P34HB producing strain and its construction method and application

Abstract

The present invention provides a strain for producing P34HB and a construction method and application thereof. The construction method comprises the following steps: S1: constructing in vitro a strain containing gene A, gene B, ogdA Genes and orf S2: introducing the recombinant vector described in S1 into the strain. The present invention uses Halomonas as the base bacteria, transforms the genes of the pathway from glucose to 4-hydroxybutyryl-CoA, mutates the specific sites of succinic semialdehyde and 4-hydroxybutyrate dehydrogenase, improves the production efficiency of the strain for P34HB, and increases the content of 4HB in P34HB, which has potential for industrialization.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a strain for producing P34HB and a construction method and application thereof. Background Art

[0002] Petrochemical plastics are closely related to people's daily lives and play a very important role. However, its degradation process takes at least 200 years to complete. Therefore, waste plastic pollution has become the third largest pollution problem after air pollution and water pollution, and it continues to threaten the living environment of human beings. Polyhydroxyalkanoates (PHA), as a fully biosynthesized and fully biodegradable bioplastic, have good material properties and are widely used in application research in many fields such as medical devices, packaging, and textiles. In recent years, the copolymer poly (3-hydroxybutyrate-co-4-hydroxybutyrate, referred to as P34HB), which is a copolymer of 3-hydroxybutyric acid (3HB) and 4-hydroxybutyric acid (4HB), has emerged. It is one of the best PHAs in the PHA family and has attracted widespread attention. Due to its good biocompatibility and thermal stability, P34HB can be used in biomedical materials, biodegradable materials, and textile fibers.

[0003] Genetic engineering is an important means to improve the efficiency of PHA production. Through gene editing technology, the metabolic pathways of microorganisms can be optimized, the activity of key enzymes can be enhanced, and the synthesis rate and yield of PHA can be increased. Although the production of PHA by microbial fermentation has broad prospects, it still faces some challenges, including high production costs, low yields of some high-quality PHAs such as P34HB and low monomer content such as 4HB, and the difficulty of metabolic engineering of microorganisms.

[0004] Therefore, there is an urgent need for a high-yield method that can simultaneously increase the yield of P34HB and the content of 4HB in P34HB, reduce the production cost of strain fermentation, and meet industrial needs. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes a strain for producing P34HB and a construction method and application thereof.

[0006] The present invention provides a method for producing poly(3-hydroxybutyrate- co -4-hydroxybutyrate) strain, comprising the following steps:

[0007] S1: In vitro construction of gene A, gene B, ogdA Genes and orfz A recombinant vector of a gene nucleotide sequence;

[0008] S2: Introduce the recombinant vector described in S1 into the strain;

[0009] Among them, gene A is 4hbd Gene or 4hbd Mutant, the 4hbd Mutants and 4hbd Genes differ in any of the following:

[0010] (i) The 77th base was replaced by G to A, and the 188th base was replaced by C to G;

[0011] (ii) The 739th base was replaced by G to A;

[0012] (iii) the 574th base was replaced by G to C;

[0013] (iiii) The 757th base was replaced by C to A;

[0014] (iiiii) The 660th base was replaced by G to T;

[0015] Gene B is sucD Gene or sucD Mutant, the sucD Mutants and sucD Genes differ in any of the following:

[0016] (1) The 56th base was replaced from 0A to C;

[0017] (2) The 270th base was replaced from A to T;

[0018] (3) The 287th base was replaced by T to C;

[0019] (4) The 543rd base was replaced from A to C;

[0020] (5) The 62nd base was replaced by T to G, and the 1223rd base was replaced by C to A;

[0021] (6) The 755th base was replaced from C to T;

[0022] Said sucD The nucleotide sequence of the gene is shown in SEQ ID NO. 23, 4hbd The nucleotide sequence of the gene is shown in SEQ ID NO. 19.

[0023] Furthermore, the strain is Halomonas.

[0024] Furthermore, the Halomonas is Halomonas lutescens MDF-9.

[0025] Furthermore, the recombinant vector described in S1 is a pSEVA vector, preferably pSEVA321.

[0026] Furthermore, the recombinant vector contains a promoter, and the promoter is a porin promoter.

[0027] The invention also provides a strain obtained by the construction method.

[0028] The present invention also provides a method for producing poly(3-hydroxybutyrate- co -4-hydroxybutyrate) method, comprising the steps of:

[0029] (1) Activation of bacterial strains;

[0030] (2) Shake flask seed culture;

[0031] (3) Shake flask fermentation culture.

[0032] Furthermore, the fermentation temperature in step (3) is 36-38°C.

[0033] Furthermore, the pH of the fermentation culture in step (3) is 8-12.

[0034] Furthermore, the fermentation culture time in step (3) is 36 to 72 hours.

[0035] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0036] The present invention uses Halomonas as the base bacteria, transforms the genes of the pathway from glucose to 4-hydroxybutyryl-CoA, mutates the specific sites of succinic semialdehyde and 4-hydroxybutyrate dehydrogenase, thereby improving the production efficiency of the strain for P34HB and increasing the content of 4HB in P34HB, and has potential for industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0038] Figure 1 Schematic diagram of the de novo synthesis pathway of the present invention;

[0039] Figure 2 pSEVA321 of Example 1 of the present invention 4hbd1-sucD1-ogdA-orfz Plasmid map;

[0040] Figure 3 pSEVA321 of Example 1 of the present invention 4hbd1-sucD2-ogdA-orfz Plasmid map;

[0041] Figure 4 This is the electrophoresis diagram of the fragment of Example 1 of the present invention;

[0042] Figure 5 pSEVA321 of Example 3 of the present invention 4hbd2-sucD1-ogdA-orfz Plasmid map;

[0043] Figure 6 pSEVA321 of Example 3 of the present invention gbd1-sucD1-ogdA-orfz Plasmid map;

[0044] Figure 7 pSEVA321 of Example 3 of the present invention yqhd-sucD1-ogdA-orfz Plasmid map;

[0045] Figure 8 This is the electrophoresis diagram of the target fragment of Example 3 of the present invention. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0047] Halomonas lutescens MDF-9 has the ability to synthesize 3HB. The present invention constructs a P34HB synthesis pathway in the MDF-9 strain by introducing an exogenous pathway from glucose to 4-hydroxybutyryl-CoA. Figure 1 As shown. By screening and mutating the enzymes in the pathway, the ability of the strain to produce P34HB was significantly improved.

[0048] in, 4hbd1 The nucleotide sequence is shown in SEQ ID NO. 19, which is derived from C. kluyveri; 4hbd2 The nucleotide sequence is shown in SEQ ID NO. 20, which is derived from Porphyromonasgingivalis TDC60; gbd1 The nucleotide sequence is shown in SEQ ID NO. 21, which is derived from Porphyromonas gingivalis W83; yqhdThe nucleotide sequence is shown in SEQ ID NO.22, which is derived from E.coli; sucD1 The nucleotide sequence is shown in SEQ ID NO. 23, which is derived from C. kluyveri; sucD2 The nucleotide sequence is shown in SEQ ID NO. 24, which is derived from Porphyromonas gingivalis WDC60; ogdA The nucleotide sequence is shown in SEQ ID NO.25, which is derived from Synechococcus sp. PCC 7002; orfz The nucleotide sequence is shown in SEQ ID NO. 26, which is derived from C. kluyveri; 4hbd1 - sucD1 - ogdA - orfz The nucleotide sequence is shown in SEQ ID NO.27.

[0049] Halomonas lutescensMDF-9: The strain is derived from patent CN113801810A.

[0050] Example 1 Succinate semialdehyde dehydrogenase ( sucD )

[0051] 1. From different sources sucD Plasmid construction of genes:

[0052] PCR was used to amplify the C. kluyveri-derived 4hbd1 , sucDx 、Synechococcus sp. PCC7002 ogdA and C. kluyveri orfz Combined junction fragments of genes 4hbd1 - sucDx - ogdA - orfz and plasmid pSEVA321-p194 backbone, where p194 is the promoter, sucDx They are sucD1 From C. kluyveri, sucD2 From Porphyromonas gingivalis WDC60. Under the action of Gibson ligase, 4hbd1 - sucDx - ogdA - orfz The fragment was recombined with the pSEVA321-p194 backbone to form a new plasmid, named pSEVA321 4hbd1-sucD1-ogdA-orfz and pSEVA321 4hbd1-sucD2-ogdA-orfz, respectively, by overlapping extension PCR with pSEVA321 4hbd1-sucD1-ogdA-orfz and pSEVA321 4hbd1-sucD2-ogdA-orfz Get the template 4hbd1 - sucD1 - ogdA - orfz Fragments and 4hbd1 - sucD2 - ogdA - orfz Fragments, take part of the product and send it to a biological company for sequencing. The plasmid information is as follows Figure 2 , 3 shown.

[0053] (a) The primer sequences for amplifying the above fragment and the pSEVA321 plasmid backbone are as follows:

[0054] 4hbd1 -F: SEQ ID NO.1;

[0055] 4hbd1 -R: SEQ ID NO.2;

[0056] sucD1 -F: SEQ ID NO. 3;

[0057] sucD1 -R: SEQ ID NO.4;

[0058] sucD2 -F: SEQ ID NO.5;

[0059] sucD2 -R: SEQ ID NO.6;

[0060] ogdA -F: SEQ ID NO.7;

[0061] ogdA -R: SEQ ID NO.8;

[0062] orfz -F: SEQ ID NO.9;

[0063] orfz -R: SEQ ID NO.10;

[0064] pSEVA321-F: SEQ ID NO. 11;

[0065] pSEVA321-R: SEQ ID NO.12.

[0066] (b) The amplification system and amplification procedure are shown in Table 1 and Table 2:

[0067] Table 1 Amplification system

[0068]

[0069] Table 2 Amplification program

[0070]

[0071] After the PCR reaction is completed, prepare agarose gel of corresponding concentration and perform electrophoresis to observe the size of DNA bands. Place the gel under ultraviolet light and quickly cut out the gel of the target DNA fragment, cutting off as much excess gel as possible.

[0072] (c) Gibson Assembly method

[0073] The concentration of the recovered DNA was tested, and the ratio of DNA addition was calculated according to the length and concentration of the target fragment and the pSEVA321 backbone, and the Gibson mixed enzyme was used for ligation. The Gibson Assembly ligation system and procedure are shown in Tables 3 and 4:

[0074] Table 3 Gibson Assembly connection system table

[0075]

[0076] Table 4 Gibson Assembly connection procedure

[0077]

[0078] 2. S17-1 E. coli transformation:

[0079] Step 1: Take out the prepared S17-1 E. coli competent cells from -80℃, thaw them on ice, and wait for the bacteria to melt after 5 minutes;

[0080] Step 2: Add 5 μL of ligation product to the competent cells and gently tap the tube wall to mix the reaction solution (do not oscillate to mix). Note: The transformation volume of the ligation product should not exceed 1 / 10 of the volume of the competent cells used;

[0081] Step 3: Place on ice for 30 min, heat shock at 42°C for 2 min, and immediately cool on ice for 2 min. Note: shaking will reduce transformation efficiency.

[0082] Step 4: Add 400 μL LB medium (without antibiotics) to the centrifuge tube, mix well, and place in a 37°C shaker at 200 rpm for 60 min.

[0083] Step 5: Centrifuge at 5000 rpm for 5 min to collect the bacteria, discard 350 μL of the supernatant, keep 100 μL of the supernatant, gently blow to resuspend the bacteria and spread it on LB medium containing the corresponding antibiotics;

[0084] Step 6: Invert the culture medium into a 37°C incubator and incubate for 12 to 16 hours.

[0085] 3. Monoclonal colony positive verification

[0086] Colonies were picked out on the corresponding resistance LB plates and verified by colony PCR. PCR products with the correct band size were sent to a biological company for sequencing.

[0087] 4. Select a single colony with the correct sequence for expansion. After 12-16 hours, combine it with Halomonas lutescensMDF-9 in a 20LB plate. After 8 hours, pick a small amount of the combined bacteria and spread them on a 60LB plate with the corresponding resistance. After 36-48 hours, perform another single clone colony verification.

[0088] 5. Sequencing and identification

[0089] The sequencing results showed that the Halomonas lutescens MDF-9 strain in this example was successfully transformed with 4hbd1 - sucD1 - ogdA - orfz Fragments and 4hbd1 - sucD2 - ogdA - orfz The recombinant strains were named MDF-9-1 and MDF-9-2, and the size of the target product was used to verify the results. Figure 4 As shown, the target fragments were 5911 bp and 5905 bp, respectively, which was in line with the expected results.

[0090] Example 2 Fermentation test of recombinant strains

[0091] (1) Culture medium:

[0092] 60LB plate culture medium: 0.5% yeast extract powder, 1% tryptone, 60% sodium chloride, 1.8 g / 100 mL agar powder, pH 8.5.

[0093] Component I: magnesium sulfate: 0.2 g / L, urea: 1.0 g / L (prepared with 50 times concentrated mother liquor: 10 g / L magnesium sulfate, 3 g / L urea);

[0094] Component II: 5.2 g / L potassium dihydrogen phosphate, prepared with 50 times mother solution of 260 g / L; 30 g / L glucose solution: prepared with 500 g / L glucose mother solution;

[0095] Component III (10 mL / L): 5 g / L ammonium ferric citrate, 1.5 g / L anhydrous calcium chloride, 41.7 ml / L 12 mol / L hydrochloric acid;

[0096] Component IV (1 mL / L): zinc sulfate heptahydrate 100 mg / L, manganese sulfate tetrahydrate 30 mg / L, boric acid 300 mg / L, copper sulfate pentahydrate 10 mg / L, sodium molybdate 30 mg / L.

[0097] Fermentation medium (50MM culture medium): hydatidiform mole 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, H2BO30.3g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L.

[0098] (2) Bacterial activation

[0099] Take the bacteria from the -80℃ refrigerator in the laboratory, pick up the bacterial liquid with a pipette tip, streak it on a plate solid culture medium (yeast powder 5 g / L; tryptone 10 g / L; sodium chloride 60 g / L, pH 8.5), and culture at 37℃ for 24 h.

[0100] ②First-level seed cultivation:

[0101] Pick a single colony and inoculate it into 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 place the culture medium in a shaker at 37°C and 220 rpm for 12 h.

[0102] ③Secondary seed cultivation:

[0103] Pipette 200 μL of the primary bacterial solution (1% inoculation volume) and inoculate it into a 150 mL conical flask (20 mL 60LB medium), and culture it in a shaker at 37°C and 220 rpm for 12 h.

[0104] (3) Fermentation culture

[0105] Inoculate 5% of the seed solution (2.5 mL) into a 500 mL conical flask and culture it in a shaker at 37°C and 220 rpm for 48 h.

[0106] (4) Determination of cell dry weight, PHA content and 4HB molar fraction

[0107] Cell dry weight (CDW): Place 30-35 mL of fermented bacterial liquid in a 50 mL centrifuge tube, centrifuge at room temperature for 6 minutes at 8000 rpm, and discard the supernatant; add appropriate amount of deionized water to restore the original volume, resuspend, ensure that the precipitate disappears completely, centrifuge under the same conditions, and discard the supernatant; seal the centrifuge tube with sealing film and freeze it in a -80℃ refrigerator for 2 h; dry the centrifuge tube in a vacuum freeze dryer for 12-16 hours; weigh and calculate the cell dry weight (g / L).

[0108] Determination of PHA content and 4HB mole fraction:

[0109] Weigh 0.05 g of the dried bacteria obtained by grinding and fermentation, place it in a well-sealed esterification tube, add 2 mL of chloroform, 1700 μL of methanol and 300 μL of concentrated sulfuric acid, react in an oil bath at 100°C for 1 h, cool to room temperature, add 1 mL of ddH2O, shake and mix thoroughly, and let stand to separate. After the aqueous phase and the organic phase are completely separated, take the chloroform layer (usually the lower layer) and filter it into a liquid phase bottle using a 0.22 μm organic filter membrane. GC detection is performed using a GC-7800 gas chromatograph, a capillary column (Rtx-5 type, 30 m long, 0.25 mm inner diameter and 0.25 μm stationary phase) and a hydrogen flame ionization detector (FID).

[0110] The carrier gas used was high-purity nitrogen. The program temperature was set as follows:

[0111] Table 5 Program temperature settings

[0112]

[0113] The injection volume was 1 μL, and the PHA content and 4HB mole fraction were calculated based on the peak area.

[0114] The fermentation results are as follows:

[0115] Table 6 sucD Screened fermentation data

[0116]

[0117] From the results in Table 6, we can see that the expression of two different sources sucD Compared with the strain with the same gene, MDF-9-1 produced a higher PHA yield and the highest 4HB ratio, which was 6.36 mol%. sucD1 Gene for 4-hydroxybutyrate dehydrogenase ( 4hbd ) for screening.

[0118] Example 3 4-Hydroxybutyrate dehydrogenase ( 4hbd )

[0119] PCR amplification 4hbd1 、Porphyromonas gingivalis TDC60 4hbd2, Porphyromonas gingivalis W83 Source gbd1, E.coli Source yqhd Respectively sucD1 , ogdA and orfz Combined junction fragments of genes 4hbd1 - sucD1 - ogdA - orfz Fragment, 4hbd2 - sucD1 - ogdA - orfz Fragment, gbd1 - sucD1 - ogdA - orfz Fragments and yqhd - sucD1 - ogdA - orfz The fragments and the plasmid pSEVA321-p194 backbone, where p194 is the promoter. Under the action of Gibson ligase, the different fragments and the pSEVA321-p194 backbone were recombined to form new plasmids, which were named pSEVA321 4hbd1-sucD1-ogdA-orfz 、 pSEVA321 4hbd2-sucD1-ogdA-orfz 、pSEVA321 gbd1-sucD1-ogdA-orfz and pSEVA321 yqhd-sucD1-ogdA-orfz , respectively, by overlapping extension PCR using the above vector plasmids as templates to obtain 4hbd2 - sucD1 - ogdA - orfz Fragment, gbd1 - sucD1 - ogdA - orfz Fragments and yqhd - sucD1 - ogdA - orfz Fragments, take part of the product and send it to a biological company for sequencing. The plasmid information is as follows Figures 5 - 7 Referring to the construction method of Example 1, the above plasmid was transformed into the Halomonaslutescens MDF-9 strain.

[0120] Verify by the size of the target product, such as Figure 8As shown in the figure, the target fragments are 5911 bp, 5911 bp and 5959 bp respectively, which is in line with the expected results. This shows that the above fragments were successfully transferred into the Halomonas lutescens MDF-9 strain in this example, and the 4hbd2 - sucD1 - ogdA - orfz The fragment was named MDF-9-3 and transferred into gbd1 - sucD1 - ogdA - orfz The fragment strain was named MDF-9-4 and transferred into yqhd - sucD1 - ogdA - orfz The strain of the fragment was named MDF-9-5.

[0121] The primer sequences are as follows (5'-3'):

[0122] 4hbd1 -F: SEQ ID NO.1;

[0123] 4hbd1 -R: SEQ ID NO.2;

[0124] 4hbd2 -F: SEQ ID NO.13;

[0125] 4hbd2 -R: SEQ ID NO.14;

[0126] gbd1 -F: SEQ ID NO.15;

[0127] gbd1 -R: SEQ ID NO.16;

[0128] yqhd -F: SEQ ID NO.17;

[0129] yqhd -R: SEQ ID NO.18;

[0130] sucD1 -F: SEQ ID NO. 3;

[0131] sucD1 -F: SEQ ID NO.4;

[0132] ogdA -F: SEQ ID NO.7;

[0133] ogdA -F: SEQ ID NO.8;

[0134] orfz -F: SEQ ID NO.9;

[0135] orfz -R: SEQ ID NO.10;

[0136] pSEVA321-F: SEQ ID NO. 11;

[0137] pSEVA321-R: SEQ ID NO.12.

[0138] Example 4 Fermentation test of recombinant strain

[0139] The strains constructed in Example 4 were subjected to fermentation test with reference to Example 2. Among them, MDF-9-1, MDF-9-3, MDF-9-4 and MDF-9-5 required the addition of 50 μL of chloramphenicol (25 mg / L).

[0140] Table 7 4hbd Screened fermentation data

[0141]

[0142] Fermentation data showed that MDF-9-1 ( 4hbd1 ) had the highest 4HB ratio, which could reach 6.28 mol%. Based on the screening results of the above enzymes, sucD1 and 4hbd1 Perform random mutation modification of enzymes.

[0143] Example 5 Succinate semialdehyde dehydrogenase ( sucD ) Random mutation transformation and fermentation test

[0144] (1) sucD1 Mutation

[0145] In order to improve the expression efficiency of succinate semialdehyde dehydrogenase, sucD1 Perform chemical random mutagenesis (such as dNTP, Tris-HCl mix) to screen out more efficient succinate semialdehyde dehydrogenase sucD1 Mutants, specific steps refer to the different sources in Example 1 sucD Gene plasmid construction.

[0146] The PCR amplification system is shown in Table 8:

[0147] Table 8 Amplification system table

[0148]

[0149] The main components of the Tris-HCl mix described in Table 7 are Tris-HCl 10 mM, KCl 50 mM, and MgCl2 2 mM. sucD1 The forward mutants are: sucD1-2, sucD1-4, sucD1-5, sucD1-8, sucD1-10, and sucD1-13.

[0150] Wild-type sucD1 Nucleotide sequence for reference:

[0151] sucD1-2: bases AAA (lysine) at positions 55-57 were replaced by ACA (threonine);

[0152] sucD1-4: GAA (glutamic acid) at bases 268-270 was replaced by GAT (aspartic acid) and GCT (alanine) at bases 433-435 was replaced by GGT (glycine);

[0153] sucD1-5: bases 286-288 GTT (valine) replaced by GCT (alanine);

[0154] sucD1-8: bases GAA (glutamic acid) at positions 541-543 were replaced by GAC (aspartic acid);

[0155] sucD1-10: TTG (leucine) at bases 61-63 was replaced by TGG (tryptophan) and ACT (threonine) at bases 1222-1224 was replaced by AAT (asparagine);

[0156] sucD1-13: The bases GCT (alanine) at positions 754 to 756 were replaced by GTT (valine).

[0157] After the mutation sucD1 (Refer to Example 1 for connection 4hbd1 - sucD1’ - ogdA - orfz The strains containing the above fragments were named MDF-9-6, MDF-9-7, MDF-9-8, MDF-9-9, MDF-9-10 and MDF-9-11.

[0158] (2) Fermentation test

[0159] The fermentation test was carried out according to the method of Example 2; wherein, MDF-9-1, MDF-9-6, MDF-9-7, MDF-9-8, MDF-9-9, MDF-9-10, and MDF-9-11 required the addition of 50 μL of chloramphenicol (25 mg / L).

[0160] Table 9 sucD 1 Fermentation data of random mutation screening

[0161]

[0162] The fermentation results showed that the dry weight, PHA content and 4HB ratio of each forward mutant strain were increased to varying degrees, among which MDF-9-7 (corresponding to the sucD1-4 mutant) had the best fermentation effect, with the cell dry weight, PHA and 4HB ratios increased to 12.58 g / L, 85.23% and 8.86 mol%, respectively.

[0163] Example 64-Hydroxybutyrate dehydrogenase ( 4hbd ) Random mutation transformation

[0164] (1) 4hbd1 Mutation

[0165] For the expression efficiency of 4-hydroxybutyrate dehydrogenase, in Example 5 sucD1 Based on random mutation transformation, 4hbd1 Perform chemical random mutagenesis (such as dNTP, Tris-HCl mix) to screen out more efficient 4-hydroxybutyrate dehydrogenase 4hbd1 For mutants, the specific steps are as follows: random mutation modification in Example 1 and Example 7.

[0166] 4hbd1-3: GGA (glycine) at bases 76-78 was replaced by GCA (alanine) and TCT (serine) at bases 187-189 was replaced by TGT (cysteine);

[0167] 4hbd1-5: GCA (alanine) at bases 739-741 was replaced by ACA (threonine);

[0168] 4hbd1-8: GTA (valine) at bases 574-576 was replaced by CTA (leucine);

[0169] 4hbd1-10: bases 757-759 CAC (histidine) replaced by AAC (asparagine);

[0170] 4hbd1-18: GAG (glutamic acid) at bases 658-660 was replaced by GAT (aspartic acid) and ACA (threonine) at bases 1096-1098 was replaced by CCA (proline).

[0171] After screening and sequencing, 4hbd1 The forward mutants are: 4hbd1-3, 4hbd1-5, 4hbd1-8, 4hbd1-10, 4hbd1-18, which will be transferred into 4hbd1Forward mutant (connected in the manner of Example 1) 4hbd1’ - sucD1 - ogdA - orfz The strains were named MDF-9-12, MDF-9-13, MDF-9-14, MDF-9-15, and MDF-9-16.

[0172] (2) Fermentation test

[0173] Among them, MDF-9-7, MDF-9-12, MDF-9-13, MDF-9-14, MDF-9-15, and MDF-9-16 need to add 50 μL of chloramphenicol (25 mg / L).

[0174] Table 10 4hbd1 Fermentation data from random mutation screening

[0175]

[0176] The fermentation results showed that the dry weight, PHA content and 4HB ratio of each forward mutant strain were increased to varying degrees, among which MDF-9-14 (corresponding to the 4hbd1-8 mutant) had the best fermentation effect, with the cell dry weight, PHA and 4HB ratios increased to 13.54 g / L, 87.86% and 10.35 mol%, respectively.

[0177] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for producing poly(3-hydroxybutyrate- co -4-hydroxybutyrate) strain, characterized in that, The steps include: S1: In vitro construction of gene A, gene B, oGD Genes and oeLhd A recombinant vector of a gene nucleotide sequence; oGD The nucleotide sequence of the gene is shown in SEQ ID NO.

25. oeLhd The nucleotide sequence of the gene is shown in SEQ ID NO.26; S2: Introduce the recombinant vector described in S1 into the strain; Among them, gene A is 4hbd Mutant, the 4hbd Mutants and 4hbd Genes differ in any of the following: (i) The 77th base was replaced by G to A and the 188th base was replaced by C to G; (ii) The 739th base was replaced by G to A; (iii) the 574th base was replaced by G to C; Gene B is sucD Mutant, the sucD Mutants and sucD The difference between the genes is that the 270th base is replaced from A to T and the 434th base is replaced from C to G; Said sucD The nucleotide sequence of the gene is shown in SEQ ID NO. 23, 4hbd The nucleotide sequence of the gene is shown in SEQ ID NO. 19; The strain is Halomonas lutescens MDF-9; The recombinant vector in S1 is pSEVA321 vector; The recombinant vector contains a promoter, and the promoter is a porin promoter.

2. A strain characterized by: Obtained by the construction method described in claim 1.

3. A method for producing poly(3-hydroxybutyrate- co -4-hydroxybutyrate) method, characterized in that The steps include: (1) Activation of bacterial strains; (2) Shake flask seed culture; (3) Shake flask fermentation culture.

4. The method according to claim 3, characterized in that The fermentation temperature in step (3) is 36-38°C.

5. The method according to claim 3, characterized in that: The pH of the fermentation culture in step (3) is 8-12.

6. The method according to claim 3, characterized in that The fermentation culture time in step (3) is 36 to 72 hours.

Images