CN105087457B

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

Info

Publication number: CN105087457B
Application number: CN201510575189.1A
Authority: CN
Inventors: 刘龙; 陈坚; 堵国成; 李江华; 宋阳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2015-09-10
Filing date: 2015-09-10
Publication date: 2018-04-10
Anticipated expiration: 2035-09-10
Also published as: CN105087457A

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 kind of method that RBS optimizations improve α-ketoisocaproic acid yield

Technical field

Optimize the method for improving α-ketoisocaproic acid yield the present invention relates to a kind of RBS, belong to genetic engineering field.

Background technology

α-ketoisocaproic acid (KIC), also known as alpha-oxo isovaleric acid, it is the mesostate of leucine synthesis and decomposition, it , can be as the nutritious supplementary pharmaceutical of nephrotic as the nitrogen-free substitute of leucine.In addition, KIC can be in feed, guarantor Strong product, the field of chemical synthesis.

KIC production method is mainly chemical synthesis at present, mainly includes diethyl oxalamide Yu Geshi reagent additions The hydrolysis of product, double carbonylation method, glycolylurea method etc..These method severe reaction conditions, are required for special construction conduct Starting material, or expensive catalyst (complex compound of such as cobalt of eight hydroxyl two and zeroth order palladium), and be required for by multistep Reaction is used as starting material, it is more difficult to is mass produced for production practices and industrialization.Microorganism conversion or enzyme transforming process, can be with Traditional chemical synthesis is substituted to produce α-ketoisocaproic acid.At present, Proteus can be come from by Bacillus coli expression Vulgaris l-amino acid deaminase, realize the high conversion process that converts high yield of the substrate leucine to ketoisocaproate.

However, yield and conversion ratio of the current α-ketoisocaproic acid in conversion process are also relatively low, not enough reach industrialization The requirement of production, so needing further to improve the yield of α-ketoisocaproic acid.It is contemplated that by ribosome bind site Optimize, the accurate translation level of l-amino acid deaminase is improved, to further improve the yield of α-ketoisocaproic acid.

The content of the invention

The invention solves first technical problem to be to provide the restructuring that a kind of l-amino acid deamination enzyme activity improves big Enterobacteria, it is by the gene of the encoded L-amino acids deaminase from proteus vulgaris (P.vulgaris), with pET28a (+) Recombinantly expressed for expression vector, the original RBS sequence areas of the carrier pET28a (+) have one or more base hair Raw mutation.

The original RBS sequences of the carrier pET28a (+) are 302bp-307bp AAGGAG sequences.

In one embodiment of the invention, the mutation is that carrier pET28a (+) original RBS series jumps are CTGCGG, CTACGG, CGGTTA or TATGGT.

The invention solves second technical problem be to provide a kind of method for building the recombination bacillus coli, be by The gene of encoded L-amino acids deaminase from proteus vulgaris (P.vulgaris), is connected with pET28a (+), obtains weight Group expression vector pET28a-lad, using pET28a-lad as template, by the method for PCR amplifications, by RBS sequence encoding mutants Afterwards, then Escherichia coli are transferred to, screening obtains recombinant bacterium.

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

It is to utilize the l-amino acid deamination enzyme activity present invention also offers a kind of method for improving α-ketoisocaproic acid yield The recombination bacillus coli that power improves obtains α-ketoisocaproic acid as whole-cell catalyst, conversion of substrate L-Leu.

In one embodiment of the invention, it is bright with the full cell of recombination bacillus coli containing 0.8g/L, 100mM/L substrates L- The system of propylhomoserin, α-ketoisocaproic acid is converted at 37 DEG C, and every 2h streams plus 100mM L-Leus, flows add 6 times altogether, altogether Conversion reaction 24h.

In one embodiment of the invention, the acquisition of the full cell of the recombination bacillus coli, it is by seed culture fluid It is inoculated into by 2% inoculum concentration in fermentation medium, 37 DEG C of cultures to OD₆₀₀For 0.6, addition final concentration 0.4mM IPTG, 37 DEG C induction 5h, collect cell.

Described seed culture and fermentation medium：Seed culture medium (/L)：Peptone 1g, dusty yeast 0.5g, NaCl 1g, distilled water are settled to 100mL.Fermentation medium (/L)：Peptone 12g, yeast extract 24g, glycerine 4mL, each component are molten Autoclaving after solution.60 DEG C are cooled to, then adds the 17mmol/L KH of 100mL sterilizings₂PO₄And 72mmol/LK₂HPO₄Solution (2.31g KH₂PO₄With 12.54g K₂HPO₄It is soluble in water, final volume 100mL, autoclaving).

The present invention is transformed expression vector pET28a-lad RBS sequences, is improved the translation efficiency of enzyme, is improved enzyme It is living, and the strain that can efficiently produce is constructed so as to improve α-ketoisocaproic acid yield.Compared with prior art, α of the invention- Ketoisocaproate yield can reach 81.41g/L, substrate conversion efficiency 88.66%.This method for transformation reaction condition is gentle, to environment Pollution is few, and substrate selective is high, and purpose product is single-minded, and reaction rate is fast, and efficiency of pcr product is high, and with short production cycle, only Need 24h.The method advantageously accounts for seriously polluted in chemical synthesis, and the problems such as complex steps, and technique is simple, is easy to Control, facilitates popularization and application.

Brief description of the drawings

The catalysis activity change of Fig. 1 RBS optimizations.

α-ketoisocaproic acid yield after Fig. 2 RBS optimizations.

Embodiment

Materials and methods

LB culture mediums：Peptone 1g, dusty yeast 0.5g, NaCl 1g, running water are settled to 100mL.

Fermentation medium (TB culture mediums)：Peptone 12g, yeast extract 24g, glycerine 4mL.High pressure after each component dissolving Sterilizing.It is cooled to 60 DEG C, then add 100mL sterilizings contain 17mmol/L KH₂PO₄With 72mmol/L K₂HPO₄Solution.

Sample preparation：The solution after 1mL conversions is taken, 10min is centrifuged under 10,000rpm, after taking supernatant to dilute, is passed through 0.45 μm of membrane filtration, filtrate feed flow analysis of hplc.

The assay of α-ketoisocaproic acid：The high performance liquid chromatographs of Agilent 1100 (matching somebody with somebody UV-vis detector) use ZORBAX SB-Aq (4.6 × 250mm, 5 μm) chromatographic column, the ammonium dibasic phosphate solution (pH2.50) that mobile phase is 0.01mol/L- Methanol solution (90:10, v/v), flow velocity 0.8mL/min, column temperature are 35 DEG C, are detected in the case where ultraviolet detection wavelength is 203nm.

The structure of embodiment 1RBS mutant libraries

With pET28a-lad, (for construction method referring to Application No. 201410050399.4, publication No. is CN 103789247A Patent application) be template, using forward primer as 5 '-NNNNNNATATACCATGGCGATATCTAGAAGAA-3 ', reverse primer It is that primer enters performing PCR reaction for 5 '-CTTAAAGTTAAACAAAATTATTTCTAGAGG-3 ', reaction product digests by Dpn I After its template, reclaimed using DNA pillars QIAquick Gel Extraction Kit, add phosphorylase and carry out terminal phosphate, add Solution I 16 DEG C of connections of ligase are overnight.Plasmid after connection is transferred in E.coli BL21 (DE3) competence, is coated on Ka Naqing On the flat board of chloramphenicol resistance.

The screening mutant that embodiment 2L- amino acid deaminases catalysis activity improves

Single bacterium colony in embodiment 1 is chosen into the 96 hole depth orifice plates containing 700 μ L LB culture mediums with sterilized toothpick In, 37 DEG C of concussions are incubated overnight, and are inoculated into 2% inoculum concentration in the 96 hole depth orifice plates containing 700 μ L fermentation mediums, 37 DEG C Concussion and cultivate is to OD₆₀₀For 0.6, add 3h after 0.4mM IPTG inductions and collect thalline, after 3400rpm centrifugations 10min, in deep hole The 1mL 100mM leucine aqueous solution is added in plate, after reacting 10 minutes, adds 100 μ L 1% FeCl₃Solution is shown Color.

The catalysis activity measure of the resting cell L-Leu of embodiment 3 and mutant sequence change

Will in embodiment 2 colour developing for black recombination bacillus coli E.coli BL21 (DE3) from LB culture mediums-turn It is connected in the triangular flask of the 250mL equipped with 25mL seed culture mediums, is incubated overnight, 50mL fermentations is inoculated into 2% inoculum concentration In culture medium, 37 DEG C, OD is arrived in 200r/min cultures₆₀₀For 0.6,0.4mM IPTG inductions are added, are collected by centrifugation after 37 DEG C of culture 5h Thalline carries out resting cell.Take 0.8g/L cells, 100mM substrate L-Leus, 37 DEG C of reaction 0.5h, 12000rpm centrifugations 2min, supernatant is taken to determine α-ketoisocaproic acid content.

According to formulaCalculate and obtain enzyme activity.(wherein C_(KIC)It is the α-ketoisocaproic acid content of measure, DCW is that dry cell weight 0.8g/L, T are reaction time 0.5h).Its mutant catalysis activity is as shown in Figure 1.Wherein RBS3, RBS4, RBS6, RBS8 relatively primitive bacterium vigor of catalysis activity improve 20% or so.

By sequence alignment, mutant RBS3, RBS4, RBS6, RBS8 RBS sequences sport CTGCGG respectively, CTACGG, CGGTTA, TATGGT.

The method that the resting cell of embodiment 4 synthesizes α-ketoisocaproic acid

After as above cultural method culture, collect RBS3, RBS4, RBS6, RBS8 thalline, 0.8g/L thalline, 100mM/L substrate L-Leus, flowed per 2h and add 100mM leucines (stream is added to 12h, 6 times altogether), under the conditions of 37 DEG C, conversion 24h.After dilution, 12000rpm centrifugation 2min centrifugations, supernatant is taken to determine content.Its determination of yield result is as shown in Figure 2.Wherein RBS4, RBS6, RBS8 yield are higher than original strain, and maximum production reaches 81.41g/L, and conversion ratio reaches 88.66%.

Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention Enclose being defined of being defined by claims.

Claims

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.