阅读说明：本技术 从磷酸二羟丙酮和甘油醛-3-磷酸产生果糖-6-磷酸的方法 (Method for producing fructose-6-phosphate from dihydroxyacetone phosphate and glyceraldehyde-3-phosphate ) 是由 A·阿拉肯亚内斯 S·布达 R·夏约 于 2019-07-26 设计创作，主要内容包括：描述了一种从磷酸二羟丙酮(DHAP)和甘油醛-3-磷酸(G3P)产生果糖-6-磷酸(F6P)的方法,所述方法包括步骤：(a)使磷酸二羟丙酮(DHAP)酶促转化成二羟丙酮(DHA)；并且(b)使如此产生的二羟丙酮(DHA)和甘油醛-3-磷酸(G3P)酶促转化果糖-6-磷酸(F6P)；或包括步骤：(a’)使甘油醛-3-磷酸(G3P)酶促转化成甘油醛；并且(b’)使如此产生的甘油醛连同磷酸二羟丙酮(DHAP)一起酶促转化成果糖-1-磷酸(F1P)；并且(c’)使如此产生的果糖-1-磷酸(F1P)酶促转化成果糖-6-磷酸(F6P)。(A method for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) is described, said method comprising the steps of: (a) enzymatically converting dihydroxyacetone phosphate (DHAP) to Dihydroxyacetone (DHA); and (b) enzymatically converting the Dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) thus produced into fructose-6-phosphate (F6P); or comprises the following steps: (a') enzymatically converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde; and (b') enzymatically converting the glyceraldehyde thus produced into fructose-1-phosphate (F1P) together with dihydroxyacetone phosphate (DHAP); and (c') enzymatically converting the fructose-1-phosphate (F1P) thus produced into fructose-6-phosphate (F6P).)

1. A method for producing fructose-6-phosphate (F6P) from dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P),

(A) the method comprises the following steps:

(a) enzymatically converting dihydroxyacetone phosphate (DHAP) to Dihydroxyacetone (DHA); and is

(b) Enzymatically converting the Dihydroxyacetone (DHA) thus produced together with glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P); or

(B) The method comprises the following steps:

(a') enzymatically converting glyceraldehyde-3-phosphate (G3P) into glyceraldehyde; and is

(b') enzymatically converting the glyceraldehyde thus produced into fructose-1-phosphate (F1P) together with dihydroxyacetone phosphate (DHAP); and is

(c') enzymatically converting the fructose-1-phosphate (F1P) thus produced into fructose-6-phosphate (F6P).

2. The method according to claim 1(a), wherein the conversion of dihydroxyacetone phosphate (DHAP) into Dihydroxyacetone (DHA) according to step (a) is effected by a phosphomonoester hydrolase (EC3.1.3. -).

3. The method according to claim 2, wherein the phosphomonoester hydrolase (EC3.1.3. -) is selected from the group consisting of:

(i) sugar phosphatases (EC 3.1.3.23);

(ii) 6-phosphogluconate phosphatase (EC 3.1.3.); (ii) a

(iii) Pyridoxal phosphate phosphatase (EC 3.1.3.74);

(iv) fructose-1-phosphate phosphatase (EC 3.1.3.); (ii) a

(v) Dihydroxyacetone phosphatase (EC3.1.3. -); (ii) a

(vi) Hexitol phosphatase (EC 3.1.3.);

(vii) acid phosphatase (EC 3.1.3.2);

(viii) alkaline phosphatase (EC 3.1.3.1);

(ix) glycerol-1-phosphate phosphatase (EC 3.1.3.21); and

(x) 3-phosphoglycerate phosphatase (EC 3.1.3.38).

4. The process according to any one of claims 1 to 3, wherein the conversion of Dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (G3P) into fructose-6-phosphate (F6P) according to step (b) is achieved by the following enzymes

(i) Aldehyde cleaving enzyme (EC 4.1.2. -); or

(ii) Transaldolase (EC 2.2.1.2).

5. The method according to claim 1(B), wherein the conversion of glyceraldehyde-3-phosphate (G3P) to glyceraldehyde according to step (a) is effected by a phosphomonoester hydrolase (EC3.1.3. -).

6. The method according to claim 5, wherein the phosphomonoester hydrolase (EC3.1.3. -) is selected from the group consisting of:

(i) glyceraldehyde 3-phosphate phosphatase (EC 3.1.3.);

(ii) alkaline phosphatase (EC 3.1.3.1);

(iii) acid phosphatase (EC 3.1.3.2);

(iv) sugar phosphatases (EC 3.1.3.23); and

(v) hexitol phosphatase (EC3.1.3. -).

7. The process according to claim 1(B) or according to claim 5 or 6, wherein the conversion of glyceraldehyde and dihydroxyacetone phosphate (DHAP) into fructose-1-phosphate (F1P) according to step (B') is effected by fructose bisphosphate aldolase (EC 4.1.2.13).

8. The process according to any one of claims 1(B) or 5 to 7, wherein the conversion of fructose-1-phosphate (F1P) into fructose-6-phosphate (F6P) according to step (c') is effected by the following enzymes

(i) Phosphoglucomutase (EC 5.4.2.2); or

(ii) Phosphomannose mutase (EC 5.4.2.8).

9. The method of any one of claims 1 to 8, which is carried out in vitro.

10. The method according to any one of claims 1(a) to 4, which is carried out in vivo in a recombinant microorganism that has been transformed with a nucleotide sequence encoding an enzyme that can catalyze the conversion process recited in step (a) according to claim 1 and a nucleotide sequence encoding an enzyme that can catalyze the conversion process recited in step (b) according to claim 1.

11. The method according to claim 1(B) or any one of claims 5 to 8, which is carried out in vivo in a recombinant microorganism which has been transformed with a nucleotide sequence encoding an enzyme which can catalyse the conversion process recited in step (a ') of claim 1(B) and a nucleotide sequence encoding an enzyme which can catalyse the conversion process recited in step (B') of claim 1 (B).

12. The method according to claim 11, wherein the microorganism is further transformed with a nucleotide sequence encoding an enzyme capable of catalyzing the transformation process as set forth in step (C') of claim 1 (C).

13. The method according to any one of claims 10 to 12, wherein the microorganism is further characterized in that it

a) Having phosphoketolase activity;

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

(ii) No phosphofructokinase activity;

and

c) (ii) (i) a Pentose Phosphate Pathway (PPP) oxidation branch which has been weakened or inactivated by inactivation of the gene coding for glucose-6-phosphate dehydrogenase or by reduction of glucose-6-phosphate dehydrogenase activity compared to the unmodified microorganism; or

(ii) No glucose-6-phosphate dehydrogenase activity.

14. The method of claim 13, wherein the microorganism is further characterized by: EMPP is further attenuated or inactivated by inactivation of the gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing glyceraldehyde 3-phosphate dehydrogenase activity compared to the unmodified microorganism.

15. Recombinant microorganism which has been transformed with the following nucleotide sequence

(a) A nucleotide sequence encoding a phosphomonoester hydrolase (EC3.1.3. -); and

(b) a nucleotide sequence encoding an enzyme selected from the group consisting of

(i) Aldehyde cleaving enzyme (EC 4.1.2. -); and/or

(ii) Transaldolase (EC 2.2.1.2).

16. Recombinant microorganism which has been transformed with the following nucleotide sequence

(a) A nucleotide sequence encoding a phosphomonoester hydrolase (EC3.1.3. -); and

(b) a nucleotide sequence encoding a fructose bisphosphate aldolase (EC 4.1.2.13);

wherein the microorganism further possesses phosphoglucomutase (EC 5.4.2.2) activity or mannosidase phosphate (EC5.4.2.8) activity.

17. The recombinant microorganism of claim 16, which has been further transformed with the following nucleotide sequence

(c) A nucleotide sequence encoding an enzyme selected from the group consisting of:

(i) phosphoglucomutase (EC 5.4.2.2); and

(ii) phosphomannose mutase (EC 5.4.2.8).

18. The recombinant microorganism of any one of claims 15-17, further characterized by: it is composed of a base, a cover and a cover

a) Having phosphoketolase activity;

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

(ii) No phosphofructokinase activity;

and

c) (ii) (i) a Pentose Phosphate Pathway (PPP) oxidation branch which has been weakened or inactivated by inactivation of the gene coding for glucose-6-phosphate dehydrogenase or by reduction of glucose-6-phosphate dehydrogenase activity compared to the unmodified microorganism; or

(ii) No glucose-6-phosphate dehydrogenase activity.

19. The recombinant microorganism of claim 18, further characterized in that: EMPP is further attenuated or inactivated by inactivation of the gene encoding glyceraldehyde 3-phosphate dehydrogenase or by reducing glyceraldehyde 3-phosphate dehydrogenase activity compared to the unmodified microorganism.

20. An enzyme combination comprising

(a) Phosphomonoester hydrolase (EC3.1.3. -); and

(b) an enzyme selected from the group consisting of

(i) Aldehyde cleaving enzyme (EC 4.1.2. -); and/or

(ii) Transaldolase (EC 2.2.1.2).

21. An enzyme combination comprising

(a) Phosphomonoester hydrolase (EC3.1.3. -);

(b) fructose bisphosphate aldolase (EC 4.1.2.13); and

(c) an enzyme selected from the group consisting of

(i) Phosphoglucomutase (EC 5.4.2.2); or

(ii) Phosphomannose mutase (EC 5.4.2.8).

22. A composition comprising a microorganism according to claim 15 or a combination of enzymes according to claim 20.

23. A composition comprising a microorganism according to claim 16 or 17 or a combination of enzymes according to claim 21.

24. Use of a microorganism according to claim 15 or of an enzyme combination according to claim 20 or of a composition according to claim 22 for the first conversion of dihydroxyacetone phosphate (DHAP) to Dihydroxyacetone (DHA) by the enzymes mentioned in (a) and subsequently further conversion of the Dihydroxyacetone (DHA) produced together with glyceraldehyde-3-phosphate (G3P) to fructose-6-phosphate (F6P) by the enzymes mentioned in (b).

25. Use of a microorganism according to claim 16 or 17 or of an enzyme combination according to claim 21 or of a composition according to claim 23 for the conversion of glyceraldehyde-3-phosphate (G3P) to glyceraldehyde first by the enzyme mentioned in (a) and subsequently further conversion of the produced glyceraldehyde together with dihydroxyacetone phosphate (DHAP) to fructose-1-phosphate (F1P) by the enzyme mentioned in (b) and subsequently further conversion of the produced fructose-1-phosphate (F1P) to fructose-6-phosphate (F6P) by the enzyme mentioned in (c).

Examples

General methods and materials

Ligation methods and transformation methods are well known in the art. The Molecular Cloning can be described in Sambrook j, et al: a Laboratory Manual, 2 nd edition, Cold Spring Harbor, n.y., 1989, and Sambrook j, the techniques found to be suitable for use in the examples below.

Materials and methods suitable for maintaining and growing bacterial cultures are well known in the art. Techniques suitable for use in the following examples can be found in the Manual of Methods for General Bacteriology (Philipp Gerhardt, R.G.E.Murray, Ralph N.Costilow, Eugene W.Nester, Willis A.Wood, Noel R.Krieg and G.Briggs Philips).

All reagents and materials used for bacterial cell growth and maintenance were obtained from Sigma-Aldrich (st. louis, MO) unless otherwise noted.

Enzyme overexpression and purification

a) Enzyme from Escherichia coli

The enzyme from E.coli has been overexpressed using a plasmid from the ASKA depository (Kitagawa, M et al DNA Res.12: 291-299 (2005)).

Strain BL21(DE3) cells (Novagen) were grown in LB medium and rendered electrocompetent. Electrocompetent BL21 cells were transformed with the corresponding plasmid expressing the desired enzyme (see Table 1) and subsequently plated on LB plates containing chloramphenicol (25 ug/ml). The plates were incubated at 30 ℃ overnight.

The transformed cells were incubated at 30 ℃ for 20 hours with shaking (160 rpm) using ZYM-5052 self-induction medium (Studier FW, prot. exp. Pur.41: 207-234 (2005)). Cells were harvested by centrifugation at 4 ℃, 4,000 rpm for 20 minutes and the pellet stored at-80 ℃.

After purification of the recombinant protein using His trap (Protino Ni-IDA 1000 kit, Macherey Nagel), expression of the recombinase was examined on a protein gel. Purification was performed according to the manufacturer's recommendations.

b) Enzymes from organisms other than E.coli

For optimal expression in E.coli, from(Invitrogen) target genes from several organisms (see table 2) were codon optimized. In addition, a His-tag was added at the 5 'end of the gene and an additional stop codon was added at the 3' end of the gene. The gene construct was flanked by NdeI and EcoRI restriction sites and was provided inside plasmid pET25b + (Merckmillipore).

Competent E.coli BL21(DE3) cells (Novagen) were transformed with these vectors according to standard heat shock procedures. The transformed cells were incubated at 30 ℃ for 20 hours with shaking (160 rpm) using ZYM-5052 self-induction medium (Studier FW, prot. exp. Pur.41: 207-234 (2005)). Cells were harvested by centrifugation at 4 ℃, 4,000 rpm for 20 minutes and the pellet stored at-80 ℃.

After purification of the recombinant protein using His trap (Protino Ni-IDA 1000 kit, Macherey Nagel), expression of the recombinase was examined on a protein gel. Purification was performed according to the manufacturer's recommendations.

Example 1: fructose-6-phosphate aldolase and fructose diphosphateEnzymeActivity inhibition assay

A series of tests were performed to determine whether AMP has an inhibitory effect on the enzymatic activity of fructose-6-phosphate aldolase and/or fructose bisphosphatase. The methods used to test enzyme activity are adapted from C.Gulerd-Hslaine, V.De Berardinis, M.Besnard-Gonnet, E.Darii, M.Debacker et al, Genome Mining for Innovative biocatalysis: new Dihydroxysceptone Aldolases for the Chemist's Toolbox. chem Cat chem, Wiley, 7: 1871-1879(2015).

a)Effect of AMP concentration on fructose bisphosphatase Activity

120 μ l of each kinetic assay contained Tris HCl buffer (50 mM; pH 7.5), 20mM NaCl, 10mM MgCl₂、1mM NADP⁺AMP (several concentrations tested), 1mM fructose 1, 6-bisphosphate (F1, 6bisP), 0.2mg/ml FBPase and accessory enzymes (glucose-6-phosphate isomerase (PGI) and NADP⁺Dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg/ml each)). The mixture was incubated at 30 ℃ for up to 20 minutes and the reaction was monitored spectrophotometrically at 340nm (measuring NADPH formation), assuming 1 molecule of reduced NADPH was produced per fructose-6-phosphate molecule. The results are shown in FIG. 1. A strong inhibitory effect of AMP on fructose bisphosphatase activity was observed.

b)Effect of AMP concentration on fructose-6-phosphate aldolase Activity of FsaA129S

120 μ l of each kinetic assay contained Tris HCl buffer (50 mM; pH 8.5), 1mM NADP⁺AMP (several concentrations tested), 200mM DHA, 3mM D, L-G3P, 0.4mg/ml FsaA129S and accessory enzymes (glucose-6-phosphate isomerase (PGI) and NADP⁺Dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg/ml each)).

The mixture was incubated at 30 ℃ for up to 20 minutes and the reaction was monitored spectrophotometrically at 340nm (measuring NADPH formation), assuming 1 molecule of reduced NADPH was produced per fructose-6-phosphate molecule. The results are shown in fig. 2. AMP inhibition with FSAA A129S was not observed.

Example 2: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) were converted in vitro to fructose-6-phosphate (F6P) via a Dihydroxyacetone (DHA) intermediate.

A series of tests were performed to determine the optimal enzyme combination for converting G3P and DHAP to F6P. These enzyme combinations should perform 2 steps:

1)DHAP→DHA

2)DHA+G3P→F6P

a)enzymes catalyzing the conversion of DHA and G3P to F6P

The methods used to test enzyme activity are adapted from C.Gulerd-Hslaine, V.De Berardinis, M.Besnard-Gonnet, E.Darii, M.Debacker et al, Genome Mining for Innovative biocatalysis: new Dihydroxysceptone Aldolases for the Chemist's Toolbox. chem Cat chem, Wiley, 7: 1871-1879(2015).

120 μ L of each kinetic assay contained Tris HCl buffer (50 mM; pH 8.5), 3mM D, L-G3P, 200mM DHA, 1mM NADP⁺0.4mg/ml enzyme and accessory enzymes (glucose-6-phosphate isomerase (PGI) and NADP)⁺Dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg/ml each)). The mixture was incubated at 30 ℃ and the reaction was monitored spectrophotometrically at 340nm (measuring NADPH formation) assuming that 1 molecule of reduced NADPH was produced per F6P molecule. The results are shown in table 3.

Table 3: production of F6P from DHA and G3P with different enzymes

a)Enzymes catalyzing the conversion of DHAP to DHA

The methods used to test enzyme activity are adapted from C.Gulerd-Hslaine, V.De Berardinis, M.Besnard-Gonnet, E.Darii, M.Debacker et al, Genome Mining for Innovative biocatalysis: new Dihydroxysceptone Aldolases for the Chemist's Toolbox. chem Cat chem, Wiley, 7: 1871-1879(2015).

120 μ l of each kinetic assay contained Tris HCl buffer (50 mM; pH 8.5), 10mM MgCl₂，100mM DHAP、0.8mM NADP⁺0.6MG/ml enzyme, 0.8MG/ml fructose-6-phosphate aldolase 1 from E.coli MG1655 (mutant FSAA A129S) and auxiliary enzymes (glucose-6-phosphate isomerase (PGI) and NADP⁺Dependent glucose-6-phosphate dehydrogenase (zwf) (0.5 mg/ml each)). The mixture was incubated at 30 ℃ and the reaction was monitored spectrophotometrically at 340nm (measuring NADPH formation) assuming that 1 molecule of reduced NADPH was produced per F6P molecule. The results are shown in table 3.

Table 4: production of DHA from DHAP with different enzymes

Example 3: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) were converted in vitro to fructose-6-phosphate (F6P) via a glyceraldehyde intermediate.

A series of assays were performed to determine the optimal enzyme combination for converting G3P and DHAP to F6P. The optimal enzyme combination should perform 3 steps:

1) G3P → glyceraldehyde

2) Glyceraldehyde + DHAP → F1P

3)F1P→F6P

a)Enzymes catalyzing the conversion of G3P to glyceraldehyde

200 μ l of each kinetic assay system contained Tris HCl buffer (50mM pH 7.5), 100mM NaCl, 10mM MgCl₂G3P (1-10-50mM), and 2mg/ml of the enzyme tested (see Table 7). The mixture was incubated at 30 ℃ overnight and the reaction was quenched with 1 volume of acetonitrile. The final product was analyzed by LCMS. LC-MS analysis was performed on an Ultimate 3000(Dionex, Thermo Fisher Scientific) coupled to a Q-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with an Electrospray (ESI) source and operated in negative ion mode. Chromatographic resolution was performed using a HILIC amide (1.9 μm, 2.1X 150mm) column (Waters) maintained at 25 ℃ run under gradient elution as follows. The mobile phase is: (A)10mM ammonium formate pH 9.45 (adjusted with ammonium hydroxide), while mobile phase (B) is 100% acetonitrile and the flow rate is 500. mu.L/min. Elution began with a 1.5 minute isocratic step at 95% B, followed by a linear gradient from 95% to 55% phase B over 7 minutes. The 55% B chromatography system was then rinsed for 2 minutes and the run ended with an 8.5 minute equilibration step.

Table 5: an enzyme catalyzing the conversion of G3P to glyceraldehyde.

a)Catalyzing the conversion of glyceraldehyde and DHAP to F1P and furtherStep (2) enzymes catalyzing the conversion of F1P to F6P

200 μ l of each kinetic assay contained Tris HCl buffer (50 mM; pH 7.5), 50mM NaCl, 5mM MgCl₂、1mM NADP⁺10mM DHAP, 10mM glyceraldehyde, 1mg/ml AldoB, 1mg/ml PGM with PMM and accessory enzymes (glucose-6-phosphate isomerase (PGI) and NADP⁺Dependent glucose-6-phosphate dehydrogenase (zwf) (1 mg/ml each)). The mixture was incubated at 30 ℃ and the reaction was monitored spectrophotometrically at 340nm (measuring NADPH formation) assuming that 1 molecule of reduced NADPH was produced per F6P molecule. The results are shown in table 6.

Table 6: an enzyme that catalyzes the conversion of glyceraldehyde and DHAP to F1P and further catalyzes the conversion of F1P to F6P. The ALDOB-encoding enzyme was incubated either along with pgm-encoding enzyme (assay 1), along with AHA _ 2903-encoding enzyme (assay 2), or along with both (assay 3).

Example 4: construction of a novel E.coli prototype producing acetone and isopropanol

Coli converts glucose to acetyl-CoA, similar to most organisms. A modified E.coli prototype in which the acetyl-CoA production yield was optimized has been previously described (WO 2013/007786). A prototype of the bacterium was constructed with the following genotype,strain A：

MG1655ΔptsHIΔzwf_edd_edaΔpfkAΔpfkB

Strain AThe plasmid-based PKT gene of phosphoketolase YP 003354041.1 from lactococcus lactis was overexpressed to produceStrain BI.e., strains with rearranged central carbon metabolism, in which the inactivated Embden-Meyerhoff-Parnas pathway (EMPP), Pentose Phosphate Pathway (PPP), and Entner Doudoroff Pathway (EDP) is replaced by a new phosphoketolase based carbon catabolism pathway. Once transformed, as compared to the wild-type MG1655 strain expressing the same acetone pathwayStrain BWith the acetone route, superior acetone yields are observed.

To construct an implementStrains which have a PKT pathway and are capable of robust growth relying on sucrose as a carbon source willStrain AFurther engineered as described below.

The PKT gene was introduced at the kdgK locus (kdgK:: P1_ RBST7_ PKT)Strain AThe chromosome (b) of (a). The resulting strain had the following genotype:

MG1655ΔptsHIΔzwf_edd_edaΔpfkAΔpfkB kdgK：：P1_RBST7_pkt

this strain was passaged for several months on minimal medium supplemented with glucose as a carbon source, while selecting successively for clones or populations with the highest growth rate until a doubling time of less than 5 hours was achieved.

Several gene deletions were performed to increase acetone and isopropanol production: Δ hemA Δ fsaA Δ fsaB.

To further increase isopropanol production, the pntAB (pyridine nucleotide transhydrogenase subunits. alpha. and. beta., Uniprot P07001 and P0AB67, NCBI reference sequences: NP-416120.1 and NP-416119.1) gene from E.coli was overexpressed by inserting a constitutively strong promoter at the pntAB locus.

The resulting strain is hereinafter referred to asStrain C。

Example 5: construction of E.coli Strain producing acetone and isopropanol from acetyl-CoA

This working example shows the production of acetone and isopropanol by recombinant E.coli strains expressing the genes constituting the acetone and isopropanol pathways.

The enzymes used in this study to convert acetyl-CoA to acetone and isopropanol are listed in table 7.

Table 7: enzymes catalyzing the conversion of acetyl-CoA to acetone and isopropanol

Expression of the acetone/isopropanol biosynthetic pathway in E.coli.

The procedure described in example 4 was usedStrain CAs a host microorganism.

All the listed genes were codon-optimized for expression in E.coli and(Thermofoisher) with the exception of the genes atoD and atoA. These last two genes were directly amplified from the genomic DNA of E.coli MG 1655.

Expression vectors containing the origin of replication pSC and a spectinomycin resistance marker are used to express the genes thlA, atoD, atoA, adc and adh. The constructed vector was named pGB 5344.

The enzymes responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to fructose-6-phosphate (F6P) were expressed in E.coli.

A modified form of pUC18(New England Biolabs) containing a modified multiple cloning site (pUC18 MCS) (WO 2013/007786) and an ampicillin resistance gene (plasmid pGB 271) was used to overexpress the genes listed in Table 8.

Table 8: an enzyme that catalyzes the conversion of DHAP and G3P to F6P.

Transformation of different combinations of plasmids intoStrain C. The strains produced in this way are summarized in table 9.

Table 9: a strain produced by converting glucose into acetone + isopropanol in vivo.

Example 6: coli strains growth and acetone/isopropanol production from acetyl-CoA

Preculture conditions

The transformed cells were then plated on LB plates supplied with ampicillin (100. mu.g/ml) and spectinomycin (100. mu.g/ml). The plates were incubated at 30 ℃ for 2 days. The isolated colonies were used to inoculate LB medium supplemented with ampicillin and spectinomycin. These precultures were incubated at 30 ℃ to achieve an optical density of 0.6.

Growth conditions are as follows:

the fermentation was carried out in a1 liter bioreactor (Multifors 2, Infors HT) with pH and temperature control. Cells using precultures were inoculated with 500ml of fermentation medium (Table 10) supplemented with ampicillin (100. mu.g/ml), spectinomycin (100. mu.g/ml), thiamine (0.6mM), glucose (1g/L) and glycerol (5g/L) to achieve an initial Optical Density (OD) of 0.05₆₀₀). During the growth phase, the temperature (T ═ 32 ℃), pH 6.5 and pO were maintained₂Constant at 5%. The glucose feed was increased from 0.1g/g DCW/hr to 0.35g/g DCW/hr. When OD is reached₆₀₀When 2, 8 and 20 are reached, 5g/L of yeast extract are added in pulses.

TABLE 10 fermentation medium composition (derived from ZYM-5052 medium (Studier FW, prot. Exp. Pur.41, (2005), 207-.

Acetone/isopropanol production stage

During this phase, the temperature T ═ 34 ℃, pH 6.5 and pO were maintained₂Constant at 5%. Glucose feeding was started at 0.50g sucrose/g DCW/hour and subsequently corrected for strain consumption. The glycerol concentration was maintained above 2 g/l.

The strains were analyzed continuously for acetone/isopropanol production using a gas chromatograph 7890a (agilent technology) equipped with a Flame Ionization Detector (FID) to measure acetone and isopropanol. Volatile organic compounds were chromatographed on a Hi-Plex H USP L17, 100x7.7mm (Agilent) using an Agilent 1260 InfinityII chromatograph. Acetone/isopropanol was quantified using a standard (Sigma).

FIG. 3 shows a comparison between the observed acetone and isopropanol specific productivities of a production strain expressing the enzymes responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHA) into fructose-6-phosphate (F6P) or, as a control, a strain not expressing the enzymes responsible for the conversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHA) into fructose-6-phosphate (F6P).

When the enzyme responsible for the conversion of glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to fructose 6-phosphate (F6P) was overexpressed (strain GBI 17553), the acetone and isopropanol specific productivity (moles produced per unit cell weight per unit time) was higher compared to strain GBI 15847.