CN117965473A

CN117965473A - Dehydrogenase system and application thereof in preparation of P34HB

Info

Publication number: CN117965473A
Application number: CN202410370527.7A
Authority: CN
Inventors: 叶健文; 吕金艳; 司徒卫; 余柳松; 林艺娜
Original assignee: Zhuhai Medfa Biotechnology Co ltd
Current assignee: Zhuhai Medfa Biotechnology Co ltd
Priority date: 2024-03-29
Filing date: 2024-03-29
Publication date: 2024-05-03
Anticipated expiration: 2044-03-29
Also published as: CN117965473B

Abstract

The invention provides a dehydrogenase system and application thereof in preparing P34HB, wherein the dehydrogenase system comprises aldehyde dehydrogenase and alcohol dehydrogenase; the aldehyde dehydrogenase is any one of aldD1, aldD, gabD4, aldH and ydcW; the alcohol dehydrogenase is any one of adhp and dhaT. The dehydrogenase system of the invention overcomes the integration problem of dehydrogenases from different sources, can play a synergistic effect, and is important for promoting the sustainability and efficiency of BDO biosynthesis. Through a dehydrogenase system, a high-efficiency metabolic pathway is constructed, so that engineering bacteria can utilize 1, 4-Butanediol (BDO) as a carbon source to produce P34HB, the synthetic efficiency of 4HB is improved through selection and optimization of the dehydrogenase, and the content of 4HB in the P34HB is further improved.

Description

Dehydrogenase system and application thereof in preparation of P34HB

Technical Field

The invention relates to the field of bioengineering, in particular to a dehydrogenase system and application thereof in preparation of P34 HB.

Background

Polyhydroxyalkanoates (PHAs) are a class of biodegradable plastics synthesized by microorganisms. Of these, 4-hydroxybutyric acid (4 HB) is an important component of PHA, and the introduction of 4HB can improve the physical properties of PHA, for example, increase the flexibility and ductility of the material, making PHA more elastic and more suitable for some specific applications, such as degradable implants in the medical field. The biosynthetic pathway for PHA production typically involves the use of renewable resources such as biomass, which, by increasing 4HB content, better enables sustainable PHA production, helping to reduce reliance on limited resources such as petroleum. Therefore, research on how to increase the 4HB content in PHA through metabolic engineering means has important application value.

PHA synthesis by metabolic pathways under the condition of glucose as a carbon source is a common production method. In order to increase the 4HB content in PHA, 1, 4-Butanediol (BDO) may be used as a precursor for 4HB, producing 4-hydroxybutyric acid during the synthesis of PHA. At present, BDO is used as a precursor for production, and in industrial application, the PHA production efficiency is low, the cost is high, and the 4HB content in the PHA is low, so that the requirement of customers on high-quality PHA cannot be met.

Alcohol dehydrogenases (Alcohol dehydrogenase, ADH) are a class of oxidases that catalyze the conversion of alcohols to aldehydes or ketones. It exists widely in organisms and participates in various physiological processes such as alcohol metabolism, alcohol degradation and the like. Aldehyde dehydrogenases (Aldehyde dehydrogenase, ALDH) are a class of oxidases that catalyze the conversion of aldehydes to acids. The enzymes are mainly involved in aldehyde metabolism in organisms and play an important role in regulating redox balance in cells and maintaining the homeostasis of intracellular aldehyde substances. Alcohol dehydrogenases and aldehyde dehydrogenases are involved in the regulatory network of metabolic pathways in organisms and thus have a wide range of applications in medicine and industry, in particular in enzyme engineering. However, because of the difference between the enzymes from different sources, the reaction conditions and the performance of the enzymes have different degrees of variation, a combination of dehydrogenases which can overcome the integration problem of the enzymes from different sources and is most suitable for biosynthesis is needed, and a more reliable technical route is provided for industrial production.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a dehydrogenase system and application thereof in preparing poly-3-hydroxybutyrate-4-hydroxybutyrate (P34 HB).

The present invention provides a dehydrogenase system comprising an aldehyde dehydrogenase and an alcohol dehydrogenase;

The aldehyde dehydrogenase is any one of aldD1, aldD, gabD4, aldH and ydcW; the nucleotide sequence of aldD1 is shown as SEQ ID No.32, and the amino acid sequence is shown as SEQ ID No. 40; the nucleotide sequence of aldD is shown as SEQ ID No.33, and the amino acid sequence is shown as SEQ ID No. 41; the nucleotide sequence of the gabD4 is shown as SEQ ID No.34, and the amino acid sequence is shown as SEQ ID No. 42; the nucleotide sequence of aldH is shown as SEQ ID No.35, and the amino acid sequence is shown as SEQ ID No. 43; the nucleotide sequence of ydcW is shown as SEQ ID No.36, and the amino acid sequence is shown as SEQ ID No. 44;

The alcohol dehydrogenase is any one of adhp and dhaT; the nucleotide sequence of adhp is shown as SEQ ID No.37, and the amino acid sequence is shown as SEQ ID No. 45; the nucleotide sequence of dhaT is shown as SEQ ID No.38, and the amino acid sequence is shown as SEQ ID No. 46.

The present invention also provides a dehydrogenase system comprising an aldehyde dehydrogenase and an alcohol dehydrogenase;

the aldehyde dehydrogenase is a gabD4 mutant, and is different from gabD4 in that the 144 th amino acid of the gabD4 mutant is alanine and the 169 th amino acid of the gabD4 mutant is alanine; the alcohol dehydrogenase is adhp.

the aldehyde dehydrogenase is a gabD4 mutant, and is different from gabD4 in that the 144 th amino acid of the gabD4 mutant is alanine and the 169 th amino acid of the gabD4 mutant is alanine;

the alcohol dehydrogenase is adhp mutant, and the difference between the alcohol dehydrogenase and adhp is any one of the following:

(i) The amino acid 201 of adhp mutant is alanine and the amino acid 263 is alanine;

(ii) The 261 st amino acid of adhp mutant is alanine and the 271 st amino acid is lysine;

(iii) The adhp mutant has alanine at amino acid 101 and lysine at amino acid 79.

The invention also provides a recombinant vector comprising a nucleotide sequence encoding the dehydrogenase system.

The invention also provides a recombinant genetically engineered bacterium, which contains the recombinant vector.

Furthermore, the recombinant genetically engineered bacterium is halomonas, preferably Halomonas lutescens MDF-9;

The Halomonas lutescens MDF-9 strain used in the present application was deposited at the microorganism strain collection in Guangdong province (GDMCC address: guangzhou City, highway 100, no. 59 building 5, ministry of microorganisms, guangdong province, post code 510070) on day 8 and 2 of 2021. Deposit number GDMCC NO:61850. the strain was designated Halomonas lutescens MDF-9 and the classification was designated as halomonas (Halomonas lutescens).

The invention also provides an application of the dehydrogenase system or the recombinant vector or the recombinant genetically engineered bacterium in preparing P34 HB.

The invention also provides a method for producing P34HB, comprising the following steps:

s1: amplifying OrfZ gene sequences in vitro, and inserting the sequences into a vector to obtain a first vector plasmid;

s2: inserting a nucleotide sequence encoding the dehydrogenase system into the first vector plasmid S1 to obtain a second vector plasmid;

s3: introducing the second vector plasmid of S2 into Salmonella;

S4: inoculating the seed solution of the halomonas in the S3 into a fermentation medium for fermentation culture; the OrfZ gene sequence is shown as SEQ ID No. 39.

Further, the fermentation culture temperature in the step S4 is 30-45 ℃.

Further, in the step S4, the pH of the fermentation culture is 6-12, and the time is 30-60 hours.

In conclusion, compared with the prior art, the invention achieves the following technical effects:

1. The dehydrogenase system of the invention overcomes the integration problem of dehydrogenases from different sources, can play a synergistic effect, and is important for promoting the sustainability and efficiency of BDO biosynthesis.

2. According to the invention, a high-efficiency metabolic pathway is constructed through a dehydrogenase system, so that engineering bacteria can utilize 1, 4-Butanediol (BDO) as a carbon source to produce P34HB, and the synthetic efficiency of 4HB is improved through the selection and optimization of the dehydrogenase, so that the content of 4HB in the P34HB is improved.

3. In the process of producing P34HB, the invention can utilize 1, 4-butanediol as a raw material carbon source, is environment-friendly, safe and nontoxic, and reduces the toxic effect of the raw material on microorganisms in the production process. The production cost of the 1, 4-butanediol is low, and the cost of the raw material for producing the P34HB is reduced.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a pathway diagram of the production of P34HB according to an embodiment of the invention;

FIG. 2 is a map of pSEVA 321-OrfZ-aldD-dhaT plasmid constructed in example 1 of the present invention;

FIG. 3 is a plasmid map of pSEVA321-OrfZ-aldD1-adhp constructed in example 1 of the present invention;

FIG. 4 is a map of pSEVA321-OrfZ-aldD2-dhaT plasmid constructed in example 1 of the present invention;

FIG. 5 is a plasmid map of pSEVA321-OrfZ-aldD2-adhp constructed in example 1 of the present invention;

FIG. 6 is a plasmid map of pSEVA321-OrfZ-gabD4-dhaT constructed in example 1 of the present invention;

FIG. 7 is a plasmid map of pSEVA321-OrfZ-gabD4-adhp constructed in example 1 of the present invention;

FIG. 8 is a map of pSEVA321-OrfZ-aldH-dhaT plasmid constructed in example 1 of the present invention;

FIG. 9 is a plasmid map of pSEVA321-OrfZ-aldH-adhp constructed in example 1 of the present invention;

FIG. 10 is a map of pSEVA321-OrfZ-ydcW-dhaT plasmid constructed in example 1 of the present invention;

FIG. 11 is a plasmid map of pSEVA321-OrfZ-ydcW-adhp constructed in example 1 of the present invention;

FIG. 12 is an electrophoresis chart of a fragment of interest according to example 1 of the present invention; wherein the No. 1 is a gabD4-adhp fragment; the fragment No. 2 is gabD 4-dhaT; the fragment 3 is aldH-adhp; the fragment No. 4 is aldH-dhaT; fragment number 5 ydcW-adhp; the ydcW-dhaT fragment No. 6; fragment number 7 aldD to adhp; aldD1-dhaT fragment number 8; fragment number 9 aldD, fragments 2-adhp; no. 10 is aldD fragment 2-dhaT.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.

In Halomonas lutescens MDF-9 strains, 10 strains which are different in enzyme combination and have the capability of producing 4HB are successfully obtained by amplifying combinations of 5 aldehyde dehydrogenases from different sources and 2 alcohol dehydrogenases from different sources, and P34HB is synthesized by taking glucose and BDO as precursors, as shown in figure 1. The 5 different sources of aldehyde dehydrogenases were aldD from the halophila strain Halomonas bluephagenesis, aldD from pseudomonas putida Pseudomonas putida, gabD4 from copper greedy Cupriavidus necator, aldH from escherichia coli ESCHERICHIA COLI, and ydcW from klebsiella pneumoniae Klebsiella pneumoniae, respectively. The 2 different sources of alcohol dehydrogenases were dhaT from pseudomonas putida Pseudomonas putida and adhp from pseudomonas halophila strain Halomonas bluephagenesis, respectively.

Subsequently 10 strains of different enzymes were combined and the strains with the ability to produce 4HB were subjected to fermentation tests, screening the highest efficiency enzyme combination system.

The method for producing P34HB by utilizing Halomonas lutescens MDF-9 modified bacteria comprises the following steps:

(1) Plate seed culture: activating strains; (2) shake flask seed culture;

(3) Dissolved oxygen and pH electrode correction; (4) setting fermentation parameters;

(5) Inoculating; (6) fermentation process control; (7) PHA extraction from the cells.

Fermenter temperature: 36-38 ℃;

pH 8～9。

EXAMPLE 1 construction of P34HB Strain synthesized with glucose and BDO as carbon sources

Since Halomonas lutescens MDF-9 strain itself lacks genes encoding the enzymes aldehyde dehydrogenase, alcohol dehydrogenase of this pathway, this example introduced BDO (1, 4-butanediol) metabolic pathway in the strain, which involved 3 enzymes aldehyde dehydrogenase, alcohol dehydrogenase and CoA transferase. To further obtain BDO-highly transformed strains, the above two enzymes were screened and tested, and specific experimental procedures were as follows:

(1) Constructing a plasmid:

The overlapping extension PCR was used to amplify the ligation of CoA transferase OrfZ to pSEVA321 backbone, and ligation was performed by Gibson ligase to construct plasmid pSEVA321-OrfZ. The 5 aldehyde dehydrogenases aldD, aldD2, gabD4, aldH, ydcW and 2 alcohol dehydrogenases dhaT, adhp and pSEVA321-OrfZ backbones were amplified by PCR. The 5 aldehyde dehydrogenase genes, 2 alcohol dehydrogenases and pSEVA321-OrfZ frameworks respectively form new plasmids under the action of Gibson ligase, 10 plasmids are total, and the information of the plasmids respectively named pSEVA321-OrfZ-aldD1-dhaT、pSEVA321-OrfZ-aldD1-adhp、pSEVA321-OrfZ-aldD2-dhaT、pSEVA321-OrfZ-aldD2-adhp、pSEVA321-OrfZ-gabD4-dhaT、pSEVA321-OrfZ-gabD4-adhp、pSEVA321-OrfZ-aldH-dhaT、pSEVA321-OrfZ-aldH-adhp、pSEVA321-OrfZ-ydcW-dhaT、pSEVA321-OrfZ-ydcW-adhp, is shown in figures 2-11.

(A) The primer sequences (5 '-3') used in the above plasmid construction procedure were as follows:

Aldehyde dehydrogenase amplification primer linked to alcohol dehydrogenase adhp:

aldD1-F1: see SEQ ID No.1;

aldD1-R1: see SEQ ID No.2;

aldD2-F1: see SEQ ID No.3;

aldD2-R1: see SEQ ID No.4;

gabD4-F1: see SEQ ID No.5;

gabD4-R1: see SEQ ID No.6;

aldH-F1: see SEQ ID No.7;

aldH-R1: see SEQ ID No.8;

ydcW-F1: see SEQ ID No.9;

ydcW-R1: see SEQ ID No.10;

adhp-F: see SEQ ID No.11;

adhp-R: see SEQ ID No.12;

aldehyde dehydrogenase amplification primer linked to alcohol dehydrogenase dhaT:

aldD1-F1: see SEQ ID No.1;

aldD1-R2: see SEQ ID No.13;

aldD2-F1: see SEQ ID No.3;

aldD2-R2: see SEQ ID No.14;

gabD4-F1: see SEQ ID No.5;

gabD4-R2: see SEQ ID No.15;

aldH-F1: see SEQ ID No.7;

aldH-R2: see SEQ ID No.16;

ydcW-F1: see SEQ ID No.9;

ydcW-R2: see SEQ ID No.17;

dhaT-F: see SEQ ID No.18;

dhaT-R: see SEQ ID No.19;

Backbone and OrfZ amplification primers:

pSEVA321-F: see SEQ ID No.20;

pSEVA321-R: see SEQ ID No.21;

OrfZ-F: see SEQ ID No.22;

OrfZ-R: see SEQ ID No.23;

pSEVA321-OrfZ-F: see SEQ ID No.24;

pSEVA321-OrfZ-R: see SEQ ID No.25.

(B) The amplification system and amplification procedure are shown in tables 1 and 2:

TABLE 1 amplification System Table

TABLE 2 amplification program Table

After the PCR reaction is completed, agarose gel with corresponding concentration is prepared, electrophoresis is carried out to observe the size of DNA bands, the gel is placed under an ultraviolet lamp, the gel of the target DNA fragment is rapidly cut off, and the redundant gel is cut off as much as possible.

(C) Gibson Assembly method connection

The concentration of the recovered DNA was measured, and the addition ratio of the DNA was calculated based on the length and concentration of the desired fragment and pSEVA321 backbone, and ligation was performed using Gibson enzyme mixture, the Gibson Assembly ligation system and the procedure are shown in Table 3 and Table 4:

TABLE 3 Gibson Assembly connection System Table

Table 4 Gibson Assembly connection procedure

(2) S17-1 E.coli transformation

Step 1: taking out competent cells of the S17-1 escherichia coli prepared in advance from the temperature of minus 80 ℃, thawing on ice, and waiting for fungus blocks to be thawed after 5 min;

Step 2: mu.L of the ligation product was added to competent cells, and the reaction was mixed with gentle vessel wall (shaking-free). And (3) injection: the ligation product conversion volume should not exceed at most 1/10 of the competent cell volume used;

step 3: ice bath for 30 min, water bath heat shock at 42 ℃ of 2 min, immediately cooling 2 min on ice, injecting: shaking can reduce conversion efficiency;

Step 4: 400 mu L of LB culture medium (without antibiotics) is added into the centrifuge tube, and the mixture is placed into a shaking table at 37 ℃ for resuscitation of 200 rpm to 60 min;

Step 5:5000 Centrifuging at rpm for 5 min to collect bacteria, discarding 350 μl of supernatant, taking 100 μl of resuspended bacteria mass, gently blowing and coating onto LB medium containing corresponding antibiotics;

step 6: and inverting the culture medium into a 37 ℃ incubator for culturing for 12-16 hours.

(3) Monoclonal colony positive verification

Colonies were picked on corresponding resistant LB plates, colony PCR verified, and PCR products with correct band sizes were sent to Bio-company for sequencing.

(4) Selecting single bacterial colony with correct sequence for expansion culture, jointing the single bacterial colony with Halomonas lutescens MDF-9 in a 20LB plate after 12-16 hours, and picking a small amount of jointed thalli to be coated on a 60LB plate with corresponding resistance after 8-h; 36-48 h followed by a further monoclonal colony validation.

(5) PCR identification results

The monoclonal colony is selected for PCR verification, the result of the electrophoresis verification is shown in figure 12, the number 1 is gabD4-adhp fragment, and the band size is 2471 bp; the fragment No. 2 gabD4-dhaT has a band size of 2627 bp; the fragment 3 is aldH-adhp, and the band size is 2532 bp; the size of the band is 2688 bp for aldH-dhaT fragment 4; fragment number 5 ydcW-adhp, band size 2471 bp; ydcW-dhaT fragment number 6, band size 2627 bp; fragment number 7 aldD to adhp, band size 2564 bp; aldD1-dhaT fragment number 8, band size 2720 bp; fragment number 9 aldD-adhp, band size 2564 bp; the aldD-dhaT fragment 10, band size 2720 bp. The results of the verification were all expected to indicate successful introduction of the 10-group enzyme combination plasmid into Halomonas lutescens MDF-9 strains. The 10 strains were designated as MDF-9-aldD1-A、MDF-9-aldD1-D、MDF-9-aldD2-A、MDF-9-aldD2-D、MDF-9-gabD4-A、MDF-9-gabD4-D、MDF-9-aldH-A、MDF-9-aldH-D、MDF-9-ydcW-A、MDF-9-ydcW-D.

EXAMPLE 2 screening test for 10 recombinant bacteria

(1) Culture medium:

60LB plate medium: yeast extract 5 g/L, tryptone 10 g/L, sodium chloride 60g/L, agar powder 1.8 g/100 mL, ampicillin 50. Mu.g/mL, kanamycin 30 g/mL, pH 8.0.

Component I: 0.2 g/L magnesium sulfate, 1.0 g/L urea (50 times concentrated mother liquor: 10 g/L magnesium sulfate, 3 g/L urea);

component II: potassium dihydrogen phosphate 5.2 g/L, 50 times mother liquor 260 g/L, glucose solution 30 g/L: glucose mother liquor is 500 g/L;

Component III (10 mL/L): ferric ammonium citrate 5 g/L, anhydrous calcium chloride 1.5 g/L,12 mol/L hydrochloric acid 41.7 mL/L;

Component IV (1 mL/L): 100 mg/L of zinc sulfate heptahydrate, 30 mg/L of manganese sulfate tetrahydrate, 300 mg/L of boric acid, 10 mg/L of copper sulfate pentahydrate and 30 mg/L of sodium molybdate.

Fermentation medium (50 MM medium): 1, 4-butanediol 5 g/L, glucose 30g/L, sodium chloride 50 g/L, yeast powder 1.2 g/L, urea 0.2-3 g/L, anhydrous magnesium sulfate 0.2 g/L, and potassium dihydrogen phosphate 1.5~5.5 g/L,Fe(III)-NH₄-Citrate 5 g/L,CaCl₂·2H₂O 2 g/L,HCl 12 mol/L,ZnSO₄·7H₂O 0.1 g/L,MnCl₂·4H₂O 0.03 g/L,H₂BO₃ 0.3 g/L,CoCl₂·6H₂O 0.2 g/L,CuSO₄·5H₂O 0.01 g/L,NiCl₂·6H₂O 0.02 g/L,NaMoO₄·2H₂O 0.03 g/L.

(2) Seed liquid preparation

Fermentation was performed using 10 bacteria constructed in example 1.

① Activating strains:

the strains were collected in a laboratory at-80℃in a refrigerator, and inoculated onto a plate solid medium (yeast powder 5 g/L; tryptone 10 g/L; sodium chloride 60 g/L; pH 8.5) by streaking with a gun head, followed by culturing at 37℃for 24. 24 h.

② Primary seed culture:

Single colonies were picked up and inoculated into 12 mL shaking tubes (5 mL 60LB medium: yeast powder 5 g/L; tryptone 10 g/L; sodium chloride 60 g/L; pH 8.5), and the culture broth was placed in a shaking table 37 ℃, 220 rpm, and cultured for 12 h.

③ Secondary seed culture:

200. Mu.L of the primary bacterial liquid (1% of the inoculum size) was aspirated, inoculated into 150 mL Erlenmeyer flasks (20 mL 60LB medium) and placed in a shaker at 37℃and 220rpm for 12 h.

(3) Fermentation medium preparation

(4) Fermentation culture

Seed solution was inoculated (2.5 mL) in 500 mL Erlenmeyer flask at 5% and incubated at shaker 37℃and 220 rpm for 48 h.

(5) Determination of cell dry weight and PHA content

Cell Dry Weight (CDW): placing 30-35 mL of fermented bacterial liquid into a 50 mL centrifuge tube, centrifuging for 6 minutes at room temperature, wherein the rotating speed is 8000 rpm, and pouring out the supernatant; adding proper deionized water to restore the original volume, re-suspending to ensure complete disappearance of the precipitate, centrifuging under the same condition, and pouring out the supernatant; placing the sealing membrane sealing centrifuge tube in a refrigerator at-80 ℃ for freezing 2 h; drying the centrifuge tube in a vacuum freeze dryer for 12-16 hours; the cells were weighed and dry weight (g/L) was calculated.

Measuring PHA content: weighing 0.05 g of dried bacterial cells of a sample obtained by fermentation after grinding, treating the standard 4-hydroxybutyric acid of 4HB with the same sample, placing the sample in an esterification pipe with good sealing property, adding 2mL chloroform, 1700 mu L methanol and 300 mu L concentrated sulfuric acid, reacting 1 h in an oil bath at 100 ℃, cooling at room temperature, adding 1mL of ddH ₂ O, fully shaking and uniformly mixing, and standing for layering. After the aqueous and organic phases were completely separated, the chloroform layer (typically the lower layer) was filtered into a liquid phase bottle using a 0.22 μm organic filter, and GC was performed using a GC-7800 gas chromatograph, a capillary column (Rtx-5 type, length 30 m, inner diameter 0.25 mm and stationary phase 0.25 μm) and hydrogen Flame Ion Detection (FID). The carrier gas is high purity nitrogen. The temperature programming settings are shown in table 5:

TABLE 5 program temperature settings

The sample injection volume is 1 mu L, the PHA is quantitatively analyzed by adopting an external standard method, and the yield of the PHA is calculated according to the peak area.

(6) Fermentation results

The fermentation results are shown in Table 6:

TABLE 6 fermentation results of 10 recombinant bacteria

The results showed that the synthesis of 4HB was able to be promoted by introducing an aldehyde dehydrogenase and an alcohol dehydrogenase in the MDF-9 strain. From the above 10 combinations of test results, it was found that MDF-9-gabD4-A had the best effect, and the cell dry weight, PHA and 4HB content were all the highest, respectively 12.68 g/L, 85.35 wt%, 12.01 mol%.

EXAMPLE 3 mutant screening of dehydrogenase

(1) Aldehyde dehydrogenase active site mutant construction

The active site of the aldehyde dehydrogenase contains conserved cysteine residues that interact directly with the aldehyde of the substrate, and in order to determine which cysteine residues are involved in substrate binding, the 2 cysteine-encoding positions (144, 169) of the aldehyde dehydrogenase are changed to alanine by site-directed mutagenesis. Alanine substitution was used because the methyl functionality of alanine was not effective in nucleophilic attack of the carbon-based carbon of the aldehyde. Specific construction procedure for site-directed mutagenesis is described in example 1.

The primers were designed as follows:

C144A-F: see SEQ ID No.26;

C144A-R: see SEQ ID No.27;

C169A-F: see SEQ ID No.28;

C169A-R: see SEQ ID No.29;

pSEVA321-OrfZ-F: see SEQ ID No.24;

pSEVA321-OrfZ-R: see SEQ ID No.25.

(2) Screening for random mutations in alcohol dehydrogenase

For more efficient expression of the dehydrogenase system, adhp was subjected to chemical random mutagenesis, and the mutants were screened for more efficient combinations with the aldehyde dehydrogenase gabD4 (C144A, C169A), for specific construction procedures, see example 1.

(A) The amplification primers were as follows:

adhp-F1: see SEQ ID No.30;

adhp-R1: see SEQ ID No.31.

(B) The amplification system is shown in Table 7:

TABLE 7 amplification System

Wherein, tris-HCl mix main component: tris-HCl 10mM, KCl 50 mM, mgCl ₂ 2 mM.

(C) Mutants of 4 strains adhp were obtained after screening and sequencing, respectively, the 44 th site of adhp was mutated from threonine to histidine and alanine (T44H, T44A), the 201 st site was mutated from aspartic acid to alanine, the 263 rd site was mutated from serine to alanine (D201A, S263H), the 261 st site was mutated from tryptophan to alanine, the 271 st site was mutated to lysine (W261A, S271K), the 101 st site was mutated from histidine to alanine, and the 79 th site was mutated to lysine (H101A, S79K).

After that, fermentation tests were carried out, and the specific fermentation medium and components are the same as in example 2, and the fermentation results are shown in table 8:

TABLE 8 fermentation results of dehydrogenase mutants

The results showed that when glutamic acid at 144 and 169 sites of aldehyde dehydrogenase gabD4 was mutated to alanine, both the cell dry weight and PHA content of the strain were increased and the synthesis of 4HB was promoted. After binding to adhp of the four mutants, MDF-9-gabD4 (C144A, C169A) -adhp (D201A, S263H) was combined with the best effect, and the cell dry weight, PHA and 4HB content was increased to 13.09 g/L, 93.11% and 13.11%, respectively.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. A dehydrogenase system, wherein the dehydrogenase system comprises an aldehyde dehydrogenase and an alcohol dehydrogenase;

the aldehyde dehydrogenase is any one of aldD1, aldD, gabD4, aldH and ydcW;

the alcohol dehydrogenase is any one of adhp and dhaT.

2. A dehydrogenase system, wherein the dehydrogenase system comprises an aldehyde dehydrogenase and an alcohol dehydrogenase;

The alcohol dehydrogenase is adhp.

3. A dehydrogenase system, wherein the dehydrogenase system comprises an aldehyde dehydrogenase and an alcohol dehydrogenase;

4. A recombinant vector comprising a nucleotide sequence encoding the dehydrogenase system of any one of claims 1 to 3.

5. A recombinant genetically engineered bacterium comprising the recombinant vector of claim 4.

6. The recombinant genetically engineered bacterium of claim 5, wherein the recombinant genetically engineered bacterium is a halomonas.

7. The dehydrogenase system according to any one of claims 1 to 3 or the recombinant vector according to claim 4 or the recombinant genetically engineered bacterium according to any one of claims 5 to 6, for use in the preparation of P34 HB.

8. A method for producing P34HB, comprising the steps of:

s2: inserting a nucleotide sequence encoding the dehydrogenase system of any one of claims 1-3 into the first vector plasmid of S1 to obtain a second vector plasmid;

s3: introducing the second vector plasmid of S2 into Salmonella;

s4: inoculating the seed solution of the halomonas in the S3 into a fermentation medium for fermentation culture.

9. The method according to claim 8, wherein the fermentation culture temperature in step S4 is 30-45 ℃.

10. The method according to claim 8, wherein the fermentation culture in step S4 has a pH of 6 to 12 for 30 to 60 hours.