CN117165617A

CN117165617A - Strain for producing P34HB by utilizing xylose as well as construction method and application thereof

Info

Publication number: CN117165617A
Application number: CN202311102843.8A
Authority: CN
Inventors: 叶健文; 吕金艳; 司徒卫; 余柳松; 林艺娜; 黄劭立
Original assignee: Zhuhai Medfa Biotechnology Co ltd
Current assignee: Zhuhai Medfa Biotechnology Co ltd
Priority date: 2023-08-29
Filing date: 2023-08-29
Publication date: 2023-12-05

Abstract

The application discloses a strain for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by utilizing xylose, a construction method and application thereof. The construction method comprises the following steps: s1: amplifying xylB-xylC-xylD-xylX target gene sequence in vitro, inserting into a vector to obtain a vector plasmid; s2: amplifying the Kivd-YqhD target gene sequence in vitro, and inserting the sequence into a vector to obtain a vector plasmid; s3: the vector plasmids of S1 and S2 are jointly introduced into a Halomonas lutescens MDF-9 strain. The method effectively reduces the price of raw materials in the process of producing P34HB, reduces the toxicity of the raw materials in the production process, and improves the production efficiency.

Description

Strain for producing P34HB by utilizing xylose as well as construction method and application thereof

Technical Field

The application relates to the field of bioengineering, in particular to a strain for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by utilizing xylose, a construction method and application thereof.

Background

White pollution increasingly attacks the earth's ecosystem, plastic restrictions are also promulgated and implemented in more and more countries, and degradable materials have become an essential part of sustainable development of human society. Polyhydroxyalkanoate (PHA for short) is a series of biodegradable and environment-friendly biopolyester with better biocompatibility, and has the characteristics of biodegradability, good biocompatibility, thermoplasticity and the like. It has now been found that over hundred PHA macromolecules, each having its own characteristics, of which poly-3-hydroxybutyrate (PHB) is the most typical representation of PHA in the early stages and is also the cheapest PHA material. In recent years, 3-hydroxybutyrate-co-4-hydroxybutyrate (P34 HB) which is a copolyester of 3-hydroxybutyrate and 4-hydroxybutyrate is a brand new and most promising PHA polymer material, the properties of which can be changed by adjusting the proportion of 4-hydroxybutyrate (4 HB) in the polymer, and P34HB has good biocompatibility, biodegradability and thermal processability of plastics, so that the novel PHA polymer material can be used as biomedical materials, biodegradable packaging materials, textile fibers and the like.

The raw materials for producing the P34HB mainly comprise glucose, 1, 4-butanediol and gamma-butyrolactone, wherein the 1, 4-butanediol and the gamma-butyrolactone are used as precursor substances for synthesizing the 4HB and mainly derived from petrochemical products, have certain toxicity to microorganisms, have high market price and are not beneficial to the industrialization of the P34HB. Glucose can be used for synthesizing 4HB by modifying glucose metabolic pathways by metabolic engineering and molecular biology, but the yield of 4HB is still low.

Lignocellulose is currently available on earth in nature, the most abundant renewable green resources, and does not compete with human available land formations. Xylose, the second most abundant sugar in lignocellulosic biomass, is not available for uptake by wild type halophila. At present, research on synthesizing PHA from xylose is mainly focused on constructing xylose metabolic pathway by means of molecular biology, the metabolic product is PHB, P34HB cannot be synthesized, and P3HB and P34HB have larger difference in physical properties, so that the application scene is very limited. Therefore, a method for producing P34HB with high yield, which can utilize xylose, reduce toxicity of raw materials, reduce production cost and improve production efficiency, is needed.

Disclosure of Invention

In order to overcome the problems of high raw material cost and low production efficiency in the P34HB synthesis process, the application provides a strain for synthesizing P34HB by utilizing xylose, a construction method and application thereof, a new metabolic pathway using xylose as a 4-hydroxybutyrate (4 HB) precursor compound is introduced by adopting a synthetic biological technology, and a pathway for synthesizing 3-hydroxybutyrate (3 HB) by using xylose as a precursor is further introduced on the basis, so that a combined metabolic pathway strain is constructed, and the aim of producing P34HB is fulfilled.

The application provides a construction method of a strain for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by utilizing xylose, which comprises the following steps:

s1: amplifying xylB-xylC-xylD-xylX target gene sequence in vitro, inserting into a vector to obtain a vector plasmid;

s2: amplifying the Kivd-YqhD target gene sequence in vitro, and inserting the sequence into a vector to obtain a vector plasmid;

s3: the vector plasmids described in S1 and S2 are jointly introduced into Salmonella choleraesuis Halomonas lutescens MDF-9, and the preservation number of the vector plasmids is GDMCC NO.61850.

The Halomonas lutescens MDF-9 strain used in the present application was deposited at the microorganism strain collection of Guangdong province (GDMCC address: guangzhou City, hirudo No. 100, no. 59, building 5, ministry of microorganisms, guangdong province, post code 510070) on day 8 of 2021. The deposit number is GDMCC No.61850. The strain was named MDF-9 and the classification was named Salmonella (Halomonas lutescens).

Further, in the step S3 strain, a vector plasmid containing a HEO-xylA-xfp objective gene sequence obtained by in vitro amplification is introduced in addition to the vector plasmids obtained in the steps S1 and S2.

Further, in the step S3 strain, a vector plasmid containing the HEO-xylA-DTE-Fuck-FucA target gene sequence obtained by in vitro amplification is introduced in addition to the vector plasmids obtained in the steps S1 and S2.

Further, the carrier used in the step S1 is pSEVA321, and the carrier used in the step S2 is pSEVA341.

Further, the amplification procedure of the target gene in the steps S1 and S2 is as follows: (1) Pre-denaturation: the temperature is 95 ℃ and the time is 3min; (2) denaturation: the temperature is 95 ℃ and the time is 15sec; (3) annealing: the temperature is 56-60 ℃ and the time is 15sec; (4) extension: the temperature is 72 ℃ and the time is 30-60sec/Kb; (5) circulating the steps (2) - (4) for 35 times; (6) complete extension: the temperature is 72 ℃ and the time is 5min.

Further, the connection system in the steps S1 and S2 is as follows: 2. Mu.L of vector fragment and 2.5. Mu. L, gibson of target gene fragment were ligated with 5. Mu.L of the enzyme cocktail.

The application also provides a strain for synthesizing the poly (3-hydroxybutyrate-co-4-hydroxybutyrate), which is obtained by the construction method.

The application also provides a method for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) with the strain, which comprises the following steps:

(1) Preparing seed liquid;

(2) Culturing seeds in shake flasks;

(3) And (5) fermenting and culturing.

Further, in the step (3), the temperature of the fermentation tank is 30-42 ℃, the pH is 6.5-11, and the time is 36-50 h.

Further, the fermentation medium component in the step (3) comprises: 30g/L xylose, 50g/L sodium chloride, 1.2g/L yeast powder, 0.2-3 g/L urea, anhydrous magnesium sulfate0.2g/L, 1.5-5.5 g/L of monopotassium phosphate, fe (III) -NH ₄ -Citrate 5g/L，CaCl ₂ ·2H ₂ O 2g/L，HCl 12mol/L，ZnSO ₄ ·7H ₂ O 0.1g/L，MnCl ₂ ·4H ₂ O 0.03g/L，H ₂ BO ₃ 0.3g/L，CoCl ₂ ·6H ₂ O 0.2g/L，CuSO ₄ ·5H ₂ O 0.01g/L，NiCl ₂ ·6H ₂ O 0.02g/L，NaMoO ₄ ·2H ₂ O0.03g/L。

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

1. the application utilizes xylose as a precursor compound to synthesize the P34HB, solves the technical problem that the xylose cannot be utilized to synthesize the P34HB, and reduces the cost of producing the P34HB raw material.

2. The application uses xylose as raw material, is safe and nontoxic, and reduces the toxic effect of the raw material on microorganisms in the production process.

3. The application constructs the combined metabolic pathway strain taking xylose as a precursor, which not only improves the yield of P34HB, but also improves the content of 4HB and improves the production efficiency.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, 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 application 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 diagram of the pathway of the application for the production of P34HB by three different xylose metabolic pathways;

FIG. 2 is a schematic diagram of pSEVA321 used in the construction procedure in example 1 of the present application _xylBCDX A plasmid map;

FIG. 3 is an electrophoresis chart of the fragment of interest of xylB-xylC-xylD-xylX in example 1 of the present application;

FIG. 4 is a schematic diagram of pSEVA341 used in the construction procedure in example 1 of the present application _Kivd-YqhD A plasmid map;

FIG. 5 is an electrophoresis chart of the Kivd-YqhD fragment of interest in example 1 of the present application;

FIG. 6 shows pRSF used in the construction process of example 2 of this application _HEO-xylA-xfp A plasmid map;

FIG. 7 is an electrophoresis chart of HEO-xylA-xfp mesh fragment in example 2 of the present application;

FIG. 8 is a diagram showing pRSF used in the construction process of example 3 of the present application _{HEO-xylA-DTE-Fuck-FucA} A plasmid map;

FIG. 9 is an electrophoresis chart of HEO-xylA-DTE-Fuck-FucA target fragment in example 3 of the present application;

FIG. 10 is a diagram showing the verification of electrophoresis of synergistic strain MDF-9-A+B in example 4 of the present application;

FIG. 11 is a diagram showing the verification of electrophoresis of synergistic strain MDF-9-A+C in example 5 of the present application.

Detailed Description

In order that those skilled in the art will better understand the present application, a technical solution in the embodiments of the present application 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 application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application.

Halomonas lutescens MDF-9 itself has a metabolic pathway for producing poly (3-hydroxy fatty acid ester), and the present application mainly increases the metabolic pathway for producing 4HB on the basis of this, and further introduces a pathway for synthesizing 3HB and 4HB using xylose as a precursor on the basis of this, and constructs a joint metabolic pathway strain (as shown in FIG. 1). After the strain is successfully constructed, the replacement of a cheap carbon source can be realized, and meanwhile, the content of 4HB is improved.

The phosphoketolase pathway (pathway B, see fig. 1) produces mainly 3HB, which can polymerize with 4HB produced by the BDO pathway to form P34HB. xylB, pta, phaA-B, phaC in this pathway is contained endogenously in halophila, and these genes are not overexpressed by the present application. According to the application, the acetyl phosphate is generated by overexpression HEO, xylA, xfp, 3-hydroxybutyryl-CoA is generated by endogenous genes Pta and PhaA-B, and then 4-hydroxybutyryl-CoA obtained by 3-hydroxybutyryl-CoA and BDO pathways is polymerized by PhaC genes to obtain the polymer P34HB.

The ribulose 1-phosphate pathway (pathway C, see figure 1) is also the primary production of 3HB. Glycolaldehyde and dihydroxyacetone phosphate are generated after HEO, xylA, DTE, fuck, fucA is over-expressed, wherein the glycolaldehyde enters the TCA cycle, which is beneficial to promoting cell growth, dihydroxyacetone phosphate and G3P generate pyruvic acid, enter an acetyl coenzyme A path, and 4-hydroxybutyryl coenzyme A obtained through PhaABC genes and BDO paths is polymerized to form P34HB.

The construction method of the engineering strain comprises the following steps:

s3: the vector plasmids of S1 and S2 are jointly introduced into a Halomonas lutescens MDF-9 strain.

In addition, the application also introduces two other approaches based on the scheme respectively: (1) In the S3 strain, a vector plasmid containing the HEO-xylA-xfp objective gene sequence was introduced in addition to the vector plasmids obtained in S1 and S2; (2) In the step S3 strain, a vector plasmid containing the HEO-xylA-DTE-Fuck-FucA target gene sequence is introduced in addition to the vector plasmids obtained in the steps S1 and S2.

Example 1 construction of 1, 4-butanediol Metabolic pathway (hereinafter referred to as A pathway)

xylB-xylC-xylD-xylX Gene expression

(1) Plasmid construction

Overlapping extension PCR amplified xylB-xylC-xylD-xylX fragment and plasmid pSEVA321 skeleton, recombining xylB-xylC-xylD-xylX fragment and pSEVA321 skeleton under the action of Gibson ligase to form new plasmid named pSEVA321 _xylBCDX pSEVA321 by PCR _xylBCDX The xylB-xylC-xylD-xylX fragment was obtained for the template, and part of the product was sent to Bio-company for sequencing, and plasmid information is shown in FIG. 2.

(a) Primer sequences (5 '-3') for amplifying xylB-xylC-xylD-xylX fragment and pSEVA321 plasmid backbone by PCR were as follows:

upstream xylB-F: see SEQ ID No.1;

downstream xylB-R: see SEQ ID No.2;

upstream xylC-F: see SEQ ID No.3;

downstream xylC-R: see SEQ ID No.4;

upstream xylD-F: see SEQ ID No.5;

downstream xylD-R: see SEQ ID No.6;

upstream xylX-F: see SEQ ID No.7;

downstream xylX-R: see SEQ ID No.8;

upstream 321-F: see SEQ ID No.9;

downstream 321-R: see SEQ ID No.10.

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.

(b) 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 3Gibson Assemblem connection System Table

TABLE 4Gibson Assemblem linker

After the reaction is completed, agarose gel with corresponding concentration is prepared, electrophoresis is carried out to observe the size of the DNA band, 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.

(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 30min, heat shock in water bath at 42 deg.C for 2min, immediately cooling on ice for 2min, and 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 at 200rpm for 60min after uniform mixing;

step 5: centrifuging at 5000rpm for 5min to collect bacteria, discarding 350 μl supernatant, collecting 100 μl of resuspended bacteria mass, gently blowing, and spreading on LB medium containing corresponding antibiotics;

step 6: the culture medium is inverted to 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 12-16h, and picking a small amount of jointed thalli to be coated on a 60LB plate with corresponding resistance after 8 h; after 36-48h, a further monoclonal colony validation was performed.

(5) Colony PCR verification

Colony PCR results show that the bacterial strain Halomonas lutescens MDF-9 successfully transfers xylB-xylC-xylD-xylX gene, which is named MDF-9-1, and the size of the target product is verified, as shown in figure 3, the target fragment is 4687bp, and the expected result is met.

Kivd-YqhD Gene expression

Constructing a plasmid:

amplifying the Kivd-YqhD and pSEVA341 frameworks by using overlap extension PCR; under the action of Gibson ligase, the Kivd-Yqhd and pSEVA341 frameworks form new plasmid and are marked as pSEVA341 _Kivd-YqhD With pSEVA341 _Kivd-YqhD Kivd-YqhD sequence was obtained for template, and part of the product was sent to Bio Inc. for sequencing (pSEVA 341 _Kivd-YqhD Transfer to Halomonas lutescens MDF-9-1, specific procedures were referenced for xylB-xylC-xylD-xylX gene expression in example 1, and plasmid information is shown in FIG. 4.

The result shows that Kivd-YqhD gene is successfully transferred into the strain Halomonas lutescens MDF-9-1 in the embodiment, and the size of the target product is verified, as shown in figure 5, the target fragment is 2856bp, and the expected result is met. The MDF-9 strain introduced into xylose metabolic pathway A will be designated MDF-9-A.

Primer sequences (5 '-3') for amplifying the target gene by PCR are as follows:

kivD-F: see SEQ ID No.11;

kivD-R: see SEQ ID No.12;

Yqhd-F: see SEQ ID No.13;

Yqhd-R: see SEQ ID No.14.

EXAMPLE 2 construction of phosphoketolase metabolic pathway (hereinafter referred to as the B pathway)

HEO-xylA-xfp Gene expression

Constructing a plasmid:

PCR amplification using overlapping extensionHEO-xylA-xfp and pRSF skeleton; HEO-xylA-xfp and pRSF skeleton under the action of Gibson ligase, HEO-xylA-xfp and pRSF skeleton form new plasmid and are marked as pRSF _HEO-xylA-xfp In pRSF _HEO-xylA-xfp HEO-xylA-xfp sequence was obtained as template and part of the product was sent to Bio Inc. for sequencing (pRSF _HEO-xylA-xfp In Halomonas lutescens MDF-9, the specific procedure is described with reference to the expression of xylB-xylC-xylD-xylX gene in example 1, and the plasmid information is shown in FIG. 6.

The result shows that HEO-xylA-xfp gene is successfully transferred into the strain Halomonas lutescens MDF-9 of the embodiment, and the size of the target product is verified, as shown in figure 7, the target fragment is 5205bp, and the expected result is met. The MDF-9 strain introduced into xylose metabolic pathway B was designated as MDF-9-B.

Primer sequences (5 '-3') for amplifying the target gene by PCR are as follows:

HEO-F: see SEQ ID No.15;

HEO-R: see SEQ ID No.16;

xylA-F: see SEQ ID No.17;

xylA-R: see SEQ ID No.18;

xfp-F: see SEQ ID No.19;

xfp-R: see SEQ ID No.20.

Example 3 construction of the ribulose 1-phosphate Metabolic pathway (hereinafter referred to as the C pathway)

HEO-xylA-DTE-Fuck-FucA Gene expression

Constructing a plasmid:

amplifying HEO-xylA-DTE-Fuck-FucA and pRSF skeleton by using overlap extension PCR; under the action of Gibson ligase, the HEO-xylA-DTE-Fuck-FucA and pRSF frameworks form a new plasmid and are marked as pRSF _{HEO-xylA-DTE-Fuck-FucA} In pRSF _{HEO-xylA-DTE-Fuck-FucA} HEO-xylA-DTE-Fuck-FucA sequence was obtained as template and part of the product was sent to Bio Inc. for sequencing (pRSF _{HEO-xylA-DTE-Fuck-FucA} In Halomonas lutescens MDF-9, the specific procedure is described with reference to the expression of xylB-xylC-xylD-xylX gene in example 1, and the plasmid information is shown in FIG. 8. The results show that this example Halomonas lutescens MDFThe HEO-xylA-DTE-Fuck-FucA gene is successfully transferred into the strain-9, and the size of a target product is verified, as shown in figure 9, the target fragment is 5836bp, and the expected result is met. The MDF-9 strain introduced into xylose metabolic pathway C will be designated MDF-9-C.

Primer sequences (5 '-3') for amplifying the target gene by PCR are as follows:

HEO-xylA-F: see SEQ ID No.21;

HEO-xylA-R: see SEQ ID No.22;

DTE-F: see SEQ ID No.23;

DTE-R: see SEQ ID No.24;

Fuck-F: see SEQ ID No.25;

Fuck-R: see SEQ ID No.26;

FucA-F: see SEQ ID No.27;

FucA-R: see SEQ ID No.28.

Example 4 construction of a synergistic Strain of pathway A+pathway B (MDF-9-A+B)

pRSF is to be used _HEO-xylA-xfp The MDF-9-A was introduced into the strain to construct a synergistic strain of the MDF-9-A+B metabolic pathway, and the specific plasmid construction method was as described in example 1. The verification method is described in example 2, and the result is shown in FIG. 10, the target fragment is 5205bp, which shows that the MDF-9-A+B combined pathway strain is successfully constructed.

Example 5 construction of a synergistic Strain of pathway A+pathway C (MDF-9-A+C)

Plasmid pRSF _{HEO-xylA-DTE-Fuck-FucA} Introduction into MDF-9-A, construction of a synergistic strain of the MDF-9-A+C metabolic pathway, specific plasmid construction methods are described in example 1, and verification methods are described in example 3. As a result, the target fragment was 5836bp, which indicates that the MDF-9-A+B combined pathway strain was successfully constructed, as shown in FIG. 11.

Example 6 three modified bacteria and method for synergistically promoting P34HB synthesis by metabolic pathways

Fermentation culture was performed using 3 strains constructed in examples 1-5:

(1) Seed liquid preparation

(1) Strain activation

The strains are taken in a refrigerator at the temperature of minus 80 ℃ in a laboratory, the strains are picked up by a gun head, streaked and inoculated on a flat solid culture medium (yeast powder 5g/L; tryptone 10g/L; sodium chloride 60g/L; pH 8.5), and cultured for 24 hours at the temperature of 37 ℃.

(2) Primary seed culture:

single colonies were picked up and inoculated into 12mL of shaking tube (5 mL of 60LB medium: 5g/L of yeast powder; 10g/L of tryptone; 60g/L of sodium chloride; pH 8.5), and the culture broth was placed on a shaking table at 37℃and 220rpm for 12 hours.

(3) Secondary seed culture:

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

(2) Fermentation medium preparation

Fermentation medium (50 MM medium): 30g/L xylose, 50g/L sodium chloride, 1.2g/L yeast powder, 0.2-3 g/L urea, 0.2g/L anhydrous magnesium sulfate, 1.5-5.5 g/L potassium dihydrogen phosphate, fe (III) -NH ₄ -Citrate 5g/L，CaCl ₂ ·2H ₂ O 2g/L，HCl 12mol/L，ZnSO ₄ ·7H ₂ O 0.1g/L，MnCl ₂ ·4H ₂ O 0.03g/L，H ₂ BO ₃ 0.3g/L，CoCl ₂ ·6H ₂ O0.2g/L，CuSO ₄ ·5H ₂ O 0.01g/L，NiCl ₂ ·6H ₂ O 0.02g/L，NaMoO ₄ ·2H ₂ O 0.03g/L。

(3) Fermentation culture

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

(4) Determination of cell dry weight and PHA content

Cell Dry Weight (CDW): placing 30-35 mL of fermented bacterial liquid into a 50mL centrifuge tube, centrifuging for 6 minutes at room temperature, and pouring out the supernatant at 8000 rpm; 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 and storing for 2 hours; drying the centrifuge tube in a vacuum freeze dryer for 12-16 hours; the cells were weighed and dry weight (g/L) was calculated.

Determination of P34PH content: weighing 0.05g of dry bacterial cells obtained by fermentation of example 4 after grindingPlacing into an esterification pipe with good sealing property, adding 2mL of chloroform, 1700 μL of methanol and 300 μL of concentrated sulfuric acid, reacting for 1h in an oil bath at 100 ℃, cooling at room temperature, and adding ddH with a volume of 1mL ₂ And O, standing for layering after fully vibrating and uniformly mixing. 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 30m, inner diameter 0.25mm 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. Establishment of a standard curve: the PHA is quantitatively analyzed by an external standard method. The PHA sample to be analyzed is subjected to methyl esterification pretreatment to form methyl 3-hydroxybutyrate and methyl 4-hydroxybutyrate, and the analysis retention time by GC program is 2.42min and 3.14min respectively. Accurately diluting a standard substance purchased from Sigma to a corresponding concentration, and respectively drawing standard curves with the sample concentration as an X axis and the peak area as a Y axis, wherein the equation of the obtained standard curves is as follows:

3-HB：Y＝19418x-12033(R ² ＝0.9999)。

4-HB：Y＝35329x-12003(R ² ＝0.9994)。

the fermentation results are shown in Table 6:

TABLE 6 fermentation results of three modified bacteria

The results show that: the modified MDF-9 can synthesize a polymer P34HB by taking xylose as a carbon source. The strain of the combined metabolic pathway further promotes the increase of the yield of P34HB, wherein the MDF-9-A+B has the best effect, the dry weight of cells reaches 8.10g/L, the content of P34HB is up to 71.34%, and the proportion of 4HB is increased to 21.81%. Toughness increases with increasing 4HB content in the P34HB copolymer material, so that the MDF-9-A+B modified strain has strong application potential in the field of industrial materials.

By combining the above examples, the application discloses a strain for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by using xylose, and a construction method and application thereof, wherein the construction method comprises the following steps: s1: amplifying xylB-xylC-xylD-xylX target gene sequence in vitro, inserting into a vector to obtain a vector plasmid; s2: amplifying the Kivd-YqhD target gene sequence in vitro, and inserting the sequence into a vector to obtain a vector plasmid; s3: the vector plasmids of S1 and S2 are jointly introduced into a Halomonas lutescens MDF-9 strain. In addition, the application also introduces two other approaches based on the scheme respectively: (1) In the S3 strain, a vector plasmid containing the HEO-xylA-xfp objective gene sequence was introduced in addition to the vector plasmids obtained in S1 and S2; (2) In the step S3 strain, a vector plasmid containing the HEO-xylA-DTE-Fuck-FucA target gene sequence is introduced in addition to the vector plasmids obtained in the steps S1 and S2.

The application utilizes the synthetic biology technology to carry out fermentation comparison on three ways of xylose utilization metabolism so as to increase the efficiency of synthesizing P34HB by Halomonas lutescens MDF-9 strain. On the basis that MDF-9 strain has the synthetic poly 3-hydroxy fatty acid ester PHB, by optimizing xylose utilization metabolic pathway, the gene required for transferring into A path in MDF-9 is found to be capable of synthesizing P34HB. Further studies have shown that by combining pathway a with pathway B and pathway C, respectively, a combination of different metabolic pathways was constructed, which further improved the yield of P34HB. The method for producing P34HB can reduce the toxic action of raw materials on microorganisms in the production process, reduce the cost of raw materials and improve the production efficiency.

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

SEQ ID No.29 is the xylB-xylC-xylD-xylX gene sequence, SEQ ID No.30 is the Kivd-YqhD gene sequence, SEQ ID No.31 is the HEO-xylA-xfp gene sequence, and SEQ ID No.32 is the HEO-xylA-DTE-Fuck-FucA gene sequence.

Claims

1. A method for constructing a strain for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) with xylose, comprising the following steps:

s3: co-introducing the vector plasmids of S1 and S2 into a strain;

the strain is Halomonas lutescens MDF-9 with the preservation number of GDMCC

NO:61850。

2. The construction method according to claim 1, wherein in addition to the vector plasmids obtained in S1 and S2, a vector plasmid comprising a HEO-xylA-xfp objective gene sequence obtained by in vitro amplification is introduced into the strain S3.

3. The construction method according to claim 1, wherein in addition to the vector plasmids obtained in S1 and S2, a vector plasmid comprising the HEO-xylA-DTE-Fuck-FucA target gene sequence obtained by in vitro amplification is introduced into the strain S3.

4. The method according to claim 1, wherein the vector used in the step S1 is pSEVA321, and the vector used in the step S2 is pSEVA341.

5. The construction method according to claim 1, wherein the amplification procedure of the target gene in the steps S1 and S2 is as follows:

(1) Pre-denaturation: the temperature is 95 ℃ and the time is 3min;

(2) Denaturation: the temperature is 95 ℃ and the time is 15sec;

(3) Annealing: the temperature is 56-60 ℃ and the time is 15sec;

(4) Extension: the temperature is 72 ℃ and the time is 30-60sec/Kb;

(5) Cycling the steps (2) - (4) for 35 times;

(6) Extending thoroughly: the temperature is 72 ℃ and the time is 5min.

6. A strain for synthesizing poly (3-hydroxybutyrate-co-4-hydroxybutyrate), which is obtained by the construction method according to any one of claims 1 to 5.

7. A method for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) using the strain of claim 6, comprising the steps of:

(1) Preparing seed liquid;

(2) Culturing seeds in shake flasks;

(3) And (5) fermenting and culturing.

8. The method according to claim 7, wherein the fermenter temperature in the step (3) is 30 to 42 ℃, the pH is 6.5 to 11, and the time is 36 to 50 hours.

9. The method of claim 7, wherein the fermentation medium composition of step (3) comprises: 30g/L xylose, 50g/L sodium chloride, 1.2g/L yeast powder, 0.2-3 g/L urea, 0.2g/L anhydrous magnesium sulfate, 1.5-5.5 g/L potassium dihydrogen phosphate, fe (III) -NH ₄ -Citrate 5g/L，CaCl ₂ ·2H ₂ O 2g/L，HCl 12mol/L，ZnSO ₄ ·7H ₂ O 0.1g/L，MnCl ₂ ·4H ₂ O 0.03g/L，H ₂ BO ₃ 0.3g/L，CoCl ₂ ·6H ₂ O 0.2g/L，CuSO ₄ ·5H ₂ O 0.01g/L，NiCl ₂ ·6H ₂ O 0.02g/L，NaMoO ₄ ·2H ₂ O 0.03g/L。