A recombinant halomonas, its construction method and application in producing polyhydroxyalkanoate

Abstract

The application provides a recombinant Halomonas, a construction method thereof and application of the recombinant Halomonas in production of polyhydroxyalkanoate. The recombinant Halomonas expresses 2-keto acid decarboxylase or a 2-keto acid decarboxylase mutant. The application constructs a 3HV synthesis path mediated by 2-keto acid decarboxylase, which can directly convert alpha-ketoglutarate in the tricarboxylic acid cycle into 3-hydroxyvaleryl coenzyme A, realizes de novo synthesis of 3HV monomers, can efficiently utilize conventional carbon sources to synthesize 3HV, breaks through the technical bottleneck of traditional dependence on exogenous propionic acid or valeric acid precursors, realizes obtaining of a higher proportion of 3HV under the condition of no need of adding exogenous precursors, and significantly simplifies the fermentation process and reduces raw material cost and production complexity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a recombinant Halomonas sp., a construction method thereof and an application of the same in producing polyhydroxyalkanoates. BACKGROUND

[0002] Polyhydroxyalkanoates (PHA) are a class of biodegradable polyesters synthesized by microorganisms, mainly composed of various hydroxy fatty acid monomers, and have good biocompatibility, biodegradability and processability. PHA is a green and environmentally friendly material that is currently recognized as a substitute for petroleum-based plastics. PHA has been widely used in packaging materials, medical devices, agricultural films and other fields. The performance of PHA depends largely on its monomer composition and proportion. Common PHA includes homopolymers such as poly-3-hydroxybutyrate (PHB), and copolymers such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV).

[0003] Among them, P3HB3HV can significantly improve the brittleness of the material, improve its flexibility and ductility by adjusting the proportion of 3-hydroxyvaleric acid (3HV) monomer; but its production usually relies on the addition of high-priced 3HV precursors (such as propionic acid or valeric acid), resulting in high production cost, which limits the breadth of its industrial application. P3HB4HB3HV introduces 4-hydroxybutyric acid (4HB) monomer to make the material have the flexibility similar to low-density polyethylene, and further improves the processing performance by 3HV, which has excellent ductility and mechanical strength. However, due to the need to accurately control the proportion of the three monomers, the synthesis process is complex, the production equipment and control strategy are high, and the supply stability of 4HB precursors (such as γ-butyrolactone) is poor and expensive, which further limits the feasibility of large-scale production.

[0004] Currently, in the research of increasing the content of 3HV in PHA copolymer, microbial metabolic engineering and enzyme engineering are mainly used. The existing metabolic engineering strategies mainly include: (1) on the basis of knocking out the propionyl coenzyme A degradation pathway, 3HV is synthesized by adding propionic acid exogenously; (2) directly adding valeric acid to increase the content of 3HV; (3) constructing a de novo synthesis pathway of 3HV, for example, introducing exogenous genes to convert succinyl coenzyme A in the tricarboxylic acid cycle (TCA) into propionyl coenzyme A, thereby improving the endogenous synthesis ability of 3HV. For the above-mentioned engineering strains, carbon source optimization, gene modification and fermentation process regulation measures are usually needed to improve the yield and proportion of 3HV. scpB

[0005] ​In the aspect of enzyme engineering, mutant libraries containing different enzyme activities are usually constructed by mutation means, and strains with different proportions of products are obtained by high-throughput screening, so as to provide diversified selection for PHA materials with different performance requirements. However, there are still many technical bottlenecks in the existing 3HV synthesis strategy: (1) the selectivity of the product regulation pathway is limited, and at present, it mainly depends on the propionic acid / valeric acid pathway, and lacks other effective synthesis means; (2) the exogenous addition of 3HV precursors often has a toxic effect on the strain while improving the synthesis efficiency of the target product, thereby inhibiting the normal growth of the strain; (3) the addition of precursors leads to a complex fermentation process and high production cost, and in the industrial amplification process, the more complex the raw materials are, the more likely it is to interfere with the process stability, which is not conducive to large-scale promotion.

[0006] Therefore, it is urgent to develop a new type of PHA copolymer synthesis strategy without the need for additional precursors, with a more efficient and stable synthesis path, strong strain tolerance and a simplified production process, so as to further reduce the industrialization threshold of PHA and promote its wide application in the field of environmental protection materials. SUMMARY

[0007] In view of the defects in the prior art, the application provides a recombinant Halomonas, a construction method thereof and application of the recombinant Halomonas in producing polyhydroxyalkanoate.

[0008] The application provides a recombinant Halomonas, wherein the recombinant Halomonas expresses any one of the following:

[0009] (1) 2-keto acid decarboxylase;

[0010] (2) 2-keto acid decarboxylase mutant;

[0011] The amino acid sequence of the 2-keto acid decarboxylase is shown in SEQ ID No. 24;

[0012] The 2-keto acid decarboxylase mutant is different from the 2-keto acid decarboxylase in any one of the following:

[0013] (a) the 99th amino acid is threonine;

[0014] (b) the 7th amino acid is serine and the 196th amino acid is histidine;

[0015] (c) the 202nd amino acid is tryptophan;

[0016] (d) the 512th amino acid is glycine;

[0017] (e) the 219th amino acid is isoleucine and the 470th amino acid is arginine;

[0018] (f) the 3rd amino acid is arginine, the 323rd amino acid is valine, and the 510th amino acid is glutamic acid.

[0019] In some embodiments, the 2-keto acid decarboxylase is derived from Lactococcus or a derivative strain thereof, , Colletes gigas or a derivative strain thereof, Atopostipes suicloacalis or a derivative strain thereof; preferably the Lactococcus lactis .

[0020] In some embodiments, the recombinant Halomonas sp. further expresses xylB gene, xylC gene, xylD gene, xylX gene and yqhD gene.

[0021] In some embodiments, the recombinant Halomonas sp. expresses xylB gene , xylC gene , xylD gene 、 xylX gene is derived from Caulobacter or a derivative strain thereof, , Pseudomonas sp. or a derivative strain thereof, Azotobacter vinelandii or a derivative strain thereof; the yqhD gene is derived from E.coli or a derivative strain thereof, Salmonella enterica or a derivative strain thereof, Klebsiella pneumoniae or a derivative strain thereof; preferably the xylB, xylC, xylD, xylX gene is derived from Caulobacter crescentus, yqhD . E.coli MG1655

[0022] In some embodiments, the Halomonas sp. is or a derivative strain thereof, Halomonas bluephagenesis or a derivative strain thereof, Halomonas campaniensis or a derivative strain thereof and Halomonas aydingkolgenesis or a derivative strain thereof.

[0023] In specific embodiments of the present application, the Halomonas sp. is preferably MDF-9, LY01, LY02, LY03, LY04.

[0024] The present application also provides a method for constructing the recombinant Halomonas sp., comprising the following steps:

[0025] (1) constructing an expression vector containing a 2-keto acid decarboxylase gene or a 2-keto acid decarboxylase mutant gene; ​

[0026] (2) introducing the expression vector constructed in (1) into Halomonas sp. to obtain the recombinant Halomonas sp.

[0027] The nucleotide sequence of the 2-keto acid decarboxylase gene is shown in SEQ ID NO. 1.

[0028] In some embodiments, the expression vector is in the form of a plasmid.

[0029] In some embodiments, the plasmid can integrate the exogenous gene into the chromosomal genome for expression or can express the exogenous gene on the plasmid; when expressed on the plasmid, the plasmid can be a high-copy plasmid or a low-copy plasmid.

[0030] In some embodiments, the high-copy plasmid is, for example, pSEVA341, ColE1, pMB1, pUC (such as pUC18, pUC19, etc.), pBluescript (such as pBluescript II KS+, pBluescript II SK+, etc.), pTZ57R, pGEM, etc.

[0031] In some embodiments, the low-copy plasmid is, for example, pRE series (such as pRE112), pSEVA321, pSC101, pACYC184, pWE15, pET series (such as pET-15b and pET-28a).

[0032] The present application also provides a method for producing polyhydroxyalkanoate, which comprises fermenting the recombinant Halomonas sp.

[0033] The method for producing polyhydroxyalkanoate can be performed in the form of batch fermentation, or in the form of continuous fermentation or fed-batch fermentation. The fermentation can be optionally performed for a desired duration, for example, 12 hours to 120 hours, such as 24 hours, 48 hours, 60 hours, 72 hours, etc., but is not limited thereto, and a person skilled in the art can appropriately select the fermentation time according to the specific circumstances. These fermentation methods are well known to those skilled in the art, and a person skilled in the art can appropriately select any one of the fermentation methods to perform the method of the present application according to the desired purpose and experimental conditions.

[0034] In the above method for producing PHA, the fermentation medium can be subjected to sterilization treatment before being added to the fermentation system, or can be directly added to the fermentation system without treatment. Furthermore, the fermentation system can be maintained in an open non-sterile condition (i.e., can be directly exposed to the environment, without the need to use closed fermentation conditions to avoid contamination by foreign bacteria), or can be maintained in a closed condition to avoid contamination by foreign bacteria.

[0035] The medium can be selected according to the characteristics of the bacteria in combination with the microbial culture techniques known in the art, and generally can include a nutrient source (such as a carbon source and / or a nitrogen source), an energy source, and essential minerals. For example, a mineral medium, LB medium, MM medium, MM-G medium, or beef extract peptone, etc. conventionally used in the art can be used, or a medium modified based on these media for a desired purpose can also be used.

[0036] The fermentation medium can be liquid, solid, or semi-solid.

[0037] The medium can include a carbon source and a nitrogen source, and / or the carbon source and / or the nitrogen source can be supplemented into the culture fermentation system during the fermentation process. The carbon source can be, for example, a saccharide, an oil, an organic acid, an organic acid ester, etc., but is not limited thereto. The nitrogen source can be an organic nitrogen source, an inorganic nitrogen source, or a mixture thereof, for example, beef extract, yeast extract, corn steep powder, cake meal powder, urea, peptone, gelatin, ammonium sulfate, ammonium chloride, potassium nitrate, etc., but is not limited thereto.

[0038] In one specific embodiment of the present application, the fermentation medium used for fermentation is a 50MM medium.

[0039] In some embodiments, the temperature of the fermentation is preferably 30-45°C, and the pH is preferably 6.5-12.

[0040] In some embodiments, the polyhydroxyalkanoate is a polyhydroxyalkanoate containing a 3-hydroxybutyric acid monomer.

[0041] In some embodiments, the polyhydroxyalkanoate (PHA) is a homopolymer or a copolymer of a monomer.

[0042] The monomer includes, but is not limited to, 3-hydroxybutyric acid (3-HB), 3-hydroxyvaleric acid (3-HV), 4-hydroxybutyric acid (4-HB), 4-hydroxyvaleric acid (4-HV), etc.

[0043] In some embodiments, the polyhydroxyalkanoate containing a 3-hydroxybutyric acid monomer is preferably poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV).

[0044] The present application also provides a method for adjusting the ratio of polyhydroxyalkanoate monomers, which comprises fermenting and culturing the recombinant Halomonas.

[0045] The present application also provides a polyhydroxyalkanoate synthesized using the recombinant Halomonas.

[0046] The application also provides the use of the recombinant Halomonas in the production of polyhydroxyalkanoate.

[0047] Compared with the prior art, the application achieves the following technical effects:

[0048] (1) The application constructs a 3HV synthesis path mediated by 2-keto acid decarboxylase (kivD), which can directly convert alpha-ketoglutarate in the tricarboxylic acid cycle into 3-hydroxyvaleryl coenzyme A, realizes de novo synthesis of 3HV monomers, and can efficiently utilize conventional carbon sources (such as glucose) to synthesize 3HV, breaking through the technical bottleneck of traditional dependence on exogenous propionic acid or valeric acid precursors; a higher proportion of 3HV can be obtained through metabolic path regulation without the need for exogenous addition of precursors, which significantly simplifies the fermentation process and reduces raw material costs and production complexity.

[0049] (2) The synthesis strategy in the application does not need to destroy the original metabolic path, simplifies the strain construction process, and improves the genetic stability and growth performance of the strain.

[0050] (3) The application further improves the content of 3HV monomers by mutating and modifying kivD, and realizes effective regulation of the proportion of 3HV in PHA. BRIEF DESCRIPTION OF DRAWINGS

[0051] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0052] Figure 1 P3HB3HV metabolic pathway map;

[0053] Figure 2 P3HB4HB3HV metabolic pathway map;

[0054] Figure 3 pSEVA341 of embodiment 1 of the application kivD Plasmid map;

[0055] Figure 4 pRE341 of embodiment 1 of the application kivD Electrophoresis verification gel map;

[0056] Figure 5 pSEVA321 of embodiment 3 of the application xylB-xylC-xylD-xylX Plasmid map;

[0057] Figure 6pSEVA321 for the present application embodiment 3 xylB-xylC-xylD-xylX Electrophoresis verification gel map;

[0058] Figure 7 pSEVA341 for the present application embodiment 3 kivD-yqhD Plasmid map;

[0059] Figure 8 pSEVA341 for the present application embodiment 3 kivD-yqhD Electrophoresis verification gel map. DETAILED DESCRIPTION

[0060] In order to enable persons skilled in the art to better understand the present application scheme, the technical solutions in the embodiments of the present application will be described clearly and completely in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative labor should belong to the scope of protection of the present application.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0062] The present application uses MDF-9 is disclosed in patent CN113801810A, the preservation number is GDMCC NO.61850, LY01 is disclosed in patent CN116396886A, the preservation number is GDMCC NO.62635, LY02 is disclosed in patent CN116925981A, the preservation number is GDMCC NO.63381, LY03 is disclosed in patent CN116970538A, the preservation number is GDMCC NO. 63382, LY04 is disclosed in patent CN117551585A, the preservation number is GDMCC NO.63383.

[0063] Example 1 3HV pathway construction

[0064] 1、3HV pathway construction

[0065] (1) Plasmid construction

[0066] Using overlap extension PCR amplification kivD Genes and pSEVA341 vectors, under the action of Gibson ligase, kivDThe gene and pSEVA341 vector constitute a new plasmid and are marked as pSEVA341 kivD . The plasmid information is shown in Table 1. Figure 3 kivD The gene sequence (nucleotide sequence is shown as SEQ ID NO. 1) is obtained by taking part of the product to a biological company for sequencing. kivD

[0067] The primer sequence (5'-3') of the PCR amplification and plasmid pSEVA341 skeleton is as follows: kivD

[0068] kivD -F: SEQ ID No. 2;

[0069] kivD -R: SEQ ID No. 3;

[0070] pSEVA341-F: SEQ ID No. 4;

[0071] pSEVA341-R: SEQ ID No. 5.

[0072] The amplification system and amplification procedure are shown in Table 1 and Table 2:

[0073] Table 1 Amplification system table

[0074]

[0075] Table 2 Amplification procedure table

[0076]

[0077] After the PCR reaction is completed, the corresponding concentration of agarose gel is prepared, electrophoresis is carried out to observe the size of the DNA band, the gel is placed under the ultraviolet lamp, the gel of the target DNA fragment is quickly cut off, and the excess gel is cut off as much as possible.

[0078] (2) Gibson Assembly method connection

[0079] The recovered DNA is detected for its concentration, and then the adding ratio of the DNA is calculated according to the length and concentration of the target fragment and the pSEVA341 skeleton, and Gibson mixed enzyme is used for connection. The Gibson Assembly connection system and procedure are shown in Table 3 and Table 4:

[0080] Table 3 Gibson Assembly connection system table

[0081] ​​​

[0082] Table 4 Gibson Assembly connection procedure

[0083]

[0084] (3) S17-1 E. coli transformation

[0085] Step 1: Take the previously prepared S17-1 E. coli competent cells out of -80°C, thaw on ice, and after 5 min, wait for the bacterial block to melt;

[0086] Step 2: Add 5 μL of the ligation product to the competent cells, gently bounce the tube wall to mix the reaction solution (do not shake to mix). Note: The volume of the ligation product should not exceed 1 / 10 of the volume of the competent cells used;

[0087] Step 3: Ice bath for 30 min, 42°C water bath heat shock for 2 min, then immediately place on ice for 2 min. Note: Shaking will reduce the transformation efficiency;

[0088] Step 4: Add 400 μL of LB medium (without antibiotics) to the centrifuge tube, mix well, and then place in a 37°C shaker at 200 rpm for 60 min;

[0089] Step 5: Centrifuge at 5000 rpm for 5 min to collect the bacteria, discard 350 μL of supernatant, and take 100 μL to gently resuspend the bacterial block and spread on LB medium containing the corresponding antibiotic;

[0090] Step 6: Invert the culture medium into a 37°C incubator and incubate for 12-16 h.

[0091] (4) Single colony positive verification

[0092] Pick the colonies on the corresponding resistant LB plate and perform colony PCR verification. Send the PCR product with correct band size to a biological company for sequencing.

[0093] (5) Select the single colony with correct sequence for expansion, after 12-16 h, perform conjugation with MDF-9 in 20 LB plates, after 8 h, pick a small amount of conjugated bacteria and spread on 60 LB plates with corresponding resistance, after 36-48 h, perform another single colony verification.

[0094] (6) Strain PCR verification

[0095] Colony PCR results show that in this example MDF-9 strain successfully conjugated pSEVA341 kivD plasmid, the size of the target product was verified by PCR, and the electrophoresis diagram is as followsFigure 4 The target fragment is 3080 bp, which is consistent with the expected result. The pSEVA341 kivD The plasmid was named MDF-9-1.

[0096] Example 2 Fermentation test of recombinant strain MDF-9-1 for producing P3HB3HV

[0097] (1) Culture medium:

[0098] 60LB plate medium: yeast extract powder 0.5%, tryptone 1%, sodium chloride 60%, agar powder 1.8 g / 100 mL, ampicillin 50 μg / mL, kanamycin 30 g / mL, pH 8.0.

[0099] Fermentation medium (50MM): glucose 30 g / 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, 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, 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.

[0100] (2) Seed liquid preparation

[0101] ① Strain activation

[0102] The strain was taken from the laboratory-80°C refrigerator, and the bacterial liquid was inoculated on the plate solid medium (yeast powder 5 g / L; tryptone 10 g / L; sodium chloride 60 g / L, pH 8.5) by gun head, and cultured at 37°C for 24 h.

[0103] ② First-stage seed culture:

[0104] A single colony was inoculated in a 12 mL shaking tube (5 mL 60LB medium: yeast powder 5 g / L; tryptone 10 g / L; sodium chloride 60 g / L; pH 8.5), and the culture was incubated at 37°C, 220 rpm for 12 h.

[0105] ③ Second-stage seed culture:

[0106] Take 200 μL of the first-stage bacterial solution (1% inoculation amount) and inoculate into a 150 mL conical flask (20 mL of 60LB medium) and place in a shaking bed at 37°C and 220 rpm for 12 h.

[0107] (3) Preparation of fermentation medium

[0108] Fermentation medium (50 MM): glucose 30 g / 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, 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, 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.

[0109] (4) Fermentation culture

[0110] Inoculate 5% (1 mL) of the seed solution into a 150 mL conical flask and place in a shaking bed at 37°C and 220 rpm for 48 h.

[0111] (5) Determination of cell dry weight and PHA content

[0112] Cell dry weight (CDW): place 10-35 mL of the bacterial solution after fermentation in a 50 mL centrifuge tube, centrifuge at room temperature for 6 min at 8000 rpm, and discard the supernatant; add deionized water to restore the original volume, resuspend, and ensure that the precipitate completely disappears, centrifuge under the same conditions, and discard the supernatant; seal the centrifuge tube with a sealing film, store in a -80°C refrigerator for 2 h, and dry in a vacuum freeze dryer for 12-16 h; weigh, and calculate the cell dry weight (g / L).

[0113] Determination of PHA content: 0.05 g of the dry bacteria obtained by fermentation was weighed, ground, and placed in an esterification tube with good sealing. 2 mL of chloroform, 1700 μL of methanol, and 300 μL of concentrated sulfuric acid were added, and the mixture was reacted at 100°C for 1 h in an oil bath. After cooling at room temperature, 1 mL of ddH2O was added, and the mixture was shaken well and allowed to stand to separate into layers. After the water phase and the organic phase were completely separated, the chloroform layer (usually the lower layer) was filtered through a 0.22 μm organic filter membrane into a liquid phase bottle, and GC detection was performed using a GC-7800 gas chromatograph, a capillary column (Rtx-5 type, 30 m in length, 0.25 mm in inner diameter, and 0.25 μm of stationary phase), and a hydrogen flame ionization detector (FID). High-purity nitrogen was used as the carrier gas. The temperature program was set as follows:

[0114] Table 5 Program temperature setting

[0115]

[0116] The injection volume was 1 μL, and the external standard method was used for quantitative analysis of PHA. The yield of PHA was calculated according to the peak area.

[0117] (6) Fermentation results

[0118] The fermentation results of the recombinant strain MDF-9-1 with a 3HV pathway are shown in Table 6:

[0119] Table 6 Fermentation results of PHA-producing strains

[0120]

[0121] The results show that, compared with the control strain, MDF-9-1 generates α-ketoglutaric acid from glucose entering the TCA cycle by expressing kivD kivD metabolism to generate 3HV monomers, with a molar ratio of 6.63%.

[0122] Example 3 Construction of 4HB 3HV pathway

[0123] Since the xylose pathway involves many genes, the genes of this pathway are expressed by two plasmids in this example. KivD has a dual function, which not only functions in the xylose metabolic pathway but also participates in the metabolism of the 3HV pathway.

[0124] 1、xyl BCDX Plasmid construction

[0125] PCR-amplified xyl BCDX and the plasmid pSEVA321 backbone, under the action of Gibson ligase, xyl BCDX ​The fragments and pSEVA321 backbone were recombined to form a new plasmid, named pSEVA321 xylBCDX , and xyl xylBCDX was obtained by overlap extension PCR using pSEVA321 BCDX as a template. Part of the product was sent to a biological company for sequencing. The specific construction and transformation steps are described in Example 1, and the plasmid information is shown in Figure 5 . The size of the target fragment was verified, and the target fragment was 5402 bp, as shown in Figure 6 , which was consistent with the expected result.

[0126] The primer sequences for amplifying the xylBCDX fragment and the pSEVA321 plasmid backbone are as follows

[0127] xylB -F: SEQ ID No. 7;

[0128] xylB -R: SEQ ID No. 8;

[0129] xylC -F: SEQ ID No. 9;

[0130] xylC -R: SEQ ID No. 10;

[0131] xylD -F: SEQ ID No. 11;

[0132] xylD -R: SEQ ID No. 12;

[0133] xylX -F: SEQ ID No. 13;

[0134] xylX -R: SEQ ID No. 14;

[0135] pSEVA321-F: SEQ ID No. 15;

[0136] pSEVA321-R: SEQ ID No. 16.

[0137] 2、 kivD - yqhD Plasmid construction

[0138] The PCR-amplified kivD - yqhD and the plasmid pSEVA341 backbone were recombined under the action of Gibson enzyme, kivD - yqhDThe fragment and the pSEVA341 backbone recombine to form a new plasmid, named pSEVA341. kivD-yqhD pSEVA341 was obtained through overlap extension PCR. kivD-yqhD Get the template kivD - yqhD (The nucleotide sequence is shown in SEQ ID No. 17). A portion of the product was sent to a biotechnology company for sequencing. Specific construction and transformation steps are described in Example 1. Plasmid information is as follows: Figure 7 As shown. Verification was performed by checking the size of the target fragment, as... Figure 8 As shown, the target fragment is 4687 bp, which is consistent with the expected result. Then, the plasmid pSEVA321... xylBCDX and pSEVA341 kivD-yqhD Join to MDF-9-BDO strain (in) Based on the MDF-9 strain, the BDO gene cluster was integrated ( aldD - dhaT-orfZ The nucleotide sequence is shown in SEQ ID No. 25 (1-1520bp is aldD, 1535-2720bp is dhaT, 3046-4336 is orfZ), and strain MDF-9--BDO-1 was obtained.

[0139] kivD -1-F: SEQ ID No. 18;

[0140] kivD -1-R: SEQ ID No. 19;

[0141] yqhD -F: SEQ ID No. 20;

[0142] yqhD -R: SEQ ID No. 21;

[0143] pSEVA341-1-F: SEQ ID No. 22;

[0144] pSEVA341-1-R: SEQ ID No. 23.

[0145] Example 4: Fermentation test of P3HB4HB3HV produced by recombinant strain MDF-9-BDO-1

[0146] (1) Culture medium

[0147] For specific steps, please refer to Example 2.

[0148] (2) Seed liquid preparation

[0149] For specific steps, please refer to Example 2.

[0150] (3) Preparation of fermentation medium

[0151] The fermentation medium (50 MM) contains glucose 20 g / L, xylose 10 g / 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, 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, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, and NaMoO4·2H2O 0.03 g / L.

[0152] (4) Fermentation culture

[0153] The specific steps refer to Example 2.

[0154] (5) Determination of cell dry weight and PHA content

[0155] The specific steps refer to Example 2.

[0156] (6) Fermentation results

[0157] The fermentation results of the recombinant strain MDF-9-BDO-1 with 4HB and 3HV pathways are shown in Table 7.

[0158] Table 7 Fermentation results of PHA production by strains

[0159]

[0160] The results show that, compared with the control strain, MDF-9-BDO-1 can perform dual-pathway metabolism by expressing xyl BCDX , kivD and yqhD exogenously, and after adding glucose and xylose simultaneously without adding precursors, the molar ratio of 3HV monomers generated from glucose entering the TCA cycle to form alpha-ketoglutarate, which is metabolized by kivD, reaches 3.01%, and the 4HB monomers are generated from xylose through the xyl BCDX pathway.

[0161] Example 5 Random mutation of kivD

[0162] ​In order to obtain different proportions of 3HV, the kivD was randomly mutated (chemically) in this embodiment, and then strains producing different proportions of 3HV monomers were screened from the mutants. The specific construction method steps are referred to Example 1. The kivD primer sequence was amplified as in Example 1.

[0163] The amplification system is shown in Table 8:

[0164] Table 8 Amplification system table

[0165]

[0166] In Table 8, the main components of Tris-HCl mix are Tris-HCl 10 mM, KCl 50 mM, and MgCl2 2 mM.

[0167] (3) The kivD mutants obtained by screening and sequencing are as follows: the 99th serine is mutated to threonine, named kivD-1 (S99T); the 7th tyrosine is mutated to serine and the 196th asparagine is mutated to histidine, named kivD-2 (Y7S, N196H); the 202nd leucine is mutated to tryptophan, named kivD-3 (L202W); the 512th alanine is mutated to glycine, named kivD-4 (A512G); the 219th phenylalanine is mutated to isoleucine and the 470th glutamine is mutated to arginine, named kivD-5 (F219I, Q470R); the 3rd threonine is mutated to arginine, the 323rd leucine is mutated to valine, and the 510th lysine is mutated to glutamic acid, named kivD-6 (T3R, L323V, K510E).

[0168] The strains of the 6 kivD mutants are named MDF-9-kivD-1 (S99T), MDF-9-kivD-2 (Y7S, N196H), MDF-9-kivD-3 (L202W), MDF-9-kivD-4 (A512G), MDF-9-kivD-5 (F219I, Q470R), and MDF-9-kivD-6 (T3R, L323V, K510E), respectively, and fermentation tests were performed. The specific fermentation steps are referred to Example 1, and the fermentation results are shown in Table 9:

[0169] Table 9 Fermentation results of kivD mutants

[0170]

[0171] The results show that by random mutation of kivD, 6 mutant strains producing different proportions of 3HV monomers are screened under the condition of ensuring the dry weight, and the molar ratios of 3HV monomers of MDF-9-kivD-1~6 are 3.71%, 7.82%, 5.56%, 9.75%, 12.77% and 8.55% respectively. The dry weight, PHA and 3HV of MDF-9-kivD-5 mutant strain are the highest, which are increased to 12.69 g / L, 84.31% and 12.77% respectively. After kivD is mutated from phenylalanine at position 219 to isoleucine and glutamine at position 470 to arginine, the cell dry weight and PHA content are increased, and the synthesis of 3HV is promoted. In the actual application of PHA, by adding different contents of 3HV monomers, the rigidity and ductility of the material can be changed accordingly. PHA with low 3HV content has high rigidity and low ductility, and is mainly applied in disposable hard packaging, agricultural mulching, 3D printing wire and the like; PHA with high 3HV content has high flexibility and high ductility, and is mainly applied in soft packaging, medical materials, high value-added products and the like. The 3HV molar ratio content obtained in this embodiment is between 3% and 12%, which can provide certain selection according to the rigidity requirement of the material in actual application.

[0172] Example 6 Fermentation test of P3HB3HV produced by other recombinant strains

[0173] The strains selected in this experimental example are Halomonas sp. LY01, LY02, LY03, LY04.

[0174] (1) 3HV pathway construction

[0175] In this experimental example, the kivD-5 mutant is selected to construct the 3HV pathway of different strains, and the recombinant strains after successful pathway construction are named as LY01-1, LY02-1, LY03-1 and LY04-1 respectively. The specific steps are referred to Example 1.

[0176] (2) Culture medium

[0177] The specific steps are referred to Example 2.

[0178] (3) Seed liquid preparation

[0179] The specific steps are referred to Example 2.

[0180] (4) Preparation of fermentation medium

[0181] The specific steps are referred to Example 2.

[0182] (5) Fermentation culture

[0183] For specific steps, please refer to Example 2.

[0184] (6) Determination of cell dry weight and PHA content

[0185] For specific steps, please refer to Example 2.

[0186] (7) Fermentation results

[0187] The fermentation results of other recombinant strains constructing the 3HV pathway are shown in Table 10:

[0188] Table 10 Fermentation results of PHA production by other recombinant strains

[0189]

[0190] The results show that the method for constructing the 3HV pathway in this invention is also applicable to other Halomonas bacteria. From the fermentation results of this experiment, the recombinant strain LY03-1 had the highest strain dry weight, PHA content, and 3HV monomer molar ratio, which were 12.53 g / L, 81.89%, and 11.51 mol, respectively.

[0191] Example 7: Fermentation test of other recombinant strains producing P3HB4HB3HV

[0192] The strains selected in this experiment were those that integrated the BDO gene cluster ( dhaT-aldD - orfZ )of MDF-9-BDO, LY01-BDO LY02-BDO LY03-BDO LY04-BDO.

[0193] (1) Construction of the 4HB3HV pathway

[0194] In this experiment, the kivD-5 mutant was selected to construct the 4HB3HV pathway for different strains. The recombinant strains after successful pathway construction were named MDF-9-BDO-2, LY01-BDO-1, LY02-BDO-1, LY03-BDO-1, and LY04-BDO-1, respectively. For specific steps, please refer to Example 3.

[0195] (2) Culture medium

[0196] For specific steps, please refer to Example 2.

[0197] (3) Seed liquid preparation

[0198] For specific steps, please refer to Example 2.

[0199] (4) Preparation of fermentation medium

[0200] The specific steps refer to Example 4.

[0201] (5) Fermentation culture

[0202] The specific steps refer to Example 2.

[0203] (6) Cell dry weight and PHA content determination

[0204] The specific steps refer to Example 2.

[0205] (7) Fermentation results

[0206] The fermentation results of the recombinant strains with 4HB and 3HV pathways are shown in Table 11:

[0207] Table 11 Fermentation results of PHA production of other recombinant strains

[0208]

[0209] The results show that the construction method of the 4HB3HV pathway in the present application is also applicable to other Halomonas. According to the fermentation results of the experimental examples, the strain dry weight, PHA content, 4HB monomer molar ratio and 3HV monomer molar ratio of the recombinant strain MDF-9-BDO-2 are the highest, which are 9.67 g / L, 69.35%, 7.72% and 6.22%, respectively. The strain dry weight, PHA content, 4HB monomer molar ratio and 3HV monomer molar ratio of the recombinant strain LY03-BDO-1 are the second, which are 9.57 g / L, 68.19%, 7.07% and 5.67%, respectively.

[0210] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant Halomonas bacteria, characterized in that, The recombinant Halomonas expresses a 2-keto acid decarboxylase mutant; The amino acid sequence of the 2-keto acid decarboxylase is shown as SEQ ID No. 24; The 2-keto acid decarboxylase mutant is distinguished from the 2-keto acid decarboxylase by any of the following: (a) the 99th amino acid is threonine; (b) the 7th amino acid is serine and the 196th amino acid is histidine; (c) the 202th amino acid is tryptophan; (d) the 512th amino acid is glycine; (e) the 219th amino acid is isoleucine and the 470th amino acid is arginine; (f) the 3rd amino acid is arginine, the 323rd amino acid is valine and the 510th amino acid is glutamic acid.

2. The recombinant Halomonas sp. of claim 1, wherein, The recombinant Halomonas sp. also expresses xylB a gene, xylC a gene, xylD a gene, xylX a gene and yqhD a gene.

3. The recombinant Halomonas sp. of claim 2, wherein, The xylB gene , xylC gene , xylD gene , xylX genes originate from Caulobacter or a derivative strain thereof , Pseudomonas sp. or a derivative strain thereof, Azotobacter vinelandii or any of the derivative strains thereof; The yqhD The gene is derived from E.coli or a derivative bacterium thereof, Salmonella enterica or a derivative bacterium thereof, Klebsiella pneumoniae or a derivative bacterium thereof.

4. The recombinant Halomonas sp. of claim 1, wherein, The salt-stressed bacteria are or a derivative thereof.

5. A method for constructing a recombinant Halomonas sp. according to claim 1, characterized in that, The method comprises the following steps: (1) constructing an expression vector containing a 2-keto acid decarboxylase mutant gene; (2) introducing the expression vector constructed in (1) into Halomonas to obtain the recombinant Halomonas.

6. A method of producing polyhydroxyalkanoates, characterized by, The method comprises fermenting and culturing the recombinant Halomonas of any one of claims 1-4.

7. The method of claim 6, wherein, The polyhydroxyalkanoate is a polyhydroxyalkanoate containing 3-hydroxybutyric acid monomers.

8. A method of adjusting the ratio of polyhydroxyalkanoate monomers, characterized by, The method comprises fermenting and culturing the recombinant Halomonas of any one of claims 1-4.

9. A polyhydroxyalkanoate synthesized by the recombinant Halomonas of any one of claims 1-4.

10. Use of the recombinant Halomonas of any one of claims 1-4 in the production of polyhydroxyalkanoate.

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