CN105087457B - A kind of method that RBS optimizations improve α ketoisocaproate yield - Google Patents

Abstract

Optimize the method for improving α ketoisocaproate yield the invention discloses a kind of RBS, belong to genetic engineering field.The present invention utilizes the genetic engineering bacterium by RBS optimizations, using leucine as substrate, whole-cell catalytic production α ketoisocaproates.With the advantages that production cost is low, working condition is gentle, impurity is less in transformation system, processing step is simple, and production operation is safe.High using α ketoisocaproates yield produced by the invention, every liter of conversion fluid contains 81.41g α ketoisocaproates.The conversion ratio of L leucines reaches 88.66%.

Description

A method of RBS optimization to improve the production of α-ketoisocaproic acid

technical field

The invention relates to a method for optimizing the production of α-ketoisocaproic acid by optimizing RBS, and belongs to the field of genetic engineering.

Background technique

α-Ketoisocaproic acid (KIC), also known as α-oxoisovaleric acid, is an intermediate metabolite in the synthesis and decomposition of leucine. As a nitrogen-free substitute for leucine, it can be used as a nutritional supplement for patients with kidney disease . In addition, KIC can be used in the fields of feed, health products, and chemical synthesis.

At present, the production methods of KIC are mainly chemical synthesis methods, mainly including the hydrolysis reaction of the addition product of diethyl oxamide and Grignard reagent, double carbonylation method, Heine method and so on. These methods reaction conditions are harsh, all need special structure as starting material, or expensive catalyst (as the complex compound of octahydroxy dicobalt and zero-valent palladium), and all need through multi-step reaction as starting material, more difficult For production practice and industrialized mass production. Microbial conversion or enzymatic conversion can replace traditional chemical synthesis to produce α-ketoisocaproic acid. At present, the L-amino acid deaminase from Proteusvulgaris can be expressed in Escherichia coli to realize a high-yield conversion process of substrate leucine to ketoisocaproic acid.

However, the yield and conversion rate of the current α-ketoisocaproic acid in the conversion process are still low, which is not enough to meet the requirements of industrial production, so it is necessary to further increase the yield of α-ketoisocaproic acid. The invention aims at improving the translation expression level of L-amino acid deaminase by optimizing the ribosome binding site, so as to further increase the output of α-ketoisocaproic acid.

Contents of the invention

The first technical problem to be solved by the present invention is to provide a recombinant escherichia coli with improved activity of L-amino acid deaminase, which is the gene encoding L-amino acid deaminase from P. pET28a(+) is an expression vector for recombinant expression, and one or more bases are mutated in the original RBS sequence region of the vector pET28a(+).

The original RBS sequence of the vector pET28a(+) is an AAGGAG sequence of 302bp-307bp.

In one embodiment of the present invention, the mutation is that the original RBS sequence of the vector pET28a(+) is mutated into CTGCGG, CTACGG, CGGTTA or TATGGT.

The second technical problem to be solved by the present invention is to provide a method for constructing the recombinant escherichia coli, which is to combine the gene encoding L-amino acid deaminase from Proteus vulgaris (P.vulgaris) with pET28a(+) The recombinant expression vector pET28a-lad was obtained by connection, and the pET28a-lad was used as a template to mutate the RBS coding sequence by PCR amplification, and then transformed into Escherichia coli, and the recombinant bacteria were screened.

In one embodiment of the present invention, the Escherichia coli is E. coli BL21(DE3).

The present invention also provides a method for increasing the production of α-ketoisocaproic acid, which is to use the recombinant Escherichia coli with improved L-amino acid deaminase activity as a whole-cell catalyst to convert the substrate L-leucine to obtain α-ketone isocaproic acid.

In one embodiment of the present invention, α-ketoisocaproic acid is transformed into α-ketoisocaproic acid at 37°C with a system containing 0.8g/L recombinant E. coli whole cells and 100mM/L substrate L-leucine, and every 100mM L-leucine was added for 2h, and the flow was added 6 times for a total of 24h.

In one embodiment of the present invention, the whole cells of recombinant Escherichia coli are obtained by inoculating the seed culture solution into the fermentation medium at an inoculum size of 2%, culturing at 37°C until the _OD600 is 0.6, and adding the final concentration of 0.4mM IPTG, induced for 5h at 37°C, and the cells were collected.

The seed culture and fermentation medium: seed medium (/L): peptone 1g, yeast powder 0.5g, NaCl 1g, distilled water to 100mL. Fermentation medium (/L): 12g of peptone, 24g of yeast extract, 4mL of glycerin, each component was dissolved and then autoclaved. Cool to 60°C, add 100mL sterilized solution of 17mmol/L KH ₂ PO ₄ and 72mmol/L K ₂ HPO ₄ (dissolve 2.31g of KH ₂ PO ₄ and 12.54g of K ₂ HPO ₄ in water, the final volume is 100 mL, autoclaved).

The invention transforms the RBS sequence of the expression vector pET28a-lad, improves the translation efficiency of the enzyme, improves the enzyme activity, and constructs a strain capable of high-efficiency production so as to increase the output of α-ketoisocaproic acid. Compared with the prior art, the yield of α-ketoisocaproic acid of the present invention can reach 81.41g/L, and the substrate conversion rate is 88.66%. The conversion method has mild reaction conditions, less environmental pollution, high substrate selectivity, specific target product, fast reaction rate, high product yield, and short production cycle, only 24 hours. The method is beneficial to solve the problems of serious pollution and cumbersome steps in the chemical synthesis method, and the process is simple, easy to control, and convenient to popularize and apply.

Description of drawings

Fig. 1 Changes in catalytic activity of RBS optimization.

Fig. 2 Production of α-ketoisocaproic acid after RBS optimization.

Detailed ways

Materials and Methods

LB medium: peptone 1g, yeast powder 0.5g, NaCl 1g, tap water to 100mL.

Fermentation medium (TB medium): peptone 12g, yeast extract 24g, glycerol 4mL. The components are dissolved and autoclaved. Cool to 60°C, and add 100 mL of a sterilized solution containing 17 mmol/L KH ₂ PO ₄ and 72 mmol/L K ₂ HPO ₄ .

Sample preparation: Take 1 mL of the converted solution, centrifuge at 10,000 rpm for 10 min, take the supernatant and dilute it, filter it through a 0.45 μm filter membrane, and use the filtrate for liquid chromatography analysis.

Determination of the content of α-ketoisocaproic acid: Agilent 1100 high performance liquid chromatography (equipped with UV-visible detector) adopts ZORBAX SB-Aq (4.6×250mm, 5μm) chromatographic column, and the mobile phase is diammonium hydrogen phosphate of 0.01mol/L Solution (pH2.50)-methanol solution (90:10, v/v), the flow rate is 0.8mL/min, the column temperature is 35°C, and the ultraviolet detection wavelength is 203nm.

The construction of embodiment 1RBS mutant library

Using pET28a-lad (see application number 201410050399.4 for the construction method, patent application publication number CN 103789247A) as a template, the forward primer is 5'- NNNNNN ATATACCATGGCGATATCTAGAAGAA-3', and the reverse primer is 5'-CTTAAAGTTAAACAAAAATTATTTCTAGAGG-3' PCR reaction was performed for primers. After the reaction product was digested with Dpn I, the template was recovered using a DNA column recovery kit, phosphorylase was added for terminal phosphorylation, and Solution I ligase was added to ligate overnight at 16°C. The ligated plasmid was transformed into the competent E.coli BL21(DE3), and spread on a kanapenicillin-resistant plate.

Example 2 L-amino acid deaminase catalytic activity improved mutant screening

Pick the single colony in Example 1 into a 96-well deep-well plate containing 700 μL LB medium with a sterilized toothpick, culture it overnight with shaking at 37° C., and inoculate it into a 96-well plate containing 700 μL fermentation medium with an inoculum size of 2%. In a deep-well plate, shake culture at 37°C until the OD ₆₀₀ is 0.6, add 0.4mM IPTG and collect the bacteria 3 hours after induction, centrifuge at 3400rpm for 10min, add 1mL of 100mM leucine aqueous solution to the deep-well plate, and react for 10 minutes , add 100 μL of 1% FeCl ₃ solution for color development.

Example 3 Determination of Catalytic Activity and Sequence Changes of Mutants Converted to L-leucine by Whole Cells

The recombinant escherichia coli E.coli BL21 (DE3) that develops the color in embodiment 2 is dark black from LB culture medium-transfer in the Erlenmeyer flask of 250mL that 25mL seed culture medium is housed, cultivate overnight, with 2% The inoculum was inoculated into 50 mL of fermentation medium, cultured at 37°C, 200r/min until the OD ₆₀₀ was 0.6, induced by adding 0.4mM IPTG, cultured at 37°C for 5h, and collected by centrifugation for whole cell transformation. Take 0.8g/L cells, 100mM substrate L-leucine, react at 37°C for 0.5h, centrifuge at 12000rpm for 2min, and take the supernatant to measure the content of α-ketoisocaproic acid.

According to the formula Enzyme activity was calculated. (Wherein C _(KIC) is the determined α-ketoisocaproic acid content, DCW is 0.8g/L of bacterium dry weight, T is the reaction time 0.5h). The catalytic activity of its mutants is shown in Figure 1. Among them, the catalytic activities of RBS3, RBS4, RBS6 and RBS8 were about 20% higher than that of the original bacteria.

After sequence alignment, the RBS sequences of mutants RBS3, RBS4, RBS6, and RBS8 were mutated into CTGCGG, CTACGG, CGGTTA, and TATGGT, respectively.

Example 4 Whole Cell Transformation Method for Synthesizing α-Ketoisocaproic Acid

After cultivating according to the above culture method, collect the thalli of RBS3, RBS4, RBS6, and RBS8, add 100 mM leucine every 2 h at 0.8 g/L thalline, 100 mM/L substrate L-leucine (flow added to 12h, 6 times in total), at 37°C, transform for 24h. After dilution, centrifuge at 12000rpm for 2min, and take the supernatant to measure the content. The yield measurement results are shown in Figure 2. Among them, the output of RBS4, RBS6 and RBS8 was higher than that of the original strain, the maximum output reached 81.41g/L, and the transformation rate reached 88.66%.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (5)

1. the recombination bacillus coli that a kind of l-amino acid deamination enzyme activity improves, it is characterised in that be by from common variation bar The gene of the encoded L-amino acids deaminase of bacterium, recombinantly expressed with pET28a (+) for expression vector, the carrier pET28a The original RBS sequence areas of (+) have one or more base to undergo mutation, and the mutation is the original of carrier pET28a (+) RBS series jumps are CTACGG, CGGTTA or TATGGT, and the Escherichia coli areE.coliBL21(DE3)。

A kind of 2. method for building recombination bacillus coli described in claim 1, it is characterised in that be by from proteus vulgaris （P. vulgaris）Encoded L-amino acids deaminase gene, be connected with pET28a (+), obtain recombinant expression carrier pET28a-lad, with pET28a-ladFor template, the method expanded by PCR, after RBS sequence encoding mutants, then it is transferred to big Enterobacteria, screening obtain recombinant bacterium.

A kind of 3. method that α-ketoisocaproic acid yield is improved using recombination bacillus coli described in claim 1, it is characterised in that will The recombination bacillus coli obtains α-ketoisocaproic acid as whole-cell catalyst, conversion of substrate L-Leu, and methods described is specific For：With the system containing the full cell of 0.8 g/L recombination bacillus colis, 100 mM/L substrate L-Leus, 37^oIt is converted under C α-ketoisocaproic acid, and every 2 h streams plus 100 mM L-Leus, flow add 6 times altogether, cotransformation reacts 24 h.

4. according to the method for claim 3, it is characterised in that the acquisition of the full cell of recombination bacillus coli, is to plant Sub- nutrient solution is inoculated into fermentation medium by 2% inoculum concentration, and 37^oC is cultivated to OD₆₀₀For 0.6, addition final concentration 0.4 mM's IPTG, 37^oC induces 5 h, collects cell.

5. according to the method for claim 4, it is characterised in that described fermentation medium contains per L：The g of peptone 12, The g of yeast extract 24, the mL of glycerine 4；Autoclaving after each component dissolving, is cooled to 60^oC, then add the 17 of 100 mL sterilizings mmol/L KH₂PO₄With 72 mmol/L K₂HPO₄Solution.