CN106367445A - Method for producing glutaric acid by whole-cell biocatalysis - Google Patents

Abstract

The invention discloses a method for producing glutaric acid by whole cell biocatalysis. In this method, the cells of the recombinant strain E.coliBL-22AB-YDT and the cells of E.coli28LGOX are first induced to express and collected, and then the cells of the recombinant strain E.coli28LGOX and the cells of the recombinant strain E.coliBL -22AB-YDT are mixed at a ratio of 1:1. ‑5 After mixing, add the substrates L‑lysine and L‑glutamic acid, make the molar ratio of L‑glutamic acid and L‑lysine 1:0.5‑4, add surfactant, and catalyze the production of whole cells glutaric acid. The method of the invention does not need to add 2-ketoglutaric acid, reduces the production cost, solves the problems of long production cycle of fermentation method, complex metabolites, low substrate conversion rate, difficult product separation and extraction, and high energy consumption, and also solves the problems of In enzymatic catalysis, the cascade catalytic process is not easy to realize, and the catalytic efficiency is improved, and the enzyme purification process is saved at the same time.

Description

A method for whole-cell biocatalytic production of glutaric acid

technical field

The invention relates to the field of biotechnology, in particular to a method for producing glutaric acid by whole cell biocatalysis.

Background technique

Glutaric acid, alias: gum acid, α,γ-propanedicarboxylic acid, 1,3-propanedicarboxylic acid, can be used as an important chemical raw material and organic intermediate, and has a wide range of applications in various fields. In industry, glutaric acid can be used to synthesize polyvinyl chloride, polyester, polyamide and polyester plasticizer of polyvinyl chloride, etc. It can also be used to synthesize liquid polyester (improving the molecular structure of PET fiber, improving PET Fiber dyeability, improve dye uptake rate). In medicine, glutaric acid can be used to synthesize various sterilization lotions and medicines. In addition, in daily life, glutaric acid can be used in the formulation of detergents, the preparation of adhesives, and the preparation of sulfur-containing flue gases. washing etc.

In the current method of synthesizing glutaric acid, there are chemical synthesis methods, such as the multi-step synthesis method using gamma-butyrolactone as raw material, the selective oxidation method using cyclopentanol-cyclopentanone as raw material, and dihydrofuran as raw material Oxidative hydrolysis ring opening, prepared by condensation of malonate and formaldehyde in the presence of diethylammonium, etc. However, the raw materials of this method are expensive and difficult to obtain, and most of them use strong oxidants, which cause great corrosion to the equipment. The reaction is more complicated, the reaction steps are many, the yield is not high, the environmental pollution is more serious, and it is limited to the laboratory, and the industrialization prospect is not great.

Si Jae Park et al. used the recombinant E. coli WL3110 strain overexpressing davAB and gabTD to ferment in a medium containing 20g/L glucose, 10g/L L-lysine and 10g/L 2-oxoglutarate , 1.7 g/L glutaric acid was obtained. This method requires exogenous addition of expensive amino acceptor 2-ketoglutarate, which greatly increases the production cost. In addition, the molar yield of glutaric acid produced by the fermentation method is low, takes a long time, the reaction system is more complicated, and the products are separated. more difficult.

People such as Jake Adkins used the recombinant Escherichia coli strain that overexpressed davAB and davDT to use glucose as a substrate, and produced 0.82g/L glutaric acid after 48 hours of fermentation. This method has a complicated reaction system and the molar yield of glutaric acid The rate is lower.

After searching, a cost-effective two-cell coupling production method of glutaric acid has not been reported yet.

Contents of the invention

Aiming at the deficiencies of the prior art, the object of the present invention is to provide a method for producing glutaric acid by whole cell biocatalysis, which saves cost and is economical and efficient.

In order to solve the problems of the prior art, the technical scheme that the present invention takes is:

A method for whole-cell biocatalytic production of glutaric acid, comprising the following steps:

A method for whole-cell biocatalytic production of glutaric acid, comprising the following steps:

Step 1, construct the bacterial strain E.coli BL-22AB-YDT that overexpresses DavBA and gabDT, then pick the single bacterium colony of recombinant bacterial strain E.coli BL-22AB-YDT from the plate and inoculate to containing 100mg/L ampicillin resistance and 35mg/L chloramphenicol-resistant 5ml LB shaking tube, cultured for 6-8h, then transferred to 100MLLB medium containing 100mg/L ampicillin resistance and 35mg/L chloramphenicol resistance, cultured to OD ₆₀₀ =0.6-0.8, collect the bacteria by centrifugation, and obtain the cells of the recombinant strain E.coli BL-22AB-YDT;

Step 2: Construct the strain E.coli 28LGOX that overexpresses LGOX, and then pick a single colony of the recombinant strain E.coli 28LGOX from the plate and inoculate it into a shake tube containing kanamycin resistance and LB. After culturing for 6-8 hours Transfer to shake flasks containing kanamycin resistance and LB, cultivate to OD ₆₀₀ =0.6-0.8, collect bacteria by centrifugation, and obtain cells of recombinant strain E.coli 28LGOX;

Step 3, mix the cells of the recombinant strain E.coli 28LGOX and the cells of the recombinant strain E.coli BL-22AB-YDT in a ratio of 1:1-5, add the substrate L-lysine (5-100g/L) and L-glutamic acid (5-100g/L), make the molar ratio of L-glutamic acid and L-lysine (5-100g/L) 1:0.5-4, add surfactant, whole cell catalysis Production of glutaric acid.

Preferably in the above method, the construction steps of overexpressing DavBA and gabDT bacterial strains in step 1 are:

The davA and davB fragments were connected with the expression vector pET-22b to obtain the recombinant plasmid pET22b-DavBA, and the recombinant plasmid pET22b-DavBA was introduced into the competent cells of E.coli BL21 to obtain the recombinant strain E.coli BL- 22BA and make the recombinant strain into competent cells, connect the gabD and gabT fragments to the expression vector pACYC-Duet to obtain the recombinant plasmid pACYC-gabDT, and introduce the recombinant plasmid into the competent cells of the recombinant strain E.coli BL-22AB, A recombinant strain E.coli BL-22AB-YDT overexpressing DavBA and gabDT was obtained.

Preferably in the above method, the method for constructing the strain E.coli 28LGOX overexpressing LGOX in step 2 is: connecting the fragment LGOX with NcoI and BamHI as restriction sites, and connecting the expression vector 28a cut with NcoI and BamHI, The recombinant plasmid 28a-LGOX was obtained, and the recombinant plasmid was introduced into the competent cells of E.coli BL21 to obtain the recombinant strain E.coli 28LGOX overexpressing LGOX.

Step 3, the process of catalyzing L-lysine in whole cells of recombinant strain E.coli BL-22AB-YDT is coupled with the process of catalyzing L-glutamic acid in whole cells of recombinant strain E.coli28LGOX

Mix the cells of recombinant E.coli 28LGOX and recombinant E.coli BL-22AB-YDT in proportion, add substrates L-lysine and L-glutamic acid, and add 0.5% X-100, at 37° C, the whole cell catalytic reaction was carried out under the condition of 200rmp, and samples were taken at regular intervals, and the amount of glutaric acid and 2-ketoglutaric acid was detected by liquid phase;

As the above method, preferably, the cell ratio of the recombinant strain E.coli 28LGOX and the recombinant strain E.coli BL-22AB-YDT in step 3 is 1:4.

As the above method, preferably, the molar ratio of the substrate L-glutamic acid and the substrate L-lysine described in step 3 is 1:2.

Preferably as the above method, the surfactant described in step 3 is one or more of SDS, Triton X-100, Tween-20, Tween-80.

Preferably as a surfactant, the concentrations of L-lysine and L-glutamic acid in step 3 are both 5-100 g/L.

Preferably, as the above method, the catalytic reaction in step 3 is carried out at 37° C. and the stirring speed is 200 rpm.

Beneficial effect

A whole-cell biocatalytic production of glutaric acid, through the overexpression of L-glutamate oxidase (LGOX), realizes the production of high-value amino acceptor 2-ketone with cheap L-glutamate as substrate Glutaric acid (2-KG), solves the economic problem of exogenously adding expensive amino acceptor 2-oxoglutarate in the process of catalyzing the conversion of L-lysine into glutaric acid in whole cells, and at the same time, by regulating The molar ratio of the substrate L-glutamic acid to the substrate L-lysine enables more efficient utilization of the substrate. In addition, by adjusting the cell OD ₆₀₀ ratio of recombinant E.coli 28LGOX and recombinant E.coli BL-22AB-YDT, the molar yield of glutaric acid reached 68.34%.

Description of drawings

Figure 1 is a schematic diagram of the production of glutaric acid by the coupling of two cells;

Fig. 2 is the SDS-PAGE diagram of the expression of LGOX induced and expressed at different temperatures.

Figure 3. Coupling of whole-cell catalyzed conversion of L-glutamate to 2-oxoglutarate and glutarate process.

detailed description

Example 1

Construction of overexpressed DavBA and gabDT strains:

(1) The recombinant strain E.coli BL-22AB overexpressing davBA was provided by our laboratory, and the recombinant strain E.coli BL-22AB was made into competent cells.

(2) gabT was synthesized by Jinweizhi, and the restriction sites were NdeI and XhoI, and it was connected to the pACYC vector to obtain the recombinant plasmid pACYC-gabT,

(3) gabD was synthesized by Jinweiweizhi, with restriction sites NcoI and HindIII, and connected to the pACYC vector to obtain the recombinant plasmid pACYC-gabD.

(4) After the recombinant plasmid pACYC-gabD was recovered by treatment with restriction endonucleases NcoI and HindIII, the fragment gabD with restriction sites of NcoI and HindIII was obtained, and the fragment was combined with the recombinant plasmid treated with the same restriction endonuclease Plasmid pACYC-gabT was ligated using T4 DNA ligase at 25°C for 30min.

(5) Transfer the above connection solution into the competent cells of Escherichia coli Trans1-T1 , spread on LB plates with 35mg/L chloramphenicol resistance, and culture overnight at 37°C.

(6) Pick a single colony grown on the plate in (5), transfer it to LB medium containing 35mg/L chloramphenicol resistance, then extract the plasmid, and then enzymatically digest it with restriction enzymes NcoI and HindIII Finally, the recombinant plasmid pACYC-gabT-gabD was obtained.

(7) Transfer the recombinant plasmid pACYC-gabT-gabD into the competent cells of the recombinant strain E.coli BL-22AB, and spread it on the culture medium with 100mg/L ampicillin resistance and 35mg/L chloramphenicol resistance. The recombinant strain E.coli BL-22AB-YDT overexpressing davBA and gabDT was obtained on the sex LB plate.

Construction of overexpressing LGOX strains:

The LGOX fragment was synthesized by Jinweizhi, with restriction sites NcoI and BamHI, and connected to the pACYC vector to obtain the recombinant plasmid pACYC-LGOX. After the recombinant plasmid pACYC-LGOX was recovered after being treated with restriction endonucleases NcoI and BamHI, a fragment LGOX with restriction sites of NcoI and BamHI was obtained, and the fragment was connected with plasmid 28a treated with the same restriction endonucleases , using T4 DNA ligase for 30 min at 25°C. The above ligation solution was transferred into the competent cells of Escherichia coli Trans1-T1 , spread on LB plate with 50mg/L kanamycin resistance, and cultured overnight at 37°C. Pick a single colony grown on the plate in the previous step, transfer it to LB medium containing 50mg/L kanamycin resistance, then extract the plasmid, and then perform digestion verification with restriction enzymes NcoI and BamHI , finally obtained the recombinant plasmid 28a-LGOX. The recombinant plasmid 28a-LGOX was transformed into the competent cells of the recombinant strain E.coliBL21 (DE3) , and spread on the LB plate with 50mg/L kanamycin resistance to obtain the overexpressed LGOX Recombinant strain E.coli 28LGOX.

Example 2

Optimum temperature screening for inducing LGOX expression

Pick a single colony of the recombinant strain E.coli 28LGOX from the plate into a 5ML LB shake tube containing 50mg/L kanamycin resistance, culture it for 6-8h and transfer it to a tube containing 50mg/L kanamycin resistance In 100ml of LB, add 0.5mmol of IPTG to OD ₆₀₀ =0.6, culture in shaker at 20°C, 25°C, 30°C, 37°C respectively, after 4h, centrifuge at 6000g for 5min to obtain overexpressed Cells of LGOX.

The obtained cells were resuspended with 5ml of 100mmol PBS, crushed using a cell disruptor, the crushing conditions were super 2s, stop 2s, the temperature was 4°C, the power was 30%, the crushing time was 10min, and then centrifuged at 7000g for 20min.

After measuring the protein concentration of the supernatant and precipitate induced under different temperature conditions, the protein concentration was 20ug, and the protein expression was obtained after SDS-PAGE analysis, as shown in Figure 2. When induced at 25°C , LGOX was significantly overexpressed in the supernatant.

Example 3

The whole cell catalyzed process of glutamate to produce 2-oxoglutarate is coupled with the process of whole cell catalyzed conversion of L-lysine to glutarate:

The recombinant strain E.coli 28LGOX cells overexpressing LGOX were cultured and collected according to the method of Example 2 as a catalyst.

At the same time, pick a single colony of the recombinant strain E.coli BL-22AB-YDT from the plate, inoculate it into a 5ml LB shaking tube containing 100mg/L ampicillin resistance and 35mg/L chloramphenicol resistance, and incubate for 6-8h Afterwards, transfer to the 100ml shake flask containing 100mg/L ampicillin resistance and 35mg/L chloramphenicol resistance, to OD ₆₀₀ =0.6, add 0.5mmol of IPTG, 20 ° C after induction culture for 12h, 7000g, Centrifuge for 5 minutes to obtain cells of the recombinant strain E.coli BL-22AB-YDT, and use it as a catalyst.

Resuspend the cells of recombinant E.coli 28LGOX and E.coli BL-22AB-YDT in 100mmol PBS, respectively.

In the reaction system, add recombinant bacteria E.coli 28LGOX and recombinant bacteria E.coli BL-22AB-YDT cells, so that in the system, the OD ₆₀₀ of E.coli 28LGOX =5, the OD of E.coli BL-22AB-YDT ₆₀₀ =20, L-sodium glutamate: 10g/L, L-lysine: 10g/L, X-100: 0.5%, sampling at regular intervals, liquid phase detection of glutaric acid and 2-ketoglutadiene Acid accumulation, the detection method is chromatographic column: Bio-Rad Aminex HPX-87H (300 mm *7.8 mm), column temperature: 55°C, mobile phase: 8 mM H ₂ SO ₄ , flow rate: 0.6 mL/min , detectors: UV, RID. After 10 hours of reaction, glutaric acid accumulated to 3.16g/L, 2-KG: 6.39g/L, and then, 2-ketoglutaric acid began to decrease. After 46 hours of reaction, 2-ketoglutaric acid was consumed, and glutaric acid The diacid accumulated to 6.14g/L, and the molar yield was 68.34%, as shown in Figure 3.

The method of the present invention is compared with the method that Si Jae Park et al. adopt the recombinant bacterial strain E. coli WL3110 that has overexpressed davAB and gabTD to obtain 1.7g/L glutaric acid (molar yield: 18.92%) by fermentation and cultivation. This method not only No exogenous addition of expensive amino acceptor 2-oxoglutaric acid is required, and the molar yield of glutaric acid is increased by 49.42%, and the product obtained by the method of the invention is easier to separate and purify.

Claims (8)

1. a kind of Whole Cell Biocatalysis produce the method for 1,3-propanedicarboxylic acid it is characterised in that comprising the following steps:

Step 1, builds the bacterial strain of overexpression davba and gabdte.coliBl-22ab-ydt, then picking restructuring from flat board Bacterial straine.coliThe single bacterium colony of bl-22ab-ydt is inoculated into and resists containing 100mg/l amicillin resistance and 35mg/l chloromycetin Property 5ml lb shake in pipe, culture 6-8h after be transferred to containing 100mg/l amicillin resistance and 35mg/l chlorampenicol resistant 100mllb culture medium in, cultivate to od₆₀₀=0.6-0.8, centrifugation collection bacterium, obtain recombinant bacterial straine.coliBl-22ab-ydt's Cell；

Step 2, builds the bacterial strain of overexpression lgoxe.coli28lgox, then picking recombinant bacterial strain from flat boarde.coliThe single bacterium colony of 28lgox is inoculated into shaking in pipe containing kalamycin resistance and lb, be transferred to after culture 6-8h containing card that In the shaking flask of chloramphenicol resistance and lb, cultivate to od₆₀₀=0.6-0.8, centrifugation collection bacterium, obtain recombinant bacterial straine.coli28lgox's is thin Born of the same parents；；

Step 3, by recombinant bacteriume.coliThe cell of 28lgox and recombinant bacteriume.coliThe cell of bl-22ab-ydt proportionally 1: 1-5 mixes, and adds substrate l- lysine and l- glutamic acid, makes l- glutamic acid be 1:0.5-4 with the mol ratio of l- lysine, adds Surfactant, whole-cell catalytic produces 1,3-propanedicarboxylic acid.

2. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: described in step 1 The bacterial strain of overexpression davba and gabdte.coliThe construction method of bl-22ab-ydt is: by fragment davb with restriction enzyme site Ndei and xhoi is connected on carrier 22b, obtains recombiant plasmid 22b-davb, by fragment davb with restriction enzyme site agei and xmai It is connected on recombiant plasmid 22b-davb, obtain recombiant plasmid 22b-davba, in addition, by fragment gabt with restriction enzyme site ndei Be connected on plasmid pacyc with xhoi, obtain recombiant plasmid pacyc-gabt, by fragment gabd with restriction enzyme site ncoi and Hindiii is connected on recombiant plasmid pacyc-gabt, obtains recombiant plasmid pacyc-gabt- gabd, by recombiant plasmid 22b- Davba and pacyc-gabt- gabd imports escherichia coliBl21(de3)In, obtain the restructuring of overexpression davba and gabdt Bacterial straine.colibl-22ab-ydt.

3. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: step 2 builds The bacterial strain of overexpression lgoxe.coliThe method of 28lgox is: by fragment lgox with ncoi and bamhi as restriction enzyme site, with warp The expression vector 28a that ncoi with bamhi enzyme action is crossed is connected, and obtains recombiant plasmid 28a-lgox, recombiant plasmid is importede.coli bl21Competent cell in, obtain the recombinant bacterium of overexpression lgoxe.coli28lgox.

4. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: weight in step 3 Group bacteriume.coliThe cell of 28lgox and recombinant bacteriume.coliThe proportionally 1:5 mixing of the cell of bl-22ab-ydt.

5. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: institute in step 3 The substrate l- glutamic acid stated and mol ratio 1:2 of substrate l- lysine.

6. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: institute in step 3 The surfactant stated is sds, triton x-100, one or more of tween-20, tween-80.

7. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: l- in step 3 The concentration of lysine and l- glutamic acid is 5-100g/l.

8. Whole Cell Biocatalysis according to claim 1 produce 1,3-propanedicarboxylic acid method it is characterised in that: urge in step 3 Change reaction at 37 DEG C, carry out under the conditions of speed of agitator 200rpm.