CN116042685B - Strain for producing P34HB by utilizing xylose as well as construction method and application thereof - Google Patents

Abstract

The application discloses a strain for producing P34HB by utilizing xylose, a construction method and application thereof. The method comprises the following steps: s1: amplifying the xylB-orfZ-aldD-yqhD target gene sequence in vitro, and inserting the xylB-orfZ-aldD-yqhD sequence into a first vector to obtain a first vector plasmid; s2: amplifying the xylD-kivD-ppdA-C-B target gene sequence in vitro, and inserting the xylD-kivD-ppdA-C-B sequence into a second vector to obtain a second vector plasmid; s3: transferring the vector plasmids obtained in the S1 and the S2 into Halomonas lutescens MDF-9 competent cells together; on the basis that MDF-9 strain has a PHB synthesis path, the application utilizes synthesis biology to add a metabolic path for synthesizing 4HB by using xylose as a precursor compound to bypass TCA cycle, thereby achieving the purpose of producing P34HB, effectively reducing raw material price, reducing toxic hazard and improving 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 biosynthesis, in particular to a strain for producing P34HB by utilizing xylose, and 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, environment-friendly and biocompatible biopolyesters. PHA is the only natural polymer material completely synthesized by microorganisms, 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. The copolyester Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (Poly (3-hydroxybutyrate-co-4-hydroxybutyrate, P34HB for short) which has appeared in recent years is a brand new and most promising PHA polymer material, the properties of which can be changed by adjusting the proportion of 4HB in the polymer, and since P34HB has good biocompatibility, biodegradability and thermal processability of plastics, the copolyester Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) 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 metabolic pathways have been engineered using metabolic engineering and molecular biology, which can synthesize 4HB using glucose, but the 4HB yield 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.

Disclosure of Invention

Aiming at the defects in the prior art, the application provides a strain for synthesizing P34HB by utilizing xylose, and a construction method and application thereof. On the basis that MDF-9 strain has a PHB synthesis path, the application utilizes a synthetic biology technology to add a metabolic path using xylose as a 4HB precursor compound, thereby achieving the purpose of producing P34HB.

The application provides a construction method of a strain for synthesizing P34HB, which comprises the following steps:

s1: amplifying xylB-yqhD-aldD-orfZ gene sequence in vitro, and inserting the xylB-yqhD-aldD-orfZ target gene sequence into a first vector to obtain a first vector plasmid;

s2: amplifying xylD-kivD-ppdA-C-B target gene sequence in vitro, and inserting the xylD-kivD-ppdA-C-B target gene sequence into a second vector to obtain a second vector plasmid;

s3: transferring the first vector plasmid obtained in the step S1 and the second vector plasmid obtained in the step S2 into Halomonas lutescens MDF-9 competent cells together; the accession number of Halomonas lutescens MDF-9 is GDMCC No.61850.

The nucleotide sequence of the xylB gene is shown as SEQ ID NO.1, the nucleotide sequence of the xylD gene is shown as SEQ ID NO.2, the nucleotide sequence of the kivD gene is shown as SEQ ID NO.3, the nucleotide sequence of the yqhD gene is shown as SEQ ID NO.4, the nucleotide sequence of the ppdA-C-B gene is shown as SEQ ID NO.5, the nucleotide sequence of the orfZ gene is shown as SEQ ID NO.6, and the nucleotide sequence of the aldD gene is shown as SEQ ID NO. 7.

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. Deposit No. GDMCC NO:61850. the strain was named MDF-9 and the classification was named Salmonella (Halomonas lutescens).

The Halomonas lutescens MDF-9 strain of the application has been disclosed in the prior application with the application number 202110929333.2 and the name of the application is a salt monad and application thereof.

Further, the amplification system of the target gene xylB-yqhD-aldD-orfZ in the step S1 and the target gene xylD-kivD-ppdA-C-B in the step S2 is as follows:

further, the amplification procedure of the target gene xylB-yqhD-aldD-orfZ in the S1 and the target gene xylD-kivD-ppdA-C-B in the step S2 is as follows:

further, in the step S1, the xylB-yqhD-aldD-orfZ gene fragment is obtained through overlap extension PCR, and the xylB-yqhD-aldD-orfZ gene fragment is inserted into the pRSF-1b vector through double enzyme digestion connection, so that the first vector plasmid is obtained.

Further, in the step S2, the xylD-kivD-ppdA-C-B gene fragment is obtained by overlap extension PCR, and the xylD-kivD-ppdA-C-B gene fragment is inserted into the pET-23a (+) vector through double enzyme digestion connection to obtain the second vector plasmid.

Further, the double enzyme digestion system comprises:

the application also provides an engineering strain for synthesizing the P34HB, which is obtained by the construction method.

The application also provides a method for producing P34HB by using the engineering strain, which comprises the following steps:

(1) Plate seed culture: activating strains;

(2) Culturing seeds in shake flasks;

(3) Dissolved oxygen and pH electrode correction;

(4) Setting fermentation parameters;

(5) Inoculating;

(6) Controlling a fermentation process;

(7) Extracting PHA from thallus.

Further, in the step (6), the temperature of the fermentation tank is controlled to be 36-38 ℃.

Further, in the step (6), the pH is controlled to 7.5 to 9.5, preferably 8.2 to 8.5.

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

1. the application uses exogenously added xylose as a precursor compound, and has the advantages of environmental protection, no pollution, low price, easy acquisition and high production safety coefficient.

2. The P4HB polyester can be prepared by biological fermentation, so that the production cost can be reduced, and the polyester is safe and nontoxic.

3. The application uses halophila MDF-9 as chassis organism, the fermentation process is low-salt and high-alkali, so the fermentation process does not need sterilization, the operation is more convenient, continuous inoculation or substrate supplementation can be realized for continuous fermentation, and compared with the strains of Escherichia, pseudomonas and Aeromonas, the application saves more energy.

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 shows the metabolic process for producing 4HB by the engineering bacterium of the present application.

FIG. 2 shows pRSF-1b constructed successfully in accordance with the present application _{bxylB-yqhD-aldD-orfZ} Carrier structure.

FIG. 3 shows the successful construction of pET-23a (+) _{xylD-kivD-ppdA-C-B} Carrier structure.

FIG. 4 shows the result of PCR verification (xylB-yqhD-aldD-orfZ) of the engineering bacterium prepared in example 1 of the present application.

FIG. 5 shows the result of PCR verification (xylD-kivD-ppdA-C-B) of the engineering bacterium prepared in example 2 of the present application.

FIG. 6 is a GC spectrum of PHB standard.

FIG. 7 is a GC spectrum of gamma-butyrolactone as a standard.

FIG. 8 is a GC spectrum of the fermentation results of the original strain.

FIG. 9 is a GC diagram of P34HB produced by fermenting the engineering bacterium constructed in the application under the condition of pH 9.0.

FIG. 10 is a GC diagram of P34HB produced by fermenting the engineering bacteria constructed in the application under the condition of pH 8.2.

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.

Xylose dehydrogenase (xylB encoding) converts xylose to xylonic acid; xylose dehydrogenase (xylD encodes) converts xylitol into 3-deoxy-glycerol-glutarate; ketoacid decarboxylase (kivD code) converts 3-deoxy-glycerol-glutarate to 3, 4-dihydroxybutanal; aldehyde reductase (yqhD encoded) converts 3, 4-dihydroxybutanal to 1,2, 4-butanetriol; diol dehydratase (ppdA-C-B encoding) converts 1,2, 4-butanetriol to 4-hydroxybutyraldehyde; aldehyde dehydrogenase (aldD encoding) converts 4-hydroxybutyraldehyde to 4-hydroxybutyrate hydrolase (orfZ encoding) converts 4-hydroxybutyrate to 4-hydroxybutyrate-CoA; pha polymerase (phaC encoding) polymerizes 4-hydroxybutyryl-CoA to poly 4-hydroxybutyrate; since MDF-9 itself has a metabolic pathway for producing poly-3-hydroxy fatty acid ester, the present application mainly adds a metabolic pathway for producing 4HB based on this metabolic pathway (as shown in FIG. 1). After the strain is successfully constructed, the purpose of producing 3HB and 4HB copolymer (P34 HB for short) can be achieved.

The technical scheme of the application is as follows:

1. genes related to the metabolism of xylose to generate 4-hydroxy butyraldehyde are introduced into MDF-9 through a vector plasmid, so that after xylose enters the MDF-9, the xylose can jump out of the TCA cycle and 4HB is produced in a new path.

2.3HB monomer (3 HB production route is MDF-9 self route) and 4HB monomer under the action of PHA polymerase PhaC, the carboxyl group of monomer and adjacent monomer can form ester bond to form binary copolymerization P34HB.

The flow of the application is as follows:

1. amplifying xylB-yqhD-aldD-orfZ target gene sequence in vitro, and inserting the xylB-yqhD-aldD-orfZ target gene sequence into a first vector to obtain a first vector plasmid;

2. amplifying xylD-kivD-ppdA-C-B target gene sequence in vitro, and inserting the xylD-kivD-ppdA-C-B target gene sequence into a second vector to obtain a second vector plasmid;

3. transferring the first vector plasmid obtained in the step S1 and the second vector plasmid obtained in the step S2 into Halomonas lutescens MDF-9 competent cells together; the accession number of Halomonas lutescens MDF-9 is GDMCC No.61850.

4. And (5) functional verification.

EXAMPLE 1 xylB-orfZ-aldD-yqhD Gene expression

(1) Constructing a plasmid: PCR amplified xylB-orfZ-aldD-yqhD; the target fragment and the vector pRSF-1b are subjected to double enzyme digestion by PmlI and NotI, and the xylB-orfZ-aldD-yqhD gene fragment and the vector pRSF-1b are recombined to form a novel plasmid which is named pRSF-1b under the action of T4 ligase _{xylB-orfZ-aldD-yqhD} The method comprises the steps of carrying out a first treatment on the surface of the pRSF-1b by overlap extension PCR _{xylB-orfZ-aldD-yqhD} The template was obtained from xylB-orfZ-aldD-yqhD and a portion of the product was sent to Bio Inc. for sequencing. Plasmid information is shown in FIG. 2.

(b) Primer sequences for amplifying xylB-orfZ-aldD-yqhD fragments

The upstream primer xylB-F (PmlI) is 5 '-CACGTGATGAACAACTTTAATCTGCACACC-3';

a downstream primer: xylB-R is 5 '-GATTTATAATACCACTTCATTCAGTGGTTGTGGC-3';

an upstream primer: orfZ-F, 5 '-GATTTATAATACCACTTCATTCAGTGGTTGTGGC-3';

a downstream primer: orfZ-R5 '-GGGGTACCTCATTTACGGTTCCTTTCC-3';

an upstream primer: aldD-F, 5 '-CGGAAAGGAACCGTAAATGAGGTACCCC-3';

a downstream primer: aldD-R5 '-GTTGTTCATTCAGAAGAGCCCGAGC-3';

an upstream primer: yqhD-F5 '-CTCGGGCTCTTCTGAATGAACAACTTTAATC-3';

a downstream primer: yqhD-R (NotI): 5 '-GCGGCCGCTTCGTATATACGGCGGC-3'.

The amplification system is as follows:

the PCR procedure was:

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) Restriction enzyme reaction of vector

Double cleavage reactions were performed according to the following system, and after all reagents were added, the products were electrophoresed in an incubator at 37℃for 3-4 hours to see if cleavage was successful.

After the PCR 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.

(d) Recovery of amplified products and carrier cleavage products

Step 1, recovering amplification products and enzyme digestion products by using an agarose gel DNA recovery kit (HiPure Gel Pure DNA mini Kit);

step 2. Gel of the target DNA fragment cut under UV lamp was put into 2ml collection tube, and 500. Mu.l Buffer GDP was added. If the gel concentration is large, the volume of Buffer GDP can be increased appropriately. Placing the gel into an oven for 10-15min, and mixing the gel upside down until the gel is completely dissolved;

step 3. HiPure DNA mini Colum is loaded into a 2ml centrifuge tube, the sol solution is transferred to the column, and if the sol solution exceeds 700 mu l, the sol solution is transferred in two times. Centrifuging at 12000rpm for 1min;

step 4. The filtrate was discarded and the column was reloaded into a 2ml collection tube. Add 300. Mu.l Buffer GDP and rest for 1min;

step 5. The filtrate was discarded and the column was reloaded into a 2ml collection tube. 600 μl Buffer DW2 (absolute ethanol has been added in advance) was added to the column. Centrifuging at 12000rpm for 1min;

step 6, repeating the steps for one time;

step 7. The filtrate was discarded and the column was reloaded into a 2ml collection tube. Centrifuging at 12000rpm for 2min;

step 8. Reloading the column into a new 1.5ml centrifuge tube. Placing in an oven for 5min, and opening the cover of the column to thoroughly remove absolute ethyl alcohol (absolute ethyl alcohol residue can affect subsequent reaction);

step 9. 15-30. Mu.l EB was added to the center of the column membrane and left at Room Temperature (RT) for 2min. Centrifuge at 12000rpm for 1min. The column can be repeated once and the eluted DNA stored at-20 ℃.

(e) Ligation of the fragment of interest with the vector

The enzyme was cloned in one step (Clon ExpressII One Step Cloning Kit) as follows:

adding into a micro-tube, mixing, and centrifuging briefly to collect the reaction solution to the bottom of the tube. The incubator was placed at 37℃for 30min. The microtubes were removed and immediately placed on ice or reduced to 4 ℃.

X (amount of carrier used) = (0.01X number of carrier bases)/carrier recovered product concentration;

y (vector usage) = (0.02×number of target gene bases)/target gene recovery product concentration.

(2) MDF-9 conversion

Step 1, taking out MDF-9 clone competent cells prepared in advance from the temperature of minus 80 ℃, thawing on ice, and waiting for fungus blocks to melt after 5 min.

And 2, adding 10 mu l of the connection product into the competent cells, mixing the reaction solution uniformly by using a gentle elastic tube wall (stirring and mixing uniformly without shaking), and standing the mixture on ice for 10-30min. And (3) injection: the ligation product conversion volume should not exceed at most 1/10 of the competent cell volume used;

step 3.42 ℃ water bath heat shock is carried out for 45-90s, then the obtained product is immediately placed on ice for cooling for 2-3min, and the conversion efficiency is reduced by shaking;

step 4, adding 700 mu l of LB culture medium (without antibiotics) into the centrifuge tube, uniformly mixing, and putting into a shaking table at 37 ℃ for resuscitation at 200rpm for 60min;

step 5.5000rpm centrifugation is carried out for 5min for bacterial recovery, 600 μl of supernatant is discarded, 100 μl of resuspended bacterial cake is gently blown off and coated on LB medium containing corresponding antibiotics;

and 6, inverting the culture medium into a 37 ℃ incubator for culturing for 12-15 hours.

(3) Plasmid small lifter

Plasmid small extract kit of Magen biological company (HiPure Plasmid Micro Kit)

Step 1, inoculating positive monoclonal colony into 5-10ml LB culture medium containing corresponding antibiotics, and placing in a shaking table at 37 ℃ for 12-16h. Preserving the bacterial liquid at-80 ℃ so as to facilitate subsequent inoculation and propagation;

step 2, 2ml of bacterial liquid is taken and put into a 2ml centrifuge tube prepared in advance, and is centrifuged at 12000rpm for 30-60s, and is collected for 2-3 times;

and 3, pouring and discarding the supernatant, and lightly beating on absorbent paper to suck the residual liquid. Adding 250 μl of precooled Buffer P1/RNase A mixed solution, and thoroughly suspending bacteria on an oscillator by high-speed vortex;

step 4. 250. Mu.l Buffer P2 was added to the 2ml tube and mixed gently upside down for 8-10 times, the solution became viscous and transparent indicating that the bacteria had been well lysed. And (3) injection: genomic DNA contamination can result if vortexing. If the number of samples is large, the operation is rapid;

step 5, adding 350 mu l Buffer P3 into the heavy suspension, immediately reversing and uniformly mixing for 8-10 times to neutralize the solution, and preventing precipitation agglomeration from affecting the neutralization effect;

step 6.12000rpm centrifugation for 10min;

step 7. HiPure DNA mini Colum II is packed into 2ml Collection Tube and the supernatant is transferred to the column. Centrifuging at 12000rpm for 30-60s;

step 8. The filtrate was discarded and 500. Mu.l Buffer PW1 was added to the column. Centrifuging at 12000rpm for 30-60s;

step 9. The filtrate was discarded and 600. Mu.l Buffer PW2, which had been diluted with absolute ethanol, was added to the column. Centrifuging at 12000rpm for 30-60s;

step 10, repeating the previous step;

step 11. The column was placed in a 1.5ml centrifuge tube prepared in advance, 15-30. Mu.l EB was added to the center of the column membrane and left at Room Temperature (RT) for 2min. Eluting DNA by centrifugation at 12000rpm for 1min;

step 12. The column was discarded and the plasmid was used for subsequent reaction storage at-20 ℃.

(4) Enzyme digestion, sequencing and identification

The result shows that the MDF-9 strain of the embodiment successfully transfers xylB-orfZ-aldD-yqhD gene, and the size of the target product is verified, as shown in figure 4, the target fragment is 4337p, and the expected result is met.

Example 2 xylD-kivD-ppdA-C-B Gene expression

Constructing a plasmid: amplifying xylD-kivD-ppdA-C-B by overlap extension PCR; notI and XhoI cleave the desired fragment xylD-kivD-ppdA-C-B and pET-23a (+); under the action of T4 ligase, xylD-kivD-ppdA-C-B and vector pET-23a (+) are recombined to form a new plasmid which is named pET-23a (+) _{xylD-kivD-ppdA-C-B} The method comprises the steps of carrying out a first treatment on the surface of the pET-23a (+C) by overlap extension PCR _{xylD-kivD-ppdA-C-B} The xylD-kivD-ppdA-C-B was obtained as a template, and a part of the product was sent to Bio Inc. for sequencing (specific procedures refer to xylB-orfZ-aldD-yqhD gene expression in example 1), and plasmid information is shown in FIG. 3.

(a) Primer sequence:

xylD-F(NotⅠ):

5`-ATAAGAATGCGGCCGCTAAACTATATGAGGTCCGCCTTGTCTAAC-3`；

xylD-R:5`-GGTAATCTCCTACTGTATACATTCAGTGGTTGTG-3`；

kivD-F:5`-CTGAATGTATACAGTAGGAGATTACCTATTAGACCG-3`；

kivD-R:5`-CTTTTCGATCTCATTTATGATTTATTTTGTTCAGC-3`；

ppdA-C-B-F:5`-GAACAAAATAAATCATAAATGAGATCGAAAAGATTTGAAGC-3`；

ppdA-C-B-R(XhoⅠ):5`-CCGCTCGAGCGGTCAAAGCGCCACGCGCAGTTCC-3`。

(b) Restriction enzyme reaction of vector

Double cleavage reactions were performed according to the following system, and after all reagents were added, the products were electrophoresed in an incubator at 37℃for 3-4 hours to see if cleavage was successful.

The result shows that the xylD-kivD-ppdA-C-B gene is successfully transferred into the MDF-9 strain of the embodiment, and the size of the target product is verified, as shown in figure 5, the target fragment is 6285bp, and the expected result is met.

EXAMPLE 3 Synthesis of P34HB by engineering Strain of the application

(1) Culture medium:

LB plate medium: yeast extract powder 0.5%; tryptone 1%; 6% sodium chloride, 1.8g/100mL agar powder, 50. Mu.g/mL ampicillin, 30. Mu.g/mL kanamycin, pH 8.0.

LB shake flask medium: yeast extract powder 0.1%; sodium chloride 6%, ampicillin 50. Mu.g/mL, kanamycin 30. Mu.g/mL, pH8.0, 30mL/250mL.

Component I: magnesium sulfate: 0.2g/L; urea: 0.6g/L; (50 times concentrated mother liquor: 10g/L magnesium sulfate, 30g/L urea);

component II: potassium dihydrogen phosphate (5.2 g/L) and 260g/L of mother liquor which is 50 times that of the potassium dihydrogen phosphate;

glucose solution (30 g/L): glucose mother liquor is prepared by 500g/L;

component III (10 mL/L): 5g/L ferric ammonium citrate, 1.5g/L anhydrous calcium chloride and 41.7 ml/L12 mol/L hydrochloric acid;

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

Fermentation medium:

36g of corn steep liquor dry powder (added after dissolution alone);

MgSO ₄ (magnesium sulfate) 0.6g;

urea 6g;

KH ₂ PO ₄ 15.6g of (dipotassium hydrogen phosphate);

C ₆ H ₁₂ O ₆ (glucose) 60g (20 g/L);

120g of NaCl (sodium chloride);

60g (20 g/L) of xylose;

ampicillin 50. Mu.g/mL;

kanamycin 30. Mu.g/mL.

Feed medium:

the pH of the culture medium is regulated by 4% NaOH, and the defoamer is added after 5% of the defoamer is prepared.

The pH is controlled between 7.5 and 9.5.

(2) Experimental protocol

2.1 plate seed culture: strain activation

The laboratory is used for taking strains in a refrigerator at the temperature of 4 ℃, the hands are sterilized by alcohol cotton, and after the hands are completely dried, an alcohol lamp is turned on. The name, date and time of the inoculum was written on the bottom of the dish. Single colonies were picked with an inoculating loop and streaked onto plates for 24 hours. The above procedure was repeated, the plate was inoculated for two stages, and cultured for 24 hours.

2.2 shake flask seed culture: first-stage bacterial liquid: taking a secondary plate, selecting a single strain, inoculating the single strain into an LB shake flask culture medium, placing the culture solution into a shaking table for culture at 37 ℃ and 220rpm, and adding 500mM IPTG when the OD is 0.1-0.2, and culturing for 12 hours.

Secondary bacterial liquid: the primary bacterial liquid was aspirated to 300. Mu.l (1% of the inoculum size), inoculated in a secondary shake flask medium, and the culture liquid was placed on a shaker at 37℃and 220rpm for 12 hours.

2.3 dissolved oxygen and pH electrode correction: the fermenter is washed clean with water, the DO electrode is marked with zero, the pH electrode is calibrated at two points (standard buffer solution is placed at normal temperature), and the fermenter is correctly installed after calibration, and the DO electrode is empty. (pH electrode and dissolved oxygen electrode do not need to be eliminated)

2.4 setting fermentation parameters: component III and component IV are prepared (dissolved in advance), and the temperature of the fermentation tank is controlled between 35 ℃ and 40 ℃. And adjusting pH to 7.5-9.5 (paying attention to opening degree of a tank inlet valve) by using alkali liquor, adding 0.3ml of defoaming agent, opening an air tank inlet valve to adjust initial air flow to 2L/min, feeding the tank, adjusting initial rotation speed to 400rpm, and calibrating to 100% after OD indication is stable.

2.5 inoculation: selecting 300ml seed solution with uniform color and few sediment, inoculating the seed solution into a fermentation tank, shaking the residual liquid and reserving 10ml bacterial liquid for measuring OD, residual sugar, and pouring into component III (30 ml) and component IV (3 ml) after inoculation.

2.6 fermentation process control: controlling the temperature of the fermentation tank to be 37+/-1 ℃, controlling the pH value to be 8.5+/-1, alternately regulating the rotating speed and the air flow to control the dissolved oxygen to be 35% -80%, controlling the initial rotating speed to be 400rpm, controlling the ventilation amount to be 2L/min, regulating the rotating speed to be 50rpm each time, regulating the maximum rotating speed to be 800rpm, regulating the air flow to be 0.5L/min each time, regulating the maximum rotating speed to be 3L/min, sampling every two hours for the first four hours, and measuring the OD and the residual sugar.

2.6.1 under normal conditions, dissolved oxygen can gradually drop, the dissolved oxygen is controlled to be more than 35% by converting gas, the condition of the liquid level in the tank is confirmed every half an hour, and the liquid level is too high, so that defoaming is needed (a small amount of times, and excessive avoidance is avoided).

2.6.2 sampling every two hours, firstly discharging for a few seconds, then taking 2ml of bacterial liquid, detecting the offline pH value of the bacterial liquid, diluting, and measuring the residual sugar and OD value.

And controlling the residual sugar to be 5-15g/L by feeding.

2.7 extraction of PHA from cells

Repeatedly adding 10000prm of water, discarding supernatant, and centrifuging for 6 times to obtain the product.

Example 4 detection of product concentration

By using gas chromatography detection, the success of engineering bacteria construction can be demonstrated as long as 3HB/4HB exists in the product.

The basic principle of the P34HB assay is to add concentrated sulfuric acid to break down the ester bonds in the PHA structure into 3-hydroxybutyric acid and 4-hydroxybutyric acid. Under the acid catalysis condition, 3-hydroxybutyric acid and 4-hydroxybutyric acid react with methanol to be converted into 3-hydroxybutyric acid methyl ester and 4-hydroxybutyric acid methyl ester, and the content of the components can be detected by Gas Chromatography (GC). The specific measurement method is as follows:

(1) Weighing 0.05g of dry thallus obtained by fermenting example 2 after grinding, placing into an esterification pipe with good sealing property, adding 2mL of chloroform, 850 μL of methanol and 150 μL of concentrated sulfuric acid, reacting for 1h in an oil bath at 100deg.C, cooling at room temperature, and adding 1ddH in mL volume 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 a hydrogen Flame Ion Detector (FID). The carrier gas is high purity nitrogen. The temperature programming settings were as follows:

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.

(2) 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＝19356x-11252(R2＝0.9999)。

4-HB：Y＝35457x-11899(R ² ＝0.9992)。

parameter results 1: the dry weight of the cells obtained in the 5L fermenter culture at pH9.0 was 71g/L, wherein the 4HB content was 11%, and the 3HB content was 55%.

Parameter results 2: the dry weight of the cells obtained in the 5L fermenter culture at pH8.2 was 80g/L, the 4HB content was 24%, and the 3HB content was 54%.

P34HB is an abbreviation of 4HB and 3HB copolymer, and 3HB and 4HB monomers form ester bonds with the hydroxyl groups of the adjacent monomers under the action of PHA polymerase PhaC, forming binary copolymer P34HB.

FIGS. 6-10 show the results of GC measurements, PHB and gamma-butyrolactone as standards, purchased from Sigma.

The GC spectrum of the standard PHB is shown in FIG. 6, 2.651min is 3HB peak, 4.049 is benzoic acid standard peak.

The GC spectrum of the standard gamma-butyrolactone is shown in FIG. 7, wherein 2.941min and 3.011 are 4HB peaks, and 4.049min is benzoic acid standard substance peak.

The GC spectrum of the fermentation result of the original strain is shown in FIG. 8, wherein 2.661min is 3HB peak, and 4.100min is benzoic acid standard substance peak.

The GC spectrum of P34HB produced by fermenting the engineering bacteria under the condition of pH9.0 is shown in figure 9, wherein 2.660min is a 3HB peak, 2.948min and 3.015 are both 4HB peaks, and 4.053min is a benzoic acid standard substance peak.

The GC spectrum of P34HB produced by fermenting the engineering bacteria under the condition of pH8.2 is shown in figure 10, 2.684min is a 3HB peak, 2.980min and 3.046 are both 4HB peaks, and 4.090min is a benzoic acid standard substance peak.

The detection result of the GC proves that 4HB can be produced by fermenting the engineering bacteria by utilizing xylose, which indicates that the strain construction is successful.

In summary, the application discloses a strain for producing P34HB by utilizing xylose, a construction method and application thereof. The method comprises the following steps: s1: amplifying the xylB-orfZ-aldD-yqhD target gene sequence in vitro, and inserting the xylB-orfZ-aldD-yqhD sequence into a first vector to obtain a first vector plasmid; s2: amplifying the xylD-kivD-ppdA-C-B target gene sequence in vitro, and inserting the xylD-kivD-ppdA-C-B sequence into a second vector to obtain a second vector plasmid; s3: the vector plasmids obtained in S1 and S2 are jointly transferred into Halomonas lutescens MDF-9 competent cells. On the basis that MDF-9 strain has a PHB synthesis path, the application utilizes a synthetic biological technology to add a metabolic path for synthesizing 4HB by using xylose as a precursor compound to bypass TCA cycle, thereby achieving the purpose of producing P34HB, effectively reducing raw material price, reducing toxic hazard risk and improving 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.

Claims (8)

1. A method for constructing a strain for producing P34HB by using xylose, comprising the steps of:

s1: amplifying xylB-orfZ-aldD-yqhD target gene sequence in vitro, inserting xylB-orfZ-aldD-yqhD sequence into a first vector, wherein the first vector is pRSF-1b, and obtaining a first vector plasmid;

s2: amplifying the xylD-kivD-ppdA-C-B target gene sequence in vitro, and inserting the xylD-kivD-ppdA-C-B sequence into a second vector, wherein the second vector is pET-23a (+), so as to obtain a second vector plasmid;

s3: transferring the first vector plasmid obtained in the step S1 and the second vector plasmid obtained in the step S2 into Halomonas lutescens MDF-9 competent cells together; the preservation number of Halomonas lutescens MDF-9 is GDMCC NO.61850;

the nucleotide sequence of the xylB gene is shown as SEQ ID NO. 1; the nucleotide sequence of the orfZ gene is shown as SEQ ID NO. 6; the nucleotide sequence of the aldD gene is shown in SEQ ID NO. 7; the nucleotide sequence of the yqhD gene is shown as SEQ ID NO. 4; the nucleotide sequence of the xylD gene is shown as SEQ ID NO. 2; the nucleotide sequence of the kivD gene is shown as SEQ ID NO. 3; the nucleotide sequence of the ppdA-C-B gene is shown as SEQ ID NO. 5.

2. The construction method according to claim 1, wherein the amplification procedure of the desired gene xylB-orfZ-aldD-yqhD in step S1 and the desired gene xylD-kivD-ppdA-C-B in step 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 ℃, the time is 30-60sec/kb, and the cycle number is 35 cycles of the steps (2) - (4);

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

(6) Maintained at 16 ℃.

3. The construction method according to claim 1, wherein in the step S1, the xylB-orfZ-aldD-yqhD gene fragment is obtained by overlap extension PCR, and the xylB-orfZ-aldD-yqhD gene fragment is inserted into pRSF-1b vector by double cleavage ligation to obtain the first vector plasmid.

4. The construction method according to claim 1, wherein in the step S2, the xylD-kivD-ppdA-C-B gene fragment is obtained by overlap extension PCR, and the xylD-kivD-ppdA-C-B gene fragment is inserted into pET-23a (+) vector by double cleavage ligation to obtain the second vector plasmid.

5. The method according to claim 3 or 4, wherein the double enzyme digestion system is:

6. an engineered strain for producing P34HB using xylose, characterized by being obtained by the construction method according to any one of claims 1 to 5.

7. A method for producing P34HB using the engineered strain of claim 6, comprising the steps of:

(1) Plate seed culture: activating strains;

(2) Culturing seeds in shake flasks;

(3) Dissolved oxygen and pH electrode correction;

(4) Setting fermentation parameters;

(5) Inoculating;

(6) Controlling a fermentation process; controlling the temperature of the fermentation tank to be 36-38 ℃ and controlling the pH value to be 7.5-9.5;

(7) Extracting PHA from thallus.

8. The method of claim 7, wherein the pH in step (6) is 8.2 to 8.5.