathway (PPP), by inactivating the gene coding for glucose-6-phosphate dehydrogenase or by reducing the glucose-6-phosphate dehydrogenase activity or by not having the glucose-6-phosphate dehydrogenase activity in comparison with unmodified organisms or microorganisms. Thus, an organism or microorganism is one which naturally has PPP comprising glucose-6-phosphate dehydrogenase but which has been modified, in particular genetically modified, such that the glucose-6-phosphate dehydrogenase activity is completely eliminated or such that it is reduced compared with a corresponding unmodified organism or microorganism, or an organism or microorganism is one which naturally does not have glucose-6-phosphate dehydrogenase activity.

"glucose-6-phosphate dehydrogenase Activity" means the combination of glucose-6-phosphate and NADP⁺Enzyme activity for conversion to 6-phosphogluconate-delta-lactone and NADPH (EC 1.1.1.49). Such enzyme activity can be measured by assays known in the art, such as, for example, described by Noltman et al (j. biol. chem. (1961)236, 1225-1230).

The term "an organism or microorganism characterized by having a reduced or inactivated oxidative branch of the Pentose Phosphate Pathway (PPP), by inactivation of a gene coding for a glucose-6-phosphate dehydrogenase or by reducing the activity of a glucose-6-phosphate dehydrogenase compared to an unmodified organism or microorganism" preferably means that an organism or microorganism in which the gene coding for a glucose-6-phosphate dehydrogenase is inactivated or in which the activity of a glucose-6-phosphate dehydrogenase is reduced compared to an unmodified organism or microorganism is achieved by genetic modification of the organism or microorganism resulting in the reduction of the inactivation.

In a preferred embodiment, the recombinant organism or microorganism of the invention is a recombinant organism or microorganism having an inactivated oxidative branch of the Pentose Phosphate Pathway (PPP) by inactivation of the gene encoding glucose-6-phosphate dehydrogenase. The inactivation of the gene coding for glucose-6-phosphate dehydrogenase in the context of the present invention means that the genes of glucose-6-phosphate dehydrogenase present in the organism or microorganism are inactivated such that they are no longer expressed and/or no longer lead to the synthesis of functional glucose-6-phosphate dehydrogenase. Inactivation can be accomplished by a number of different approaches known in the art. Inactivation can be achieved, for example, by disruption of the gene encoding glucose-6-phosphate dehydrogenase or by complete deletion of said gene by introduction of a selection marker. Alternatively, the promoter of the gene encoding glucose-6-phosphate dehydrogenase may be mutated in such a way that the gene is no longer transcribed into mRNA. Other ways of inactivating the gene encoding phosphofructokinase known in the art are: expressing a polynucleotide encoding RNA having a nucleotide sequence complementary to a transcript of the glucose-6-phosphate dehydrogenase gene such that the mRNA is no longer translated into protein, to express a polynucleotide encoding RNA that represses expression of the gene through an RNAi effect; expressing a polynucleotide encoding an RNA having an activity of specifically cleaving a transcript of the gene; or expressing a polynucleotide encoding an RNA that represses expression of the gene through co-suppression. These polynucleotides may be incorporated into vectors which may be introduced into organisms or microorganisms by transformation to effect inactivation of the gene encoding glucose-6-phosphate dehydrogenase.

Examples

Example 1: construction of novel Escherichia coli bases (chassis) for isobutene production

Like most organisms, E.coli converts glucose to acetyl-CoA. An improved E.coli locus has been previously described in which the production and flux of acetyl-CoA has been increased to optimize the production of acetyl-CoA (WO 2013/007786). Wherein a bacterial base with the following genotype, strain a, was constructed:

MG1655ΔptsHIΔzwf_edd_edaΔpfkAΔpfkB

plasmid-based overexpression of the PKT gene from lactococcus lactis phosphoketolase YP 003354041.1 in strain a produced strain B. This strain B is characterized by "rewiring" the central carbon metabolism, wherein the inactive Embden-Meyerhoff-Parnas pathway (EMPP), Pentose Phosphate Pathway (PPP) and Entner Doudoroff Pathway (EDP) are replaced by a new phosphoketolase based carbon catabolic pathway. Upon introduction of the acetone pathway into strain B, higher acetone production was observed compared to the wild-type MG1655 strain expressing the same acetone pathway.

In order to construct a strain with PKT pathway and robust growth on sucrose as carbon source, strain a was further engineered as described below.

The 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:

MG1655ΔptsHIΔzwf_edd_edaΔpfkAΔpfkB kdgK::P1_RBST7_pkt

the strain is passaged for several months on minimal medium supplemented with glucose as carbon source, while continuously selecting the clone or population with the highest growth rate until a doubling time of less than 5 hours is reached.

To allow for efficient sucrose consumption, the CscA, CscB and CscK encoding genes were inserted into the chromosome at the zwf locus (Δ zwf:: P1_ cscA _ cscB _ cscK _ FRT). CscA (Unit part O86076; NCBI reference sequence WP _000194515.1), CscB (Unit part Q7WZY 9; NCBI reference sequence WP _001197025.1) and CscK (Unit part Q7WZY 7; NCBI reference sequence WP _001274855.1) encoding sucrose hydrolase, non-PTS sucrose permease (non-PTS sucrose permease) and fructokinase respectively allow uptake of sucrose, hydrolysis to glucose and fructose and phosphorylation of fructose to fructose-6-phosphate, thus efficiently metabolizing fructose (for review see Biotechnology Advances 32(2014)905 919). These modifications bring about strains with the PKT pathway and are capable of efficient growth on sucrose.

To further increase sugar consumption, the glk (glucokinase gene, Uniprot P0A6V 8; NCBI reference sequence: NP-416889.1) gene from E.coli was overexpressed by inserting an additional copy under the control of the PN25 promoter of the pfkA locus.

The resulting strain is hereinafter referred to as strain C.

Example 2: construction of E.coli strains for the production of isobutene from acetyl-CoA

This working example shows the production of isobutene by a recombinant strain of escherichia coli expressing the following genes: (i) an isobutene pathway was constructed. (ii) Encodes pantothenate kinase, CoaA.

The enzymes used in this study to convert acetyl-CoA to Isobutene (IBN) via 3-methylcrotonic acid (FIG. 6) are listed in Table A. The pantothenate kinase is derived from the coaA gene from the E.coli MG1655 strain (Uniprot accession number: P0A6I 3).

TABLE A

Expression of the isobutene biosynthetic pathway in E.coli

The strain C described in example 1 was used as a host microorganism.

All listed genes were codon-optimized for expression in E.coli and(Thermofisiher) or Twist Bioscience, except for the genes Ydii, UbiX and CoaA. Finally these were directly amplified from the genomic DNA of E.coli MG 1655. Mutants of CoaA (R106A) were then constructed by site-directed mutagenesis. The genes thlA and AibA/B integrate into the ssrS and mgsA loci, respectively, in the bacterial chromosome, producing strain D.

Expression vectors containing the origin of replication pSC and a tetracycline resistance marker were used to express the genes MvaS, ECH, Ydii and UbiD. The constructed vector was named pGB 12762.

pUC18, which contains a modified multiple cloning site (pUC18 MCS) (WO2013/007786) and a modified version of the ampicillin resistance gene (New England Biolabs), was used for overexpression of the UbiX and Ydii genes (plasmid pGB6546) or the combination of the UbiX, Ydii and CoaA (R106A) genes (plasmid pGB 13095). Different combinations of the above plasmids were transformed into strain D by electroporation. The strains produced in this way, strains E and F, are summarized in table B.

TABLE B

Example 3: growth of E.coli strains and production of isobutene from acetyl-CoA

Preculture conditions

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

Growth conditions

The fermentation was carried out in a 1L bioreactor (Multifors 2, Infors HT) with pH and temperature control. The precultured cells were used to inoculate 500 ml of fermentation medium (Table C), supplemented with ampicillin (100. mu.g/ml), tetracycline (10. mu.g/ml), thiamine (0.6mM), glutamic acid (50mM), pantothenic acid (5mM), sucrose (1g/l) and glycerol (5g/l), to achieve an initial Optical Density (OD) of 0.05₆₀₀). During growth, temperature (T ═ 32 ℃), pH ═ 6.5, and pO₂5% remains constant. The sucrose feed was maintained at 0.1g/g DCW/h. 5g/L yeast extract was added at 16, 24 and 30 hours.

At the end of the growth phase (t 40h), the cell density of strain E was lower than that of strain F, 13g/l for strain E and 15.5g/l for strain F (fig. 7).

IBN production phase

During this phase, the temperature T ═ 34 ℃, pH 6.5 and pO₂5% remains constant. The sucrose feed was started at 0.30g sucrose/g DCW/h and then adjusted for strain consumption. The glycerol concentration was kept above 2 g/l.

Strains E and F were analyzed continuously for Isobutene (IBN) production using a gas chromatograph 7890a (agilent technology) equipped with a Flame Ionization Detector (FID) to measure IBN. Volatile organic compounds were chromatographed on a PoraBond Q column (25 m.times.0.25 mm. times.0.35 mm) (Agilent) and IBN quantification was performed using standard gas (Sigma).

FIG. 8 shows a comparison of IBN volumetric productivity (mass of IBN produced per volume of fermentation broth and per unit time) of strains E and F during a fermentation run. The volumetric productivity of strain F (mass of IBN produced per unit volume of fermentation broth and per unit time) was significantly higher throughout the production phase than strain E, which had a maximum volumetric productivity in the range of 66% of the maximum volumetric productivity reached by strain F.

Furthermore, this higher volumetric productivity is not only due to the higher biomass concentration of strain F, but also to the higher yield per unit biomass. As shown in fig. 9, the specific productivity (quality of isobutene produced per unit biomass and per unit time) of strain F was higher compared to the reference strain E during most of the production phase (more precisely, from t-42 h to the end of the run). The maximum specific IBN productivity of strain E ranged from 82% of the maximum specific productivity achieved by strain F.

Table C: fermentation Medium composition (derived from ZYM-5052 medium (Studier FW, prot. Exp. Pur.41, (2005), 207-234)).

Example 4: quantification of coenzyme A and thioester intermediate thereof

Extraction of

120 μ L of bacterial culture was sampled directly from the fermentor and filtered through a membrane filter under vacuum. Immediately after filtration, the filter was placed in aluminum foil and immersed in liquid nitrogen to stop all metabolic reactions. The aluminum foil with filter was then stored at-80 ℃ until extraction. In a Falcon tube at-80 ℃ with 2mL cold MeOH/H₂The O (80/20) mixture extracted intracellular metabolites (from the membrane filter) within 15 minutes. After this extraction time, Falcon tubes were centrifuged at-9 ℃ for 20 minutes. The supernatant was then removed and transferred to a new tube. Second extraction on Membrane Filter Using 1mL of Cold MeOH/H₂The O-mixture was carried out according to the same procedure as described above (except that the extraction time at-80 ℃ and the centrifugation step were both reduced to 5 min). The combined supernatant (ca. 3mL) was filtered through a 0.2 μm syringe filter and 2.4mL was evaporated to dryness by a Speed-Vac concentrator. Then, 120. mu.L of ACN/H was used₂O (50/50, v/v) redissolving the dry extract, again passing through a 0.2 μm syringe filterFiltered and transferred to HPLC vials for LC-MS analysis.

LC-MS analysis

The analysis was performed in negative ionization mode on a UHPLC system coupled to a Q-exact mass spectrometer (ThermoFisher Scientific, Massachusetts, USA). For the LC fraction, a column of BEH amide (1.7 μm, 2.1X 100mm, Waters) adjusted at 25 ℃ was used. The flow rate was set at 0.5mL/min and 2. mu.L of sample was injected. The mobile phase consisted of a binary gradient (A: ammonium formate (10mM) + 0.1% ammonium hydroxide and B: acetonitrile) starting from 95% B in 1.5min, then dropping to 55% B until 55% B in 8.5 min and remaining at 55% B in 2min, then returning to the initial conditions. The total analysis time was 19 min. At the mass level, the analysis was performed in the negative ionization mode in Full MS + ddMS2 mode. If desired, ion packets between 80< m/z <1200Da are considered and all ions with an ion intensity greater than 1e5 are fragmented using a collision energy of 35eV to obtain additional structural information. Calibration curves for the target compounds were recorded under the same LC/MS conditions. Data analysis was performed using Xcalibur v3.0.63 software (ThermoFisher Scientific). The LC-MS characteristics of the target compounds are summarized in table D.

Table D: LC-MS characterization of coenzyme A and thioesters thereof

Compound (I)1	Molecular formula	Molecular weight (g/mol)	RT(min)	Observed m/z
					CoA	C21H36N7O16P3S	767.535	6.96	766.1096[M-H]-；382.5509[M-H]2+
AcCoA	C23H38N7O17P3S	809.570	6.83	808.1204[M-H]-；403.5561[M-H]2+
					AcAcCoA	C25H40N7O18P3S	851.607	6.78	850.1310[M-H]-；424.5618[M-H]2+
HMG-CoA	C27H44N7O20P3S	911.659	7.27	910.1526[M-H]-；454.5722[M-H]2+
					MC-CoA	C26H42N7O17P3S	849.635	6.53	848.1515[M-H]-；423.5719[M-H]2+

¹CoA: coenzyme A; AcCoA: acetyl-coenzyme a; AcAcAcCoA: acetoacetyl-coenzyme a; HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme a; MC-CoA: 3-methylcrotonyl-coenzyme A

HPLC analysis

There are two LC methods available for the detection and quantification of CoA-derived compounds.

The first LC method ("method 1") was performed by an HPLC 1260 system coupled to a multi-wavelength detector (MWD) (Agilent, Santa Clara, USA). The separation was carried out by Zorbax Eclipse coupled to a C18 column (3.5 μm, 4.6X 100mm, Agilent) adjusted to 30 ℃. The mobile phase consisted of isocratic elution with acetonitrile (5%) and phosphate buffer (100mM) at pH5 (95%) over 10 min. The flow rate was set at 1.5mL/min and the amount of sample was 5. mu.L.

The second method ("method 2") was performed using an HPLC 1260 system coupled to a Diode Array Detector (DAD) (Agilent, Santa Clara, USA). A ZorbaxsbAq column (5 μm, 4.6X 250mm, Agilent) adjusted at 30 ℃ was used for the isolation of the metabolites. 5 μ L of sample was injected. The flow rate was set at 1.5 mL/min. The mobile phase consisted of a binary gradient (A: acetonitrile; and B: H2 SO48.4mM), starting from 100% B, then dropping to 30% B until 8min and remaining at 30% B for 1 min. Within this 1min of 30% B, the flow rate was increased to 2 mL/min. After 9min, the binary gradient returned to the original condition (i.e., 100% B and a flow rate of 1.5 mL/min) within 2min and remained at 100% B within 3 min. The total run time was 14 min.

In both methods, the detection of the target metabolite is carried out at λ 260nm and calibration curves of the pure compound are recorded under the LC conditions used for each. ChemStation software (Agilent) was used for data analysis.

Table E: residence time of coenzyme A and thioesters thereof in HPLC-based quantitative method

¹CoA: coenzyme A; AcCoA: acetyl-coenzyme a; AcAcAcCoA: acetoacetyl-coenzyme a; HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme a; MC-CoA: 3-methylcrotonyl-coenzyme A

Example 5: conversion of 3-methylcrotonic acid to isobutene by recombinant Clostridium ljunii expressing ubiDHav6

This working example shows the production of isobutene from 3-methylcrotonic acid by recombinant Clostridium ljunii expressing the genes encoding (i) 3-methylcrotonic acid decarboxylase ubiD HAv6 and (ii) prenyltransferase ubiX. The 3-methylcrotonic acid decarboxylase converts 3-methylcrotonic acid to isobutylene, which is derived from Hypocrea atroviride (Trichoderma atroviride), and is further engineered to achieve efficient IBN production. Prenyltransferases convert FMN to isoprene-FMN, thereby providing a cofactor for isoprene decarboxylase. The source of this enzyme is E.coli MG 1655.

Expression of 3-methylcrotonic acid decarboxylase and prenyltransferase in Clostridium youngii DSM13528

These genes were codon optimized for expression in c. Expression vectors containing the pCB102 replicon for maintenance in clostridium ljunii and the erythromycin resistance gene for selection were used for gene expression. Expression was driven by the thl gene promoter from clostridium acetobutylicum ATCC 824 (which binds to the ribosome binding site from phage T7) and terminated by the rrnB terminator from escherichia coli MG 1655.

Culture conditions

From the transformation of cell frozen culture inoculated with 20g/L fructose and 5 u g/mL clarithromycin 2YT medium, with 2 bar CO₂And (4) pressurizing. Cells were grown to mid-exponential phase. The pH of the culture was adjusted to maintain the pH between 5 and 6. The culture was carried out in serum bottles at 150rpm and 37 ℃. Cells were harvested by centrifugation and stored at-80 ℃ or resuspended directly at an OD600 of 13 in PETC1754 medium supplemented with 20g/L fructose. Cells were grown in glass vials at 150rpm and 37 ℃ for 72 h. Amount of IBNDetermined by GC-MS analysis of the headspace.

The strain containing 3-methylcrotonic acid decarboxylase and prenyl transferase (SGP244) was compared with the strain containing the corresponding empty vector (SGP184) (FIG. 10). Compared to SGP184, SGP244 increased isobutene accumulation by a factor of 201.

Example 6: conversion of 3-methylcrotonic acid to isobutene by recombinant Clostridium ljunii expressing FDCSs5v2

This working example shows the production of isobutene from 3-methylcrotonic acid by recombinant Clostridium ljunii expressing the genes encoding (i) 3-methylcrotonic acid decarboxylase FDCSs5v2 and (ii) prenyltransferase ubiX. The 3-methylcrotonic acid decarboxylase converts 3-methylcrotonic acid to isobutene and is derived from streptomyces and further engineered to achieve efficient IBN production. Prenyltransferases convert FMN to isoprene-FMN, thereby providing a cofactor for isoprene decarboxylase. The source of this enzyme is E.coli MG 1655.

Expression of 3-methylcrotonic acid decarboxylase and prenyltransferase in Clostridium youngii DSM13528 these genes were codon optimized for expression in Clostridium youngii and synthesized by Genscript. Expression vectors containing the pCB102 replicon for maintenance in clostridium ljunii and the erythromycin resistance gene for selection were used for gene expression. Expression was driven by the thl gene promoter from clostridium acetobutylicum ATCC 824 (which binds to the ribosome binding site from phage T7) and terminated by the rrnB terminator from escherichia coli MG 1655.

Culture conditions

From the transformation of cell frozen culture inoculated with 20g/L fructose and 5 u g/mL clarithromycin 2YT medium, with 2 bar CO₂And (4) pressurizing. Cells were grown to mid-exponential phase. The pH of the culture was adjusted to maintain the pH between 5 and 6. The culture was carried out in serum bottles at 150rpm and 37 ℃. Cells were harvested by centrifugation and stored at-80 ℃ or resuspended directly at an OD600 of 13 in PETC1754 medium supplemented with 20g/L fructose. Cells were grown in glass vials at 150rpm and 37 ℃ for 72 h. The amount of IBN was determined by GC-MS analysis of the headspace.

The strain containing 3-methylcrotonic acid decarboxylase and prenyl transferase (SGP 339) was compared with the strain containing the corresponding empty vector (SGP184) (FIG. 11). Compared to SGP184, SGP244 showed a 2093-fold increase in isobutene accumulation.

Example 7: for removal of CO/CO from CO/CO via acetyl-CoA₂/H₂Construction of a Clostridium-Yang Strain for gas production of isobutene

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

TABLE F

Expression of the isobutene biosynthetic pathway in Clostridium youngyi DSM13528

These genes were codon optimized for expression in c. Expression vectors containing the pCB102 replicon for maintenance in clostridium ljunii and the erythromycin resistance gene for selection were used to express the genes divided into two operons. Expression of both operons was driven by the promoter of the thl gene of C.acetobutylicum ATCC 824 and terminated by the rrnB terminator of E.coli MG 1655.

Culture conditions

For preculture, a frozen culture of transformed cells was inoculated with PETC1754 medium supplemented with 20g/L fructose and 5. mu.g/mL clarithromycin, and 2 bar CO₂And (4) pressurizing. Cells were grown overnight in serum flasks at 150rpm and 37 ℃. For main culture, PETC1754 medium supplemented with 5. mu.g/mL clarithromycin was inoculated from the preculture and supplemented with 55% CO, 25% H at2 bar₂And 20% CO₂(all volume percents) of gasesThe mixture is pressurized. The pH of the culture was adjusted to maintain the pH between 5 and 6 and gas was refilled daily to maintain a pressure of 2 bar. The culture was carried out in serum bottles at 150rpm and 37 ℃ for 6 days. The amount of IBN was determined by GC-MS analysis of the headspace.

The strain containing the isobutene pathway (SGP353) was compared with the strain containing the corresponding empty vector (SGP348) (fig. 12). While SGP353 produced a large amount of isobutylene, only trace amounts were found in SGP348 (fig. 13). Thus, by introducing the isobutene pathway in clostridium ljunii, the signal of isobutene was increased 1971-fold compared to the strain without the isobutene pathway.