CN114480235B

CN114480235B - Method for preparing alpha-ketoisovalerate by fermenting metabolic engineering escherichia coli

Info

Publication number: CN114480235B
Application number: CN202210069994.7A
Authority: CN
Inventors: 周哲敏; 周丽; 朱滢; 袁中喆; 刘光庆; 孙梓晋; 杜士宇; 刘贺
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2023-08-25
Anticipated expiration: 2042-01-21
Also published as: CN114480235A

Abstract

The invention discloses a method for preparing alpha-ketoisovalerate by fermenting metabolic engineering escherichia coli, belonging to the field of bioengineering. The invention constructs a recombinant strain of escherichia coli, and carries out transformation optimization on the host strain to obtain the recombinant strain capable of efficiently producing alpha-ketoisovalerate by a fermentation method. The recombinant strain can ferment with cheap glucose as a substrate to generate alpha-ketoisovalerate with higher added value, and the yield of the alpha-ketoisovalerate can reach 55.8g/L after 26h of fermentation.

Description

Method for preparing alpha-ketoisovalerate by fermenting metabolic engineering escherichia coli

Technical Field

The invention relates to a method for preparing alpha-ketoisovalerate by fermenting metabolic engineering escherichia coli, belonging to the field of bioengineering.

Background

Alpha-ketoisovalerate is a precursor substance for synthesizing amino acids such as valine, leucine, isoleucine and the like, and participates in the metabolic process of a living body. In the aspect of medical treatment, the alpha-keto acid tablet is one of main components, and can be used for treating chronic uremia; in terms of feed, can be used to stimulate muscle growth in livestock. Therefore, the alpha-ketoisovaleric acid has wide application prospect in the fields of medicines, foods, cosmetics and the like.

At present, the synthesis of alpha-ketoisovalerate by a biological fermentation method is still in an exploration stage, and is mainly carried out by corynebacterium glutamicum or escherichia coli. In 2010, krause et al (Applied and Environmental Microbiology,2010,76 (24): 8053-8061) performed high density fermentation using Corynebacterium glutamicum, the yield of alpha-ketoisovalerate reached 21.8.+ -. 3.2g/L, and the conversion was 0.47.+ -. 0.05mol/mol glucose. In 2020, li Yating overexpresses a key enzyme IlvC, alsS, ilvD in the synthesis process of alpha-ketoisovalerate, knocks out an catabolic pathway ilvE, competes for metabolism leuA, balances reducing force, and finally ferments and synthesizes 19.2g/L of alpha-ketoisovalerate in escherichia coli in a shaking flask manner, wherein the conversion rate reaches 0.8mol/mol, which is higher than that of corynebacterium glutamicum. However, the research level has a great gap from industrial application. Further inhibiting competing metabolic pathways, for example: the tricarboxylic acid cycle (TCA cycle) is expected to further improve the yield, conversion and production strength of alpha-ketoisovaleric acid.

Acetolactate synthase (AlsS) can decarboxylate pyruvic acid to acetolactate to further synthesize α -ketoisovalerate, but the enzyme also has the activity of catalyzing the synthesis of isobutyraldehyde from α -ketoisovalerate, which is further reduced to form isobutanol. Researchers have mainly used this nonspecific enzymatic activity to ferment and synthesize isobutanol, α -ketoisovalerate as a major byproduct of this fermentation process. However, in the process of synthesizing alpha-ketoisovalerate by fermentation, when the concentration of the isobutanol by-product is high, not only is the conversion rate of the alpha-ketoisovalerate reduced, but also the growth performance of thalli is inhibited, and finally the synthesis level of the alpha-ketoisovalerate is severely reduced. The enzyme is modified by Shota Atsumi (Applied and Environmental Microbiology,2009,75 (19): 6306-6311), so that the enzyme activity of catalyzing and synthesizing isobutyraldehyde can be reduced, the enzyme activity of synthesizing alpha-ketoisovalerate is not affected obviously, and the substrate specificity is improved. However, the enzymatic synthesis of alpha-ketoisovalerate using this mutant has not been reported.

The escherichia coli has the characteristics of clear genetic background, easy genetic operation, high growth rate, low nutrition requirement and the like, and is used as a cell factory for fermentation production of various products. However, there is no report on the fermentation and synthesis of alpha-ketoisovalerate in E.coli. Constructing recombinant escherichia coli synthesized by alpha-ketoisovalerate, improving recombinant strains and fermentation processes thereof, being beneficial to further improving the yield of the alpha-ketoisovalerate, reducing the cost and promoting the large-scale fermentation production of the alpha-ketoisovalerate.

Disclosure of Invention

The invention aims to provide recombinant escherichia coli with improved conversion efficiency of alpha-ketoisovalerate, which can be used for producing the alpha-ketoisovalerate by taking low-cost substrate glucose as a raw material through fermentation; the recombinant escherichia coli is subjected to at least one improvement of (a) to (d):

(a) Expressing heterologous acetolactate synthase AlsS; the heterologous acetolactate synthase takes an amino acid sequence shown as SEQ ID NO.1 as a starting sequence;

(b) Replacing the RBS sequence of the heterologous acetolactate synthase with any one of SEQ ID NO. 2-4;

(c) Adding a DAS+4 degradation tag after an AceF subunit of Pyruvate Dehydrogenase (PDH); the DAS+4 degradation tag has a sequence shown as SEQ ID NO. 5.

In one embodiment, the heterologous acetolactate synthase is mutated at least one of positions 424, 487, 488 based on SEQ ID NO. 1.

In one embodiment, the heterologous acetolactate synthase mutates glutamine at position 424, 487 or 488 to tryptophan based on SEQ ID No. 1.

In one embodiment, the key enzyme of the α -ketoisovalerate synthesis pathway is overexpressed using the T7 promoter.

In one embodiment, the mutated acetolactate synthase AlsS is RBS-optimized to enhance synthesis of α -ketoisovalerate.

In one embodiment, the recombinant escherichia coli takes escherichia coli B0016-050T4 as a host, the escherichia coli B0016-050T4 is the prior art, and the escherichia coli is disclosed in paper metabolic engineering modification escherichia coli production alpha-ketoisovalerate, and the strain deletes a byproduct acetic acid anabolic pathway coding gene ackA-pta, a lactic acid anabolic pathway coding gene ldhA, an ethanol anabolic pathway coding gene adhE, a succinic acid anabolic pathway coding gene frdA and a valine anabolic pathway editing gene ilvE on a chromosome, expresses a T7 RNA polymerase coding gene T7 RNAP and strengthens the expression of an NADPH coenzyme cycle metabolic pathway coding gene pntAB. The ackA-pta has Gene IDs 946775 and 946778 at NCBI; the ldhA has a Gene ID of 946315 at NCBI; the adhE has a Gene ID 945837 at NCBI; the frdA has a Gene ID of 948667 at NCBI; the ilvE has a Gene ID of 948278 at NCBI; the T7 RNAP is accession number M38308.1 at NCBI and the pntAB is accession numbers 946628 and 946144 at NCBI.

The invention provides a method for producing alpha-ketoisovalerate, which uses recombinant escherichia coli as a fermentation strain to produce the alpha-ketoisovalerate.

In one embodiment, the method uses glucose as a carbon source.

In one embodiment, the process is carried out under aerobic conditions.

In one embodiment, the method employs two-stage fermentation:

the first stage: in the growth stage of the thalli, the temperature is controlled to be 37 ℃, and the stirring rotation speed and the ventilation quantity are regulated to control the dissolved oxygen concentration to be more than or equal to 30 percent.

And a second stage: when OD is ₆₀₀ When the value reaches 20, the alpha-ketoisovaleric acid synthesis stage is carried out, IPTG with the final concentration of 0.8mM is added for induction, the temperature is reduced to 30 ℃, and the dissolved oxygen concentration is controlled to be less than 15%.

In one embodiment, the method is also specific for culturing to OD ₆₀₀ The bacterial suspension of 2.5 was induced with IPTG.

In one embodiment, the strain is aerobically cultured in a fermenter to OD ₆₀₀ The value reaches 20, and IPTG is added for induction.

The invention also protects the application of the recombinant escherichia coli or the method for producing the alpha-ketoisovalerate in the fields of medicines, foods, cosmetics and the like.

The beneficial effects are that: the invention constructs a recombinant escherichia coli strain for efficiently synthesizing alpha-ketoisovalerate, and carries out transformation optimization on the host strain to obtain recombinant bacteria capable of producing the alpha-ketoisovalerate by a fermentation method and a fermentation method. The recombinant strain can ferment with cheap glucose as a substrate to generate alpha-ketoisovalerate with higher added value for 26 hours, the yield of the alpha-ketoisovalerate can reach 55.8g/L, the conversion rate of the alpha-ketoisovalerate in the whole fermentation process reaches 0.85mol/mol glucose, the conversion rate in the second stage is improved to 0.99mol/mol glucose and is close to 1mol/mol theoretical glucose, and the volume production intensity is 2.14g/L h. The accumulation amount of the byproduct isobutanol is obviously reduced to 1.51g/L, and the yield is only 0.04mol/mol glucose.

Drawings

FIG. 1 shows strain 050T4/pC _T SD _T The effect of alpha-ketoisovalerate yield and isobutanol in a 5L fermenter; (A) Strain 050T4/pC _T SD _T Fermenting the yield of alpha-ketoisovalerate; (B) an isobutanol concentration gradient growth curve.

FIG. 2 shows the effect of AlsS mutants on the synthesis of alpha-ketoisovalerate and accumulation of isobutanol.

FIG. 3 is the effect of RBS sequence optimization of AlsS on alpha-ketoisovalerate synthesis and isobutanol accumulation.

FIG. 4 is a graph showing the effect of attenuating tricarboxylic acid cycle on synthesis of alpha-ketoisovalerate; (a) PDH enzyme activity comparison; (B) optimizing the rotating speed; and (C) optimizing the turning time in two stages.

FIG. 5 strain 050TY/pC _T SD _T Synthesis of alpha-ketoisovaleric acid in Q487S-RBS 55L fermenter.

Detailed Description

Culture medium:

LB liquid medium: firstly, weighing 10g of Tryptone (Tryptone), 5g of Yeast powder (Yeast extract) and 10g of sodium chloride (NaCl) in a beaker by using an electronic balance, then adding deionized water into the beaker to fix the volume to 1L, and finally, carrying out damp-heat sterilization for 20min at 121 ℃ in a high-pressure steam sterilization pot.

LB solid medium: 20g of agar powder is weighed and added into 1L of LB liquid medium, and then the mixture is put into a high-pressure steam sterilization pot for sterilization at 121 ℃ under moist heat for 20min.

M9-2 Medium (g/L): glucose 36, yeast powder 5, KH ₂ PO ₄ 3，Na ₂ HPO ₄ 6，NaCl 0.3，NH ₄ Cl 1，MgSO ₄ ·7H ₂ O0.49, trace element liquid 0.1% (v/v).

M9-4 Medium (g/L): glucose 30, yeast powder 4, KH ₂ PO ₄ 13.5 peptone 4, citric acid monohydrate 1.7, mgSO ₄ ·7H ₂ O 0.49，(NH4) ₂ HPO ₄ 4, microelement liquid 0.1% (v/v).

Microelement liquid: mnSO ₄ ·4H ₂ O 0.5g/L，FeSO ₄ ·7H ₂ O 10.0g/L，CaCl ₂ 2.0g/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ 0.1g/L，CuSO ₄ ·5H ₂ O 3.0g/L，Na ₂ B ₄ O ₇ ·10H ₂ O 0.23g/L，ZnSO ₄ ·7H ₂ O5.25 g/L, formulated with 0.1mol/L HCl.

Corresponding antibiotics are added into the culture medium according to the requirement, and the addition amount of the antibiotics is as follows: the final kanamycin concentration was 50. Mu.g/mL and the final ampicillin concentration was 120. Mu.g/mL.

The method for fermenting the alpha-ketoisovaleric acid by shaking bottles comprises the following steps:

(1) Pre-culture of strains

The recombinant strain was streaked on LB plate medium and cultured at 37℃for 24 hours. The single colony of the flat plate is inoculated in LB liquid culture medium and cultured for 10 hours at 37 ℃ and 200r/min to obtain pre-culture bacterial liquid.

(2) Fermentation culture

50mL of the bacterial liquid prepared in the example (1) was inoculated into 50mL of M9-2 medium containing 36g/L glucose, and shake culture was performed at 37℃with a 200r/min shaker. Thallus OD ₆₀₀ When the value reaches 2.5, the added IPTG inducer reaches a final concentration of 0.4mmol/L, and the shake flask is placed at 30 ℃ and shake-induced by a shaking table at 200r/min until the culture time reaches 36h. During this period, the pH was measured every 4 hours with pH paper and the pH of the broth was adjusted to neutral with ammonia.

The method for synthesizing the alpha-ketoisovaleric acid by fermenting in a 5L fermentation tank comprises the following steps:

single colony is selected to 30mL LB culture medium, cultured for 8h at 37 ℃ at 200r/min, and then the culture solution is inoculated to 50mL seed solution culture medium, and cultured for 12h at 37 ℃ at 200 r/min. 100mL of the seed solution was inoculated into a 5L fermenter containing 2L of M9-4 fermentation medium, and the initial glucose concentration was 30g/L. In the growth stage of thallus, the temperature is controlled to be 37 ℃, and the temperature is regulatedThe stirring speed and the ventilation quantity control the dissolved oxygen concentration to be more than or equal to 30 percent. When OD is ₆₀₀ When the value reaches 20, the alpha-ketoisovalerate synthesis stage is carried out, IPTG with the final concentration of 0.8mM is added for induction, and the temperature is reduced to 30 ℃ to synthesize the alpha-ketoisovalerate. Ammonia is used to control pH value to 7 in the whole fermentation stage.

The method for measuring the alpha-ketoisovaleric acid comprises the following steps:

alpha-ketoisovaleric acid was detected by High Performance Liquid Chromatography (HPLC). The detection conditions are as follows: chromatographic column Prevail Organic Acid (250 mm. Times.4.6 mm,5 μm) with mobile phase KH at 25mmol/L at pH 2.5 ₂ PO ₄ The flow rate of the solution is 1mL/min, the column temperature is 40 ℃, the wavelength of an ultraviolet detector is 210nm, and the sample injection amount is 10 mu L.

Construction of recombinant plasmids by the Gibbsen Assembly method: see Gibson et al Enzymatic assembly of DNA molecules up to several hundred Kilobases Nat. Methods 2009,6 (5): 343-5 for specific procedures.

Modifying a chromosome gene by using a Red recombination method: see K.A. Datsenko et al, one-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.Proc.Natl.Acad.Sci.U.S.A, 2000,97,6640-6645.

The enzyme activity determination method comprises the following steps:

enzyme activity determination: the method adopts a biological engineering (Shanghai) kit and refers to a "Pyruvate Dehydrogenase (PDH) activity detection kit colorimetric method" for determination.

Example 1: starting strain 050T4/pC _T SD _T Two-stage fermentation production of alpha-ketoisovaleric acid

Strain 050T4/pC _T SD _T (disclosed in paper, "method for preparing alpha-ketoisovalerate by metabolic engineering Escherichia coli"): plasmid pC for electrotransduceing strain 050T4 with the overexpression of the genes T7 RNAP and pntAB encoding T7 RNA polymerase, from which formate, acetate, ethanol, lactate, succinate, valine and/or other key enzymes in the alpha-ketoisovalerate pathway were knocked out _T SD _T Transferring into the strain, fermenting in a 5L fermentation tank, wherein the specific steps are as follows:

recombinant E.coli 050T4/pC was selected _T SD _T Single colonyIn 30mL LB medium, the culture medium is cultivated for 8 hours at 37 ℃ and 200r/min, and then the culture medium is inoculated in 50mL seed liquid medium, and cultivated for 12 hours at 37 ℃ and 200 r/min. 100mL of the seed solution was inoculated into a 5L fermenter containing 2L of M9-4 fermentation medium, and the initial glucose concentration was 30g/L. In the growth stage of the thalli, the temperature is controlled to be 37 ℃, and the stirring rotation speed and the ventilation quantity are regulated to control the dissolved oxygen concentration to be more than or equal to 30 percent. When OD is ₆₀₀ When the value reaches 20, the alpha-ketoisovalerate synthesis stage is carried out, IPTG with the final concentration of 0.8mM is added for induction, and the temperature is reduced to 30 ℃ to synthesize the alpha-ketoisovalerate. Ammonia is used to control pH value to 7 in the whole fermentation stage.

As shown in FIG. 1 (A), the yield of α -ketoisovaleric acid was 45g/L, whereas the accumulation of isobutanol was 13g/L. As shown in fig. 1 (B), the growth rate of escherichia coli and the final cell concentration were significantly reduced with the increase of the isobutanol content. The addition of isobutanol above 1.5g/L can significantly inhibit cell growth. The results indicate that isobutanol is potentially toxic to microorganisms and its accumulation can significantly reduce cell growth performance, ultimately reducing the yield and conversion of the desired product. Therefore, there is a need to further reduce strain 050T4/pC _T SD _T Is a byproduct of isobutanol accumulation level.

Example 2: construction and expression of acetolactate synthase mutants

Acetolactate synthase (AlsS) is a key enzyme in the synthesis pathway of α -ketoisovalerate, which, in addition to synthesizing acetolactate from two pyruvate molecules, catalyzes the conversion of α -ketoisovalerate to isobutyraldehyde, further reducing to isobutanol using the dehydrogenase in the cell. The present example modifies plasmid pC _T SD _T The AlsS enzyme coding gene constructs Q424S, Q487S and Q488S mutants to reduce the accumulation of isobutanol, and the specific steps are as follows:

with pC _T SD _T The plasmid is used as a template, the P1/P2, P3/P4 and P5/P6 are respectively used as an upstream primer and a downstream primer (the primers are shown in the table 1) to be amplified by a whole plasmid PCR, the whole plasmid is transformed into E.coli DH5 alpha competent cells, the transformant is selected to carry out colony PCR verification, and the recombinant plasmid pC respectively carrying SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 is obtained after sequencing _T SD _T Q424S、pC _T SD _T Q487S、pCT _S D _T Q488S。

TABLE 1 primers used for Point mutation

Recombinant plasmid pC to be constructed and obtained _T SD _T Q424S、pC _T SD _T Q487S、pCT _S D _T Q488S was transformed into 050T4 strain, respectively, to obtain recombinant strain. Culturing the obtained recombinant strain in M9-2 medium at 37deg.C and 200r/min respectively, and growing to OD ₆₀₀ At 2.5, the recombinant strain expressing the Q424S and Q487S mutants was fermented for 36 hours to the end of sugar consumption, with 0.05g/L and 0.25g/L respectively for isobutanol accumulation, and 1.3g/L for wild type, by fermentation with 0.4mM IPTG and lowering the temperature to 30℃for 200 r/min. It can be seen that the mutant expressing strain had significantly lower levels of isobutanol accumulation than the wild type AlsS (figure 2). The accumulation of isobutanol of the Q488S mutant is 0.3g/L, which is almost the same as that of the Q487S mutant, and the catalytic functions of the two amino acid sites are similar. And strain 050T4/pC _T SD _T In contrast, strain 050T4/pC _T SD _T The accumulation level of isobutanol of Q487S was reduced to 19.2%, while the yield of alpha-ketoisovaleric acid was 14.87g/L, which was reduced by 22.5%.

Example 3: alsS ribosome binding site sequence optimisation

In order to minimize the accumulation of isobutanol and maximize the production of alpha-ketoisovalerate, strain 050T4/pC of example 2 _T SD _T On the basis of Q487S, the RBS sequence of AlsSQ487S was modified. RBS calculator (version 2.1) software was used to design RBS sequences of different intensities. With pC _T SD _T The Q487S plasmid is used as a template, and the P7/P8, P9/P10 and P11/P12 are respectively used as an upstream primer and a downstream primer (shown in Table 2) for full plasmid PCR amplification, and RBS sequences positioned at the upstream of AlsS enzyme are respectively modifiedFor a strength of 6.8X10 ⁴ 、41.7×10 ⁴ 、55.3×10 ⁴ RBS sequences of Au, recombinant plasmid pC was obtained, respectively _T SD _T Q487S-RBS6.8、pC _T SD _T Q487S-RBS42、pC _T SD _T Q487S-RBS55。

TABLE 2 primers used for RBS optimization

The recombinant plasmids obtained by construction are respectively transformed into strains 050T4, which are 050T4/pC respectively _T SD _T Q487S-RBS6.8、050T4/pC _T SD _T Q487S-RBS42、050T4/pC _T SD _T Q487S-RBS55. Picking single colony into 5mL LB culture medium, culturing at 37deg.C at 200r/min for 8 hr, transferring into 50mL M9-2 culture medium according to 2% inoculum size, culturing at 37deg.C at 200r/min to OD ₆₀₀ 2.5, then 0.4mM IPTG is added for induction, the temperature is changed to 30 ℃ and the fermentation is carried out at 200r/min, and the fermentation is carried out for 36 hours until the sugar consumption is finished. Every 4 hours, the pH was adjusted to 7.0 with ammonia.

Strain 050T4/pC _T SD _T The yield of the synthesized alpha-ketoisovaleric acid by Q487S-RBS55 (highest RBS intensity 55.3X104 Au) is 19.2g/L at most, which is the same as that of strain 050T4/pC _T SD _T The yield of (a) was kept at the same level (fig. 3). Although, strain 050T4/pC _T SD _T The accumulation of isobutanol in Q487S-RBS55 increased to 0.6g/L, which was also greater than that of strain 050T4/pC _T SD _T 56.3% lower, indicating successful reduction of isobutanol accumulation without affecting α -ketoisovalerate synthesis.

Example 4: weakening tricarboxylic acid cycle

1) AceF-added degradation tag DAS+4 on chromosome

The tricarboxylic acid cycle competes with the common precursor substance pyruvate with the alpha-ketoisovalerate anabolic pathway. In order to increase the conversion rate of the synthesis of alpha-ketoisovalerate, the present example further weakens the activity of the key enzyme (PDH) of the TCA cycle by fusing the das+4 degradation tag at the C-terminal of the AceF subunit of PDH enzyme on the chromosome of strain 050T4, reduces the amount of carbon source consumed for growing the bacterial cells, and promotes the synthesis of alpha-ketoisovalerate, and the primer sequences used are shown in table 3.

The pACYC-Kan-DAS+4 plasmid shown in SEQ ID No.9 is used as a template, a P13+P14 primer is used for PCR amplification of a target gene fragment, and the target gene fragment is integrated at an AceF gene stop codon TAA on a strain B0016-050T4 chromosome. The transformant is verified by PCR with a P15+P16 primer, the length of the gene fragment after wild type amplification is 620bp, and the length of the gene fragment after integration reaction is 740bp, which indicates that the AceF gene is knocked out successfully, and the correct strain is verified to be 050TY.

TABLE 3 primers used for chromosomal Gene engineering

The PDH enzyme activity of the constructed strain was measured. The method comprises the following specific steps: pCTSDTQ487S-RBS55 was transformed into strains 050T4 and 050TY, respectively, 050T4/pCTSDTQ487S-RBS55 and 050TY/pCTSDTQ487S-RBS55. Picking single colony into 5mL LB culture medium, culturing at 37deg.C at 200r/min for 8 hr, transferring into 50mL M9-2 culture medium with 2% inoculum size, culturing at 37deg.C at 200r/min to OD ₆₀₀ PDH enzyme activity was measured by adding 0.4mM IPTG to induce, culturing at 30℃for 12 hours at 200r/min, sampling. The results showed that the Pdh activity of 050TY strain was significantly lower than 050T4, indicating that das+4 degradation tag was effective (fig. 4A).

Further verifying the influence of strain transformation on the fermentation synthesis of alpha-ketoisovalerate. The method comprises the following specific steps: the plasmid pCTSDTQ487S-RBS55 constructed in example 3 was transformed into strain 050TY, 050TY/pCTSDTQ487S-RBS55. Picking single colony into 5mL LB culture medium, culturing at 37deg.C at 200r/min for 8 hr, transferring into 50mL M9-2 culture medium according to 2% inoculum size, culturing at 37deg.C at 200r/min to OD ₆₀₀ 2.5, then 0.4mM IPTG was added for induction, and the mixture was incubated at 30℃and fermented at 50, 100, 150 and 200r/min, respectively, until the fermentation was completed, and the pH was maintained at 7.0 with ammonia water at 4h intervals. The influence on the synthesis of alpha-ketoisovaleric acid after adding degradation label DAS+4 is compared at different rotating speeds.

Fermentation results at different rotational speeds show that the strain is similar to the strain 050T4/pC _T SD _T Compared with Q487S-RBS55, the strain 050TY/pC with weakened TCA cycle _T SD _T Q487S-RBS55 was fermented at 100rpm until the end of consumption of sugar, with a yield of 19.79g/L for 36h, a total conversion of 0.93mol/mol glucose, a conversion of 1mol/mol glucose after the second stage induction, and a significant increase in both total and second stage conversion (FIG. 4B). Further optimizing the turning time of the bacterial growth stage and the alpha-ketoisovalerate synthesis stage, showing that the bacterial growth to OD ₆₀₀ For 4, the yield was 22.1g/L at maximum, the total conversion increased to 0.94mol/mol glucose (approaching 1mol/mol glucose from the theoretical conversion) and the second stage conversion reached 1mol/mol glucose (FIG. 4C).

The strain 050TY/pCTSDTQ487S-RBS55 has the highest yield and conversion rate under the microaerophilic condition, and the possible reasons are that after weakening the TCA cycle, the synthesis amount of NADH is reduced, less oxygen is needed as a final electron acceptor to realize the cycle of reduced coenzyme, the redox force is balanced, and the synthesis path of alpha-ketoisovalerate is smoother; meanwhile, the conversion rate of alpha-ketoisovalerate is obviously improved by inhibiting competitive metabolic pathways.

Example 5: bacterial strain 050TY/pC _T SD _T Fermenting Q487S-RBS55 in 5L fermenter

The two-stage fermentation process is adopted to synthesize alpha-ketoisovaleric acid. The first stage is an aerobic mode, and bacterial growth is performed. Amplifying according to the optimal culture condition of shake flask, that is, the concentration of thallus grows to OD ₆₀₀ And when the fermentation liquid is 20 degrees, the fermentation liquid is converted into second-stage microaerobic fermentation. The method comprises the following specific steps:

the first stage: inoculating 050TY/pCTSDTQ487S-RBS55 seed solution cultured at 37deg.C and 200r/min for 8 hr into M9-4 culture medium at a ratio of 5% (v/v), and culturing at 37deg.C with dissolved oxygen concentration of 30% or more to OD ₆₀₀ Reaching 20, and entering a second stage;

and a second stage: adding 0.8mM IPTG for induction, cooling to 30deg.C, regulating ventilation volume to 1L/min, and controlling rotation speed of stirring paddle to 400r/min to maintain dissolved oxygen below 15%.

After two-stage fermentation for 26h, alpha-ketoneThe yield of isovaleric acid reaches 55.8g/L, the conversion rate of the whole fermentation process reaches 0.85mol/mol glucose, the conversion rate of the second stage is increased to 0.99mol/mol glucose and is close to 1mol/mol theoretical glucose, and the volume production strength is 2.14g/L h. And strain 050T4/pC _T SD _T In contrast, strain 050TY/pC _T SD _T The yield, volume production intensity and production intensity of the second stage of Q487S-RBS55 were increased by 30%, 20.2% and 45.4%, respectively. The accumulation of isobutanol is obviously reduced to 1.51g/L, and the yield is only 0.04mol/mol glucose, which indicates that AlsS modification effectively reduces the accumulation of by-product isobutanol. At the same time, strain 050TY/pC _T SD _T Cell density ratio strain 050T4/pC of Q487S-RBS55 _T SD _T 13.9% lower, indicating that weakening the tricarboxylic acid cycle reduces overgrowth of the cells, helping to increase the conversion of α -ketoisovalerate.

TABLE 4 comparison of fermenter fermentation results

Use of strain 050TY/pC _T SD _T The yield, volume strength and total conversion of the fermentative synthesis of alpha-ketoisovalerate by Q487S-RBS55 are 2.6, 4 and 1.8 times higher than those reported in the literature (Applied and Environmental Microbiology,2010,76 (24): 8053-8061). This is also the highest level of alpha-ketoisovalerate synthesis reported to date.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Gln Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg

210 215 220

Pro Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu

225 230 235 240

Pro Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu

245 250 255

Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly

260 265 270

Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp

275 280 285

Pro Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr

290 295 300

Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln

305 310 315 320

Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile

325 330 335

Glu His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile

340 345 350

Leu Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala

355 360 365

Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu

370 375 380

Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser

385 390 395 400

His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr

405 410 415

Leu Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp

420 425 430

Ala Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val

435 440 445

Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala

450 455 460

Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr

465 470 475 480

Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser

485 490 495

Ala Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe

500 505 510

Gly Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val

515 520 525

Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro

530 535 540

Val Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys

545 550 555 560

Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu

565 570

<210> 2

<211> 19

<212> DNA

<213> artificial sequence

<400> 2

aaggagatat accatggat 19

<210> 3

<211> 27

<212> DNA

<213> artificial sequence

<400> 3

gggtatccaa aaccaaagga ggtttaa 27

<210> 4

<211> 29

<212> DNA

<213> artificial sequence

<400> 4

gctcgagaaa tctactaagg aggctatta 29

<210> 5

<211> 48

<212> DNA

<213> artificial sequence

<400> 5

gcagctaatg atgaaaatta cagcgaaaat tacgcagatg ccagctaa 48

<210> 6

<211> 1716

<212> DNA

<213> artificial sequence

<400> 6

atgttaacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60

gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120

attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180

gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240

gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300

actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360

cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420

gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480

gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540

aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600

atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660

aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720

ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780

tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840

gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900

ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960

cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020

gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080

catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140

gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200

cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260

aacggtatgt ctacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320

ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380

ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440

tatgacatgg ttgcattcca gcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500

ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560

tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620

atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680

gaattcgggg aactcatgaa aacgaaagct ctctag 1716

<210> 7

<211> 1716

<212> DNA

<213> artificial sequence

<400> 7

atgttaacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60

gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120

attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180

gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240

gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300

actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360

cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420

gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480

gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540

aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600

atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660

aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720

ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780

tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840

gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900

ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960

cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020

gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080

catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140

gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200

cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260

aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320

ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380

ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440

tatgacatgg ttgcattctc tcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500

ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560

tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620

atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680

gaattcgggg aactcatgaa aacgaaagct ctctag 1716

<210> 8

<211> 1716

<212> DNA

<213> artificial sequence

<400> 8

atgttaacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60

gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120

attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180

gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240

gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300

actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360

cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420

gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480

gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540

aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600

atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660

aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720

ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780

tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840

gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900

ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960

cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020

gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080

catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140

gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200

cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260

aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320

ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380

ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440

tatgacatgg ttgcattcca gtctttgaaa aaatataacc gtacatctgc ggtcgatttc 1500

ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560

tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620

atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680

gaattcgggg aactcatgaa aacgaaagct ctctag 1716

<210> 9

<211> 5152

<212> DNA

<213> artificial sequence

<400> 9

attccgggca gctaatgatg aaaattacag cgaaaattac gcagatgcca gctaaggatc 60

cgtcgacctg cagttcgaag ttcctattct ctagaaagta taggaacttc agagcgcttt 120

tgaagctcac gctgccgcaa gcactcaggg cgcaagggct gctaaaggaa gcggaacacg 180

tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc 240

tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg 300

cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg 360

ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg 420

atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc gtttcgcatg 480

attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 540

tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 600

caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 660

gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 720

gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 780

ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 840

cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 900

gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 960

catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 1020

gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 1080

cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 1140

gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 1200

gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 1260

gagttcttct aataagggga tcttgaagtt cctattccga agttcctatt ctctagaaag 1320

tataggaact tcgaagcagc tccagcctac acatttccta atgcaggagt cgcataaggg 1380

agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat 1440

agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg 1500

tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc 1560

acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc 1620

ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct 1680

ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc 1740

aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag 1800

cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg 1860

atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg 1920

atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac 1980

tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat 2040

taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc 2100

aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa 2160

ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga 2220

tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct 2280

cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca 2340

tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc 2400

agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca 2460

ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 2520

tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt 2580

tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc 2640

agctccttcc ggtgggcgcg gggcatgact aacatgagaa ttacaactta tatcgtatgg 2700

ggctgacttc aggtgctaca tttgaagaga taaattgcac tgaaatctag aaatatttta 2760

tctgattaat aagatgatct tcttgagatc gttttggtct gcgcgtaatc tcttgctctg 2820

aaaacgaaaa aaccgccttg cagggcggtt tttcgaaggt tctctgagct accaactctt 2880

tgaaccgagg taactggctt ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc 2940

cttaaccggc gcatgacttc aagactaact cctctaaatc aattaccagt ggctgctgcc 3000

agtggtgctt ttgcatgtct ttccgggttg gactcaagac gatagttacc ggataaggcg 3060

cagcggtcgg actgaacggg gggttcgtgc atacagtcca gcttggagcg aactgcctac 3120

ccggaactga gtgtcaggcg tggaatgaga caaacgcggc cataacagcg gaatgacacc 3180

ggtaaaccga aaggcaggaa caggagagcg cacgagggag ccgccagggg aaacgcctgg 3240

tatctttata gtcctgtcgg gtttcgccac cactgatttg agcgtcagat ttcgtgatgc 3300

ttgtcagggg ggcggagcct atggaaaaac ggctttgccg cggccctctc acttccctgt 3360

taagtatctt cctggcatct tccaggaaat ctccgccccg ttcgtaagcc atttccgctc 3420

gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga ggaagcggaa tatatcctgt 3480

atcacatatt ctgctgacgc accggtgcag ccttttttct cctgccacat gaagcacttc 3540

actgacaccc tcatcagtgc caacatagta agccagtata cactccgcta gcgctgatgt 3600

ccggcggtgc ttttgccgtt acgcaccacc ccgtcagtag ctgaacagga gggacagctg 3660

atagaaacag aagccactgg agcacctcaa aaacaccatc atacactaaa tcagtaagtt 3720

ggcagcatca cccgacgcac tttgcgccga ataaatacct gtgacggaag atcacttcgc 3780

agaataaata aatcctggtg tccctgttga taccgggaag ccctgggcca acttttggcg 3840

aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc 3900

actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag ctaaaatgga 3960

gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt 4020

tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac 4080

ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat 4140

tcttgcccgc ctgatgaatg ctcatccgga gttccgtatg gcaatgaaag acggtgagct 4200

ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt 4260

ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca 4320

agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat 4380

gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa 4440

tatggacaac ttcttcgccc ccgttttcac tatgggcaaa tattatacgc aaggcgacaa 4500

ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc tgtgatggct tccatgtcgg 4560

cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaattttt 4620

ttaaggcagt tattggtgcc cttaaacgcc tggtgctacg cctgaataag tgataataag 4680

cggatgaatg gcagaaattc gaaagcaaat tcgacccggt cgtcggttca gggcagggtc 4740

gttaaatagc cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg 4800

tgaccgtgtg cttctcaaat gcctgaggtt tcagcaaaaa acccctcaag acccgtttag 4860

aggccccaag gggttatgct agttattgct cagcggtggc agcagcctag gttaattaac 4920

gtgcttcctt tatgtgaaaa tctaataatg tatatcaaat gcatcttata aaaataccct 4980

tgcattgtaa atggatcttc tctgctttac gttatggagg taacaacgtg aaaaatctgc 5040

atcacaaagc tgaaaagaaa tccgttgaaa ttcgtcaggc tctcgttcag gaaaccctta 5100

tctgacgcat aggtaatcgt ttgcgtaaaa acctttgtca agacctgtta tc 5152

Claims

1. Alpha-ketoneRecombinant escherichia coli with improved isovaleric acid conversion efficiency takes escherichia coli B0016-050T4 as a host and pC _T SD _T As a vector, the T7 promoter is utilized to overexpress key enzymes of the synthesis pathway of alpha-ketoisovalerate, and the recombinant escherichia coli is modified as follows:

(a) Expressing acetolactate synthase AlsS mutant; the heterologous acetolactate synthase takes an amino acid sequence shown as SEQ ID NO.1 as a starting sequence, and on the basis of the SEQ ID NO.1, the 487 th glutamine is mutated into serine;

(b) Replacing the RBS sequence of acetolactate synthase with the sequence shown in SEQ ID NO. 4;

(c) Fusing a DAS+4 degradation tag at a C-terminal termination codon TAA of a pyruvate dehydrogenase enzyme AceF subunit on a chromosome of the strain 050T 4; the nucleotide sequence of the DAS+4 degradation tag is shown as SEQ ID NO. 5.

2. A method for producing α -ketoisovalerate, characterized in that the recombinant escherichia coli of claim 1 is fermented in a medium containing glucose; the method adopts two-stage fermentation:

the first stage: the temperature is controlled to be 35-37 ℃, and the dissolved oxygen concentration is controlled to be more than or equal to 30%.

And a second stage: when OD is ₆₀₀ And after the value reaches 20, adding IPTG into the culture system, cooling to 28-32 ℃, and controlling the dissolved oxygen concentration to be less than 15% to continue fermentation.

3. A method according to claim 2, wherein the induction is carried out in the second stage by adding IPTG at a final concentration of 0.7-0.9 mM.

4. A method according to claim 2 or 3, characterized in that the medium used for fermentation is M9-4 medium, in g/L: glucose 30, yeast powder 4, KH ₂ PO ₄ 13.5 peptone 4, citric acid monohydrate 1.7, mgSO ₄ ·7H ₂ O 0.49，(NH4) ₂ HPO ₄ 4 and 0.1 percent of microelement liquid; the microelement liquid contains: mnSO ₄ ·4H ₂ O 0.5 g/L，FeSO ₄ ·7H ₂ O 10.0 g/L，CaCl ₂ 2.0 g/L，(NH ₄ ) ₆ Mo ₇ O ₂₄ 0.1 g/L，CuSO ₄ ·5H ₂ O 3.0 g/L，Na ₂ B ₄ O ₇ ·10H ₂ O 0.23 g/L，ZnSO ₄ ·7H ₂ O 5.25 g/L。