CN114480235A - Method for preparing alpha-ketoisovalerate through fermentation of escherichia coli modified by metabolic engineering - Google Patents

Abstract

The invention discloses a method for preparing alpha-ketoisovalerate through fermentation of escherichia coli modified by metabolic engineering, and belongs to the field of bioengineering. The invention constructs a recombinant strain of escherichia coli, and modifies and optimizes the host strain to obtain the recombinant strain capable of efficiently producing alpha-ketoisovalerate by a fermentation method. The recombinant strain can use cheap glucose as a substrate to ferment and generate alpha-ketoisovalerate with higher added value, and the yield of the alpha-ketoisovalerate can reach 55.8g/L after fermentation for 26 hours.

Description

Method for preparing alpha-ketoisovalerate through fermentation of escherichia coli modified by metabolic engineering

Technical Field

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

Background

Alpha-ketoisovalerate is a precursor substance for synthesizing amino acids such as valine, leucine and isoleucine, and participates in the metabolic process of a living body. In the aspect of medical treatment, the alpha-ketonic acid tablet is one of the main components of the alpha-ketonic acid tablet and can be used for treating chronic uremia; in the feed aspect, it can be used for stimulating the muscle growth of livestock. Therefore, the alpha-ketoisovalerate has wide application prospects 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 using 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 with an α -ketoisovalerate production of 21.8. + -. 3.2g/L and a conversion of 0.47. + -. 0.05mol/mol glucose. In 2020, lyattine over-expresses key enzymes IlvC, AlsS and IlvD in the synthesis process of alpha-ketoisovalerate, simultaneously knocks out catabolic pathway ilvE and competitive metabolism TouA, balances reducing power, and finally synthesizes 19.2g/L of alpha-ketoisovalerate in a shake flask in escherichia coli through fermentation, wherein the conversion rate reaches 0.8mol/mol and is higher than that of corynebacterium glutamicum. The above research level is still far from industrial application. Further inhibiting competing metabolic pathways, such as: and the tricarboxylic acid cycle (TCA cycle) is expected to further improve the yield, the conversion rate and the production intensity of the alpha-ketoisovalerate.

Acetolactate synthase (AlsS) can decarboxylate pyruvate to acetolactate to further synthesize α -ketoisovalerate, but the enzyme also has the activity of catalyzing α -ketoisovalerate to synthesize isobutyraldehyde, which is further reduced to isobutanol. Researchers have primarily utilized this non-specific enzymatic activity for the fermentative synthesis of isobutanol, α -ketoisovalerate as a major byproduct of the fermentation process. However, in the alpha-ketoisovalerate fermentation synthesis process, when the concentration of the isobutanol byproduct is high, the conversion rate of the alpha-ketoisovalerate is reduced, the growth performance of thalli is inhibited, and finally the synthesis level of the alpha-ketoisovalerate is seriously reduced. The enzyme is modified by Shota Atsumi (Applied and Environmental Microbiology,2009,75(19):6306-6311), so that the enzyme activity for catalyzing and synthesizing isobutyraldehyde can be reduced, the enzyme activity for synthesizing alpha-ketoisovalerate is not obviously influenced, and the specificity of a substrate is improved. However, no report on the enzymatic synthesis of alpha-ketoisovalerate by using the mutant is available at present.

The Escherichia coli has the characteristics of clear genetic background, easy genetic operation, high growth rate, low nutritional 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 α -ketoisovalerate in E.coli. The construction of the recombinant escherichia coli synthesized by the alpha-ketoisovalerate and the improvement of the recombinant strains and the fermentation process thereof are 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 alpha-ketoisovalerate conversion efficiency, which can ferment and produce alpha-ketoisovalerate by taking glucose as a cheap substrate as a raw material; the recombinant Escherichia coli is improved by at least one of (a) to (d):

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

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

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

In one embodiment, the heterologous acetolactate synthase is mutated at least one of positions 424, 487, 488 according to SEQ ID No. 1.

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

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

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

In one embodiment, the recombinant Escherichia coli is prepared by using Escherichia coli B0016-050T4 as a host, and Escherichia coli B0016-050T4 is a prior art, and is disclosed in the paper "metabolic engineering Escherichia coli producing alpha-ketoisovalerate", wherein the strain deletes a byproduct acetate anabolism pathway encoding gene ackA-pta, lactate anabolism pathway encoding gene ldhA, ethanol anabolism pathway encoding gene adhE, succinate anabolism pathway encoding gene frdA and valine synthetic pathway editing gene ilvE on a chromosome, expresses T7 RNA polymerase encoding gene T7 RNAP, and enhances the expression of NADPH coenzyme cyclic metabolic pathway encoding gene pntAB. The ackA-pta has Gene IDs 946775 and 946778 at NCBI; the ldhA at NCBI has a Gene ID of 946315; the adhE has a Gene ID of 945837 at NCBI; the frdA has Gene ID 948667 at NCBI; the ilvE at NCBI has a Gene ID of 948278; the T7 RNAP accession number M38308.1 at NCBI and the pntAB Gene ID 946628 and 946144 at NCBI.

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

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

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

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

the first stage is as follows: in the thallus growth stage, the temperature is controlled at 37 ℃, the stirring speed and the ventilation volume are adjusted, and the dissolved oxygen concentration is controlled to be more than or equal to 30 percent.

And a second stage: when OD is reached₆₀₀When the value reaches 20, the synthesis stage of alpha-ketoisovalerate is started, 0.8mM IPTG 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 further comprises culturing to OD₆₀₀The 2.5 bacterial solution was induced with IPTG.

In one embodiment, the strain is grown aerobically in a fermentor to OD₆₀₀The value reached 20 and induction was performed by addition of IPTG.

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

Has the advantages that: the invention constructs a recombinant escherichia coli strain for efficiently synthesizing alpha-ketoisovalerate, and modifies and optimizes the host strain to obtain a recombinant bacterium capable of producing the alpha-ketoisovalerate by a fermentation method and the fermentation method. The recombinant strain can use cheap glucose as a substrate, alpha-ketoisovalerate with higher added value is generated by fermentation, the yield of the alpha-ketoisovalerate can reach 55.8g/L after fermentation for 26 hours, 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 increased to 0.99mol/mol glucose, the theoretical glucose is close to 1mol/mol 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_TSD_TThe yield of alpha-ketoisovalerate and the influence of isobutanol in a 5L fermentation tank; (A) strain 050T4/pC_TSD_TThe yield of the fermented alpha-ketoisovalerate; (B) isobutanol concentration gradient growth curve.

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

FIG. 3 is a graph showing the effect of RBS sequence optimization of AlsS on α -ketoisovalerate synthesis and isobutanol accumulation.

FIG. 4 is a graph of attenuating the effect of tricarboxylic acid cycles on alpha-ketoisovalerate synthesis; (A) comparison of PDH enzyme activities; (B) optimizing the rotating speed; (C) the two-stage turning time is optimized.

FIG. 5 shows strain 050TY/pC_TSD_TThe alpha-ketoisovalerate is synthesized in a Q487S-RBS 555L fermentation tank.

Detailed Description

Culture medium:

LB liquid medium: 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, adding deionized water in the beaker to a constant volume of 1L, and finally performing moist heat sterilization at 121 ℃ for 20min in a high-pressure steam sterilization pot.

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

M9-2 medium (g/L): glucose 36, yeast powder 5, KH₂PO₄ 3，Na₂HPO₄ 6，NaCl 0.3，NH₄Cl 1，MgSO₄·7H₂O0.49 and trace element liquid 0.1% (v/v).

M9-4 medium (g/L): 30 portions of glucose, 4 portions of yeast powder and KH₂PO₄13.5 peptone 4, citric acid monohydrate 1.7, MgSO₄·7H₂O 0.49，(NH4)₂HPO₄And 4, 0.1% (v/v) of trace element liquid.

Trace element liquid: MnSO₄·4H₂O 0.5g/L，FeSO₄·7H₂O 10.0g/L，CaCl₂ 2.0g/L，(NH₄)Mo₇O₂₄ 0.1g/L，CuSO₄·5H₂O 3.0g/L，Na₂B₄O₇·10H₂O 0.23g/L，ZnSO₄·7H₂O5.25 g/L, prepared 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: kanamycin to a final concentration of 50. mu.g/mL, ampicillin to a final concentration of 120. mu.g/mL.

The fermentation method of the alpha-ketoisovalerate in a shake flask comprises the following steps:

(1) pre-culture of the Strain

The recombinant strain was streaked on an LB plate medium and cultured at 37 ℃ for 24 hours. And inoculating the plate single colony to an LB liquid culture medium, and culturing at 37 ℃ at 200r/min for 10h to obtain a pre-culture bacterial liquid.

(2) Fermentation culture

50mL of M9-2 medium containing 36g/L glucose was inoculated with 2mL of the bacterial suspension prepared in example (1) and shake-cultured at 37 ℃ and 200r/min with a shaker. Bacterial body OD₆₀₀When the value reached 2.5, IPTG inducer was added to a final concentration of 0.4mmol/L, and the flask was placed in a 30-liter flaskShaking and inducing the culture at the temperature of 36h by a shaking table at 200 r/min. During the period, pH value is measured by pH test paper every 4h, and the pH value of the fermentation liquor is adjusted to be neutral by ammonia water.

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

picking single bacterial colony into 30mL LB culture medium, culturing for 8h at 37 ℃ and 200r/min, inoculating the culture solution into 50mL seed liquid culture medium, and culturing for 12h at 37 ℃ and 200 r/min. 100mL of the seed solution was inoculated into a 5L fermentor containing 2L M9-4 fermentation medium at an initial glucose concentration of 30 g/L. In the thallus growth stage, the temperature is controlled at 37 ℃, the stirring speed and the ventilation volume are adjusted, and the dissolved oxygen concentration is controlled to be more than or equal to 30 percent. When OD is reached₆₀₀When the value reaches 20, the synthesis stage of the alpha-ketoisovalerate is started, the final concentration of the alpha-ketoisovalerate is added to be induced by 0.8mM IPTG, and meanwhile, the temperature is reduced to 30 ℃ to synthesize the alpha-ketoisovalerate. The pH value is controlled to 7 by ammonia water in the whole fermentation stage.

The method for measuring the alpha-ketoisovalerate comprises the following steps:

detecting alpha-ketoisovalerate by High Performance Liquid Chromatography (HPLC). The detection conditions are as follows: column of Prevail Organic Acid (250 mm. times.4.6 mm, 5 μm) with mobile phase KH at 25mmol/L and pH 2.5₂PO₄The 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.

Constructing a recombinant plasmid by a Gibbson assembly mode: see Gibson et al, enzymic assembly of DNA molecules up to a partial and cloned killbased, Nat. methods,2009,6(5): 343-5.

Transforming chromosome genes 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-.

The enzyme activity determination method comprises the following steps:

and (3) enzyme activity determination: the determination is carried out by adopting a biological engineering (Shanghai) kit according to a colorimetric method of Pyruvate Dehydrogenase (PDH) activity detection kit.

Example 1: starting strain 050T4/pC_TSD_TTwo-stage fermentation production of alpha-ketoisovalerate

Strain 050T4/pC_TSD_T(disclosed in a paper of 'a method for preparing alpha-ketoisovalerate by transforming escherichia coli through metabolic engineering)') fermentation on a tank: the strain 050T4 which is knocked out of formate, acetate, ethanol, lactic acid, succinic acid and valine and integrates T7 RNA polymerase coding genes T7 RNAP and pntAB overexpression electrically transforms plasmid pC of key enzyme overexpressed in alpha-ketoisovalerate pathway_TSD_TTransferring the strain into a 5L fermentation tank for fermentation, and specifically comprises the following steps:

selecting recombinant Escherichia coli 050T4/pC_TSD_TThe single colony is cultured in 30mL LB culture medium at 37 ℃ and 200r/min for 8h, and then the culture solution is inoculated in 50mL seed liquid culture medium at 37 ℃ and 200r/min for 12 h. 100mL of the seed solution was inoculated into a 5L fermentor containing 2L M9-4 fermentation medium at an initial glucose concentration of 30 g/L. In the thallus growth stage, the temperature is controlled at 37 ℃, the stirring speed and the ventilation volume are adjusted, and the dissolved oxygen concentration is controlled to be more than or equal to 30 percent. When OD is reached₆₀₀When the value reaches 20, the synthesis stage of the alpha-ketoisovalerate is started, the final concentration of the alpha-ketoisovalerate is added to be induced by 0.8mM IPTG, and meanwhile, the temperature is reduced to 30 ℃ to synthesize the alpha-ketoisovalerate. The pH value is controlled to 7 by ammonia water in the whole fermentation stage.

As shown in FIG. 1(A), the yield of alpha-ketoisovalerate reached 45g/L, whereas the accumulation of isobutanol reached 13 g/L. As shown in fig. 1(B), the growth rate of escherichia coli and the final obtained cell concentration significantly decreased as the isobutanol content increased. Addition of isobutanol above 1.5g/L can significantly inhibit cell growth. This result indicates 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, it is desirable to further reduce the strain 050T4/pC_TSD_TAccumulation level of isobutanol as a by-product of (a).

Example 2: construction and expression of acetolactate synthase mutants

Acetolactate synthase (AlsS) is a key enzyme in the synthesis pathway of alpha-ketoisovalerate, can synthesize acetolactate from two pyruvate molecules, can catalyze the conversion of alpha-ketoisovalerate into isobutyraldehyde, and can further reduce the acetolactate into isobutanol by utilizing dehydrogenase in cells. The embodiment is modifiedConstructed plasmid pC_TSD_TThe AlsS enzyme coding gene constructs Q424S, Q487S and Q488S mutants so as to reduce the accumulation amount of isobutanol, and the specific steps are as follows:

in the form of pC_TSD_TPlasmid is used as a template, P1/P2, P3/P4 and P5/P6 are respectively used as upstream and downstream primers (the primers are shown in table 1) for full-plasmid PCR amplification, the full-plasmid PCR amplification is converted into E.coli DH5 alpha competent cells, transformants are selected for colony PCR verification, and after sequencing, recombinant plasmid pC carrying SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 is obtained_TSD_TQ424S、pC_TSD_TQ487S、pCT_SD_TQ488S。

TABLE 1 primers used for Point mutations

The recombinant plasmid pC obtained by construction_TSD_TQ424S、pC_TSD_TQ487S、pCT_SD_TQ488S is transformed into 050T4 strains respectively to obtain recombinant strains. Culturing the obtained recombinant strains in M9-2 culture medium at 37 deg.C and 200r/min, respectively, and growing to OD₆₀₀At 2.5, the recombinant strains expressing the mutants Q424S and Q487S were fermented for 36h to the end of sugar consumption, with isobutanol accumulation amounts of 0.05g/L and 0.25g/L, respectively, compared with the wild type at 1.3g/L, induced with 0.4mM IPTG and fermented at 200r/min with the temperature lowered to 30 ℃. It can be seen that the strain expressing the mutant had significantly lower levels of isobutanol accumulation than the wild-type AlsS (figure 2). The isobutanol accumulation amount of the Q488S mutant is 0.3g/L and is almost the same as that of the Q487S mutant, which indicates that the catalytic functions of the two amino acid sites are similar. With strain 050T4/pC_TSD_TIn contrast, strain 050T4/pC_TSD_TThe accumulation level of the isobutanol of Q487S is reduced to 19.2%, and the yield of the alpha-ketoisovalerate is 14.87g/L, which is reduced by 22.5％。

Example 3: AlsS ribosome binding site sequence optimization

In order to minimize the accumulation of isobutanol and maximize the production of a-ketoisovalerate, strain 050T4/pC was used in example 2_TSD_TBased on Q487S, the RBS sequence of AlsSQ487S was modified. RBS sequences of varying strengths were designed using RBS calculator (version2.1) software. In the form of pC_TSD_TQ487S plasmid was used as a template, and PCR was performed using P7/P8, P9/P10, and P11/P12 as upstream and downstream primers (shown in Table 2), respectively, to modify the RBS sequence located upstream of the AlsS enzyme to have an intensity of 6.8X 10⁴、41.7×10⁴、55.3×10⁴RBS sequence of Au to obtain recombinant plasmid pC_TSD_TQ487S-RBS6.8、pC_TSD_TQ487S-RBS42、pC_TSD_TQ487S-RBS55。

TABLE 2 primers used for RBS optimization

The recombinant plasmids obtained by construction are respectively transformed into a strain 050T4 which is 050T4/pC respectively_TSD_TQ487S-RBS6.8、050T4/pC_TSD_TQ487S-RBS42、050T4/pC_TSD_TQ487S-RBS 55. Selecting single colony to 5mL LB culture medium, culturing at 37 deg.C and 200r/min for 8h, transferring to 50mL M9-2 culture medium according to 2% inoculum size, culturing at 37 deg.C and 200r/min to OD₆₀₀At 2.5, 0.4mM IPTG was added for induction, and the mixture was fermented at 30 ℃ and 200r/min for 36 hours until sugar consumption was completed. Every 4h, the pH was maintained at 7.0 with ammonia.

Strain 050T4/pC_TSD_TQ487S-RBS55 (maximum RBS intensity 55.3X 104Au) synthesized alpha-ketoisovalerate with the yield up to 19.2g/L, and strain 050T4/pC_TSD_TThe yield of (2) was maintained at the same level (FIG. 3). Although, strain 050T4/pC_TSD_TThe accumulation of isobutanol in Q487S-RBS55 increased to 0.6g/L, which is also higher than strain 050T4/pC_TSD_TLow 56.3%, indicating that the accumulation of isobutanol can be successfully reduced without affecting the synthesis of alpha-ketoisovalerate.

Example 4: weakening tricarboxylic acid cycle

1) AceF added degradation tag DAS +4 on chromosome

The tricarboxylic acid cycle competes with the alpha-ketoisovalerate anabolic pathway for the common precursor pyruvate. In order to increase the conversion rate of α -ketoisovalerate synthesis, the DAS +4 degradation tag was fused to the C-terminus of the AceF subunit of PDH enzyme on the chromosome of strain 050T4, so that the activity of the key enzyme (PDH) of TCA cycle was further weakened, the amount of carbon source consumed by the growing cells was reduced, and α -ketoisovalerate synthesis was promoted, and the sequences of the primers used were as shown in table 3.

A target gene fragment is amplified by PCR by using a pACYC-Kan-DAS +4 plasmid shown in SEQ ID NO.9 as a template and a primer P13+ P14, and is integrated at an AceF gene stop codon TAA on a chromosome of a strain B0016-050T 4. The transformant is verified by PCR (polymerase chain reaction) of a primer P15+ P16, the length of a gene fragment after wild type amplification is 620bp, the length of a gene fragment after integration is 740bp, the AceF gene is successfully knocked out, and a strain which is verified to be correct is named as 050 TY.

TABLE 3 primers used for chromosomal Gene modification

The PDH enzyme activity of the constructed strain was measured. The method comprises the following specific steps: pCTSDTQ487S-RBS55 were transformed into strains 050T4, 050TY, 050T4/pCTSDTQ487S-RBS55, 050TY/pCTSDTQ487S-RBS55, respectively. Selecting single colony to 5mL LB culture medium, culturing at 37 deg.C and 200r/min for 8h, transferring to 50mL M9-2 culture medium at 2%, culturing at 37 deg.C and 200r/min to OD₆₀₀2.5, then adding 0.4mM IPTG for induction, culturing at 30 ℃ and 200r/min for 12h, sampling, and determining PDH enzyme activity. The results show that Pdh activity of the 050TY strain is significantly lower than 050T4, suggesting that DAS +4 degradation signature is effective (fig. 4A).

Further verifying the influence of strain modification on the fermentation synthesis of alpha-ketoisovalerate. In particular toThe method comprises the following steps: plasmid pCTSDTQ487S-RBS55 constructed in example 3 was transformed into strain 050TY, 050TY/pCTSDTQ487S-RBS 55. Selecting single colony to 5mL LB culture medium, culturing at 37 deg.C and 200r/min for 8h, transferring to 50mL M9-2 culture medium according to 2% inoculum size, culturing at 37 deg.C and 200r/min to OD₆₀₀And 2.5, adding 0.4mM IPTG for induction, culturing at 30 ℃, fermenting at the rotating speeds of 50 r/min, 100 r/min, 150 r/min and 200r/min respectively until sugar consumption is finished, and adjusting the pH value to be 7.0 by using ammonia water at intervals of 4 h. And (3) comparing the influence on the synthesis of alpha-ketoisovalerate after adding the degradation label DAS +4 at different rotating speeds.

The fermentation results at different rotating speeds show that the strain is 050T4/pC_TSD_TStrain 050TY/pC after weakening of TCA cycle compared with Q487S-RBS55_TSD_TQ487S-RBS55 fermented at 100rpm until sugar consumption is finished, the yield of the fermentation was 19.79g/L for 36h, the total conversion rate was 0.93mol/mol glucose, the conversion rate after the second stage induction was 1mol/mol glucose, and the total conversion rate and the conversion rate in the second stage were both significantly increased (FIG. 4B). Further optimizing the turning time of the thallus growth stage and the alpha-ketoisovalerate synthesis stage, showing that the thallus grows to OD₆₀₀When the conversion rate is 4, the alpha-ketoisovalerate synthesis stage is started, the yield can reach 22.1g/L at most, the total conversion rate is increased to 0.94mol/mol glucose (approximate to the theoretical conversion rate of 1mol/mol glucose), and the conversion rate of the second stage reaches 1mol/mol glucose (figure 4C).

The strain 050TY/pCTSDTQ487S-RBS55 has the highest yield and conversion rate under the microaerobic condition, and the possible reason is that after TCA cycle is weakened, the synthesized amount of NADH is reduced, and less oxygen is needed to be used as a final electron acceptor to realize the cycle of reduced coenzyme, so that the redox force is balanced, and the synthesis path of alpha-ketoisovalerate is smoother; and simultaneously, the conversion rate of the alpha-ketoisovalerate is obviously improved by inhibiting a competitive metabolic pathway.

Example 5: strain 050TY/pC_TSD_TQ487S-RBS55 fermentation in 5L fermentor

The alpha-ketoisovalerate is synthesized by a two-stage fermentation method. The first stage is aerobic mode, and the thallus grows. Amplifying in equal proportion according to the optimum culture conditions of the shake flask, i.e. the thallus concentration grows to OD₆₀₀When the fermentation temperature is 20 ℃, the fermentation is switched to the second stage of microaerobic fermentation. The method comprises the following specific steps:

the first stage is as follows: inoculating seed solution of 050TY/pCTSDTQ487S-RBS55 cultured at 37 deg.C and 200r/min for 8h at a ratio of 5% (v/v) into M9-4 culture medium, and culturing at 37 deg.C with dissolved oxygen concentration of 30% or more to OD₆₀₀Reaching 20, and entering a second stage;

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

After two-stage fermentation for 26 hours, the yield of the alpha-ketoisovalerate reaches 55.8g/L, the conversion rate in the whole fermentation process reaches 0.85mol/mol glucose, the conversion rate in the second stage is improved to 0.99mol/mol glucose, the theoretical glucose is close to 1mol/mol glucose, and the volume production intensity is 2.14g/L h. With strain 050T4/pC_TSD_TIn contrast, strain 050TY/pC_TSD_TThe yield, volumetric production strength and second stage production strength of Q487S-RBS55 were improved by 30%, 20.2% and 45.4%, respectively. The accumulation amount of isobutanol is obviously reduced to 1.51g/L, and the yield is only 0.04mol/mol glucose, which indicates that the AlsS modification effectively reduces the accumulation of isobutanol as a byproduct. At the same time, strain 050TY/pC_TSD_TCell Density ratio of Q487S-RBS55 Strain 050T4/pC_TSD_TThe content is 13.9 percent lower, which indicates that the over growth of thalli is reduced by weakening tricarboxylic acid cycle, and the conversion rate of alpha-ketoisovalerate is improved.

TABLE 4 comparison of fermentation results in fermentors

The strain 050TY/pC was used_TSD_TThe yield, the volume green strength and the total conversion rate of the alpha-ketoisovalerate synthesized by Q487S-RBS55 fermentation are 2.6, 4 and 1.8 times of those reported in the literature (Applied and Environmental Microbiology,2010,76(24): 8053-8061). This is also the highest level of α -ketoisovalerate synthesis reported to date.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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

<110> university of south of the Yangtze river

<120> method for preparing alpha-ketoisovalerate by fermenting escherichia coli transformed by metabolic engineering

<130> BAA211869A

<160> 9

<170> PatentIn version 3.3

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Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn

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

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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 (10)

1. Recombinant escherichia coli with improved alpha-ketoisovalerate conversion efficiency is characterized by having the capability of producing alpha-ketoisovalerate by fermenting glucose serving as a raw material; the recombinant Escherichia coli is improved by at least one of (a) to (d):

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

(b) replacing an RBS sequence of acetolactate synthase with a sequence shown in any one of SEQ ID No. 2-4;

(c) adding a DAS +4 degradation tag behind an AceF subunit of pyruvate dehydrogenase; the DAS +4 degradation label has a nucleotide sequence shown in SEQ ID NO. 5.

2. The recombinant Escherichia coli of claim 1, wherein said acetolactate synthase mutant has a mutation in at least one of glutamine 424, 487, and 488 as compared to that in SEQ ID NO. 1.

3. The recombinant E.coli of claim 1 or 2, wherein a T7 promoter is used to overexpress key enzymes of the α -ketoisovalerate synthesis pathway.

4. The recombinant Escherichia coli according to any one of claims 1 to 3, wherein the recombinant Escherichia coli is a host Escherichia coli B0016-050T 4.

5. The recombinant Escherichia coli of claim 1, wherein the recombinant Escherichia coli is pC_TSD_TThe method is characterized in that an acetolactate synthase mutant shown in any one of SEQ ID NO. 6-8 is expressed as a vector, an RBS sequence of the acetolactate synthase mutant is replaced by a sequence shown in any one of SEQ ID NO. 2-4, and a DAS +4 degradation label is added behind an AceF subunit of pyruvate dehydrogenase.

6. A method for producing alpha-ketoisovalerate, characterized in that the recombinant Escherichia coli according to any one of claims 1 to 5 is fermented in a glucose-containing medium.

7. The method according to claim 6, characterized in that it employs a two-stage fermentation:

the first stage is as follows: 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 reached₆₀₀And 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.

8. The method of claim 7, wherein the second stage is induced by the addition of IPTG at a final concentration of 0.7-0.9 mM.

9. The method according to any one of claims 6 to 8, wherein the medium used for the fermentation is M9-4 medium containing, in g/L: 30 portions of glucose, 4 portions of yeast powder and KH₂PO₄13.5, peptone 4, citric acid monohydrate 1.7, MgSO₄·7H₂O0.49，(NH4)₂HPO₄4 and 0.1 percent of trace element liquid; the trace element liquid contains: MnSO₄·4H₂O 0.5g/L，FeSO₄·7H₂O 10.0g/L，CaCl₂ 2.0g/L，(NH₄)Mo₇O₂₄ 0.1g/L，CuSO₄·5H₂O 3.0g/L，Na₂B₄O₇·10H₂O 0.23g/L，ZnSO₄·7H₂O 5.25g/L。

10. Use of the recombinant Escherichia coli according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9 in the fields of medicine, food or cosmetics.

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