Application of xylose transporter in production of P34HB

Abstract

The invention discloses the application of xylose transporter in producing P34HB. 4hbd ‑ sucD ‑ ogdA‑ orfz and integrated into the Halomonas lutescens MDF‑9 strain and introduced xylB ‑ xylC ‑ xylD ‑ xylX , a strain producing P34HB was obtained, and the strain was used to screen an efficient exogenous xylose transporter, and the xylose transporter was mutated to achieve efficient utilization of xylose. The new method of preparing P34HB using the xylose transporter system and the exogenous expression of xylose degradation pathway solves the problem of the utilization efficiency of cheap carbon sources and increases the content of 4HB in P34HB.

Description

Technical Field

[0001] The invention relates to the field of biosynthesis, and in particular to the application of xylose transporter in the production of P34HB. Background Art

[0002] In today's society, the demand for sustainable development and environmentally friendly materials is increasing. Biodegradable plastics have received extensive attention and research as a viable alternative to traditional plastics. co 4-Hydroxybutyrate (P34HB) is a biodegradable polymer with great potential in the plastics industry, but its production process still faces challenges.

[0003] Xylose is a cheap non-food carbon source and is widely present in nature. However, many microorganisms do not actively utilize xylose, which limits its application in the synthesis of biodegradable plastics. Halomonas usually do not have the ability to effectively utilize xylose, so it is necessary to improve the utilization rate of xylose by exogenously expressing the xylose metabolic pathway. However, the efficiency of existing technologies in this regard is still limited and has not yet reached the ideal level, resulting in low yields of xylose converted into 4-hydroxybutyrate (4HB). Screening and modifying xylose transporters are key steps to improve the efficiency of xylose utilization by halomonas. However, existing technologies have certain limitations in selecting and modifying xylose transporters, and their selectivity and efficiency need to be further improved. Enhancing the metabolic capacity of halomonas for xylose by exogenously expressing the xylose metabolic pathway is an important means to increase the production of 4-hydroxybutyrate. However, existing technologies may have insufficient precision in regulating metabolic pathways, resulting in low efficiency of metabolic pathways or unsatisfactory product distribution. In order to overcome this challenge, it is urgent to construct an effective xylose transporter and use genetic engineering technology to regulate related metabolic pathways to improve the efficiency of xylose utilization by microorganisms, solve the problem of cheap carbon source utilization, and increase the 4HB content in P34HB. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes the application of xylose transporter in the production of P34HB. Based on the fact that Halomonas lutescens MDF-9 can synthesize 3-hydroxybutyrate (3HB) from glucose, the present invention introduces exogenous xylose degradation pathway genes and their transporters into the tricarboxylic acid cycle (TCA), and then exogenously integrates and expresses 4hbd-sucD - oGD - oeLhd The target gene sequence promotes the production of poly(3-hydroxybutyrate- co -4 hydroxybutyrate) (P34HB).

[0005] The present invention provides a method for producing poly(3-hydroxybutyrate- co -4-hydroxybutyrate), wherein the xylose transporter is any one of the following:

[0006] (1) Any one of xylE, xylFGH, and HEO;

[0007] (2) Any of the HEO mutants;

[0008] The amino acid sequence of the HEO is shown in SEQ ID No. 1;

[0009] The HEO mutant is different from HEO in any of the following:

[0010] i) Glutamic acid at position 105 and Leucine at position 112 were both mutated to aspartic acid;

[0011] ii) both serine at position 210 and aspartic acid at position 235 were mutated to alanine;

[0012] iii) valine at position 65 was mutated to alanine, and glycine at position 96 was mutated to cysteine;

[0013] iiii) glycine at position 326 was mutated to cysteine;

[0014] iiiii) Alanine at position 348 was mutated to aspartic acid and Threonine at position 351 was mutated to lysine.

[0015] The present invention also provides a DNA molecule, wherein the DNA molecule is a nucleotide sequence encoding the HEO mutant.

[0016] The invention also provides a recombinant vector, which contains the DNA molecule.

[0017] The present invention also provides a method for producing poly(3-hydroxybutyrate- co -4-hydroxybutyrate), the engineered bacteria express 4hbd , sucd, ogdA, orfZ , xyB , xy , xyD , xy Gene and any of the following:

[0018] a. expressing the xylose transporter;

[0019] b. expressing the DNA molecule;

[0020] c. comprising the recombinant vector.

[0021] Furthermore, the engineered bacteria is Halomonas; preferably, the Halomonas is Halomonas lutescens MDF-9, with a deposit number of GDMCC NO: 61850.

[0022] The Halomonas lutescens MDF-9 strain used in this application has been deposited in Guangdong Microbiological Culture Collection Center (GDMCC for short, address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, zip code 510070) on August 2, 2021. The deposit number is GDMCC NO.61850. The strain name is Halomonas lutescens MDF-9, and the classification name is Halomonas lutescens.

[0023] The present invention also provides a method for constructing the engineering bacteria, comprising the following steps:

[0024] S1: In vitro expansion 4hbd-sucd-ogdA-orfZ Gene sequence, xyB - xy - xyD - xy Gene sequence, introduced into the strain;

[0025] S2: amplify the DNA molecule in vitro and introduce it into the strain described in S1 to obtain an engineered bacterium; wherein the 4hbd- sucd-ogdA-orfZ The target gene sequence is shown in SEQ ID No.5; xyB - xy - xyD - xy The target gene sequence is shown as SEQ ID No.6.

[0026] Furthermore, the 4hbd - sucd-ogdA-orfZ The gene sequence was introduced using the pRE112 vector; and / or xyB - xy - xyD - xy The gene sequence was introduced using the pSEVA321 vector.

[0027] The present invention also provides a method for producing poly (3-hydroxybutyrate- co -4-hydroxybutyrate) method, comprising the steps of:

[0028] 1) Seed solution preparation;

[0029] 2) Shake flask seed culture;

[0030] 3) Fermentation culture.

[0031] Furthermore, the fermentation temperature is 30-38° C.; and / or the fermentation pH is 7-11.

[0032] Furthermore, the fermentation time is 36 to 49 hours.

[0033] Furthermore, the culture medium components of the fermentation culture include a carbon source, and the carbon source is preferably glucose and xylose.

[0034] Furthermore, the addition ratio of glucose to xylose is (1-2):1, preferably 2:1.

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

[0036] 1. The xylose transporter system provided by the present invention can efficiently select xylose, thereby improving the utilization efficiency of xylose by microorganisms.

[0037] 2. The present invention constructs a new metabolic pathway for producing P34HB through the xylose transporter system, so that the engineered bacteria can use xylose and glucose as carbon sources to produce P34HB, thereby increasing the content of 4HB in P34HB.

[0038] 3. The engineered bacteria provided by the present invention can efficiently utilize cheap carbon sources to produce P34HB, thereby reducing the cost of raw materials for producing P34HB.

[0039] 4. The present invention further increases the content of 4HB in P34HB by optimizing the ratio of glucose to xylose in the fermentation medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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.

[0041] Figure 1 A metabolic pathway for producing P34HB by the Halomonas lutescens MDF-9 strain of the present invention;

[0042] Figure 2 pRE112 constructed in Example 1 of the present invention 4hbd-sucd-ogdA-orfZ Plasmid map;

[0043] Figure 3 In Example 1 of the present invention 4hbd-sucd-ogdA-orfZ Integrated electrophoresis verification diagram;

[0044] Figure 4 pSEVA321 constructed in Example 2 of the present invention xylBCDX Plasmid map;

[0045] Figure 5 pSEVA321 in Example 2 of the present invention xylBCDX Electrophoresis verification of successful conjugation into the MDF-9-4sor strain;

[0046] Figure 6 pSEVA341 constructed in Example 3 of the present invention xylE 、pSEVA341 xylFGH 、pSEVA341 HEO Plasmid map;

[0047] Figure 7 In Example 3 of the present invention xy , xyF , HEO Electropherogram of target fragment. DETAILED DESCRIPTION

[0048] 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.

[0049] This application constructs a new metabolic pathway for producing P34HB, such as Figure 1 HalomonaslutescensMDF-9 can use glucose to synthesize 3-hydroxybutyric acid (3HB). Based on this, the present invention introduces exogenous xylose degradation pathway genes xyB - xy - xyD - xy and its transporter, enter the tricarboxylic acid cycle, and then express exogenously 4hbd-sucD - oGD - oeLhd The target gene sequence promotes the production of P34HB by the Halomonas lutescens MDF-9 strain. Further, by mutating the xylose transporter, the utilization rate of xylose by Halomonas lutescens MDF-9 is improved. The third part of the present invention further improves the content of 4HB in the P34HB produced by the strain and the utilization rate of xylose by optimizing the ratio of carbon sources glucose and xylose in the fermentation medium.

[0050] Example 1 4hbd-sucd-ogdA-orfZ Pathway construction

[0051] Using homologous recombination 4hbd-sucd-ogdA-orfZ The gene expression cluster was inserted into the genome to ensure stable expression of downstream genes of the TCA cycle, and upstream genes were expressed using plasmids for transporter screening.

[0052] 1. 4hbd-sucd-ogdA-orfZ Genome integration

[0053] (1) Plasmid construction

[0054] Overlap extension PCR 4hbd-sucd-ogdA-orfZ and pRE112 vector with upstream and downstream homology arms of the G7 insertion site, where the G7 site is a common insertion site for genomic expression in Halomonas. Under the action of Gibson ligase, 4hbd-sucd-ogdA-orfZ and pRE112 vector to form a new plasmid and mark it as pRE112 4hbd-sucd-ogdA-orfZ , plasmid information such as Figure 2 As shown. 4hbd-sucd-ogdA-orfZ Get the template 4hbd-sucd-ogdA-orfZ Sequence, and take part of the product to send to a biological company for sequencing.

[0055] PCR amplification 4hbd-sucd-ogdA-orfZ The primer sequences (5'-3') for the backbone of plasmid pRE112 are as follows:

[0056] 4hbd -F: see SEQ ID No.7;

[0057] 4hbd -R: see SEQ ID No.8;

[0058] sucd -F: see SEQ ID No.9;

[0059] sucd -R: see SEQ ID No.10;

[0060] oGD -F: see SEQ ID No.11;

[0061] oGD -R: see SEQ ID No.12;

[0062] f -F: see SEQ ID No.13;

[0063] f -R: see SEQ ID No.14;

[0064] pRE112-F: see SEQ ID No.15;

[0065] pRE112-R: see SEQ ID No.16.

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

[0067] Table 1 Amplification system

[0068] Reagents Volume (μL) Template DNA 1 2× Rapid Taq Master Mix 10 Upstream primer (10 μM) 1 Downstream primer (10 μM) 1 <![CDATA[ddH2O]]> To 20

[0069] Table 2 Amplification program

[0070] Cycle steps Temperature (℃) time Pre-denaturation 95 3 min transsexual 95 15 sec annealing 56~60 15 sec extend 72 30-60 sec / Kb Number of cycles 35 Complete extension 72 5 min 16 ∞

[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, while cutting off the excess gel as much as possible.

[0072] (2) Gibson Assembly method connection

[0073] The concentration of the recovered DNA was tested, and the proportion of DNA added 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] (3) S17-1 Escherichia 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] (4) 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] (5) Select a single colony with the correct sequence for expansion. After 12 to 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 it on a 60LB plate with corresponding resistance. After 36 to 48 hours, perform another single clone colony verification.

[0088] (6) Screening of genome-integrated strains

[0089] Select a single colony with the correct sequence for expansion, and after 12-16 hours, conjugate with Halomonas lutescensMDF-9 in a 20LB plate. After 8 hours, pick a small amount of conjugated bacteria and spread them on a 60LB plate with the corresponding resistance. After 36-48 hours, perform the first verification. If the verification is successful, the resistance can be removed.

[0090] (7) Antibiotic withdrawal

[0091] In a deep-well plate 60LB, 42℃, subculture for 3 to 7 generations (24 hours for one generation, plate verification starts from the 3rd generation), take a small amount of bacterial liquid after removing the antibiotics and add 60LB (about 10 5 ~10 6Dilute) and spread evenly on 60LB plate. When subcultured, transfer to new 60LB medium (without resistance) at a ratio of 0.02%. After a single colony grows on the plate, take a single colony and streak it on 60LB and 60LB (cm) plates respectively. After culturing for 12 hours, if it can grow on 60LB but not on 60LB (cm), it means that the resistance is successfully removed. Select the colony with successful resistance removal for PCR verification, and then sequence it after successful verification.

[0092] (8) Strain PCR verification

[0093] Verify by the size of the target product, such as Figure 3 As shown in Figure 2, the target fragment is 5523 bp, which is consistent with the expected results. The results show that the Halomonas lutescens MDF-9 strain genome has been successfully inserted 4hbd-sucd-ogdA-orfZ gene expression cluster, and the strain was named MDF-9-4sor.

[0094] Example 2 Xylose degradation pathway xyB - xy - xyD - xy Plasmid construction

[0095] Plasmid construction: PCR amplification xyB - xy - xyD - xy and plasmid pSEVA321 backbone, under the action of Gibson ligase, xyB - xy - xyD - xy The fragment was recombined with the pSEVA321 backbone to form a new plasmid, named pSEVA321 xylBCDX , by overlap extension PCR with pSEVA321 xylBCDX Get the template xyB - xy - xyD - xy ( xylBCX ), take part of the product and send it to a biological company for sequencing. For the specific construction and transformation steps, refer to Example 1. The plasmid information is as follows: Figure 4 By verifying the size of the target fragment, as shown in Figure 5 As shown, the target fragment is 4687 bp, which is in line with the expected result. The plasmid was then joined to the MDF-9-4sor strain to obtain the strain MDF-09-4sor-1.

[0096] Amplification xylBCX The primer sequences (5'-3') of the fragment and pSEVA321 plasmid backbone are as follows:

[0097] xyB -F: see SEQ ID No.17;

[0098] xyB -R: see SEQ ID No.18;

[0099] xy -F: see SEQ ID No.19;

[0100] xy -R: see SEQ ID No.20;

[0101] xylD- F: see SEQ ID No. 21;

[0102] xyD -R: see SEQ ID No.22;

[0103] xy -F: see SEQ ID No.23;

[0104] xy -R: see SEQ ID No.24;

[0105] pSEVA321-F: see SEQ ID No. 25;

[0106] pSEVA321-R: see SEQ ID No.26.

[0107] Example 3 Construction of transport protein plasmid

[0108] Plasmid construction: Amplification of three transporters using overlap extension PCR xylE, xylFGH, HEO and pSEVA341 backbone; xylE, xylFGH, HEO The pSEVA341 backbone was ligated with Gibson ligase. xylE, xylFGH, HEO Combined with the pSEVA341 backbone to form a new plasmid and labeled as pSEVA341 xylE 、 pSEVA341 xylFGH 、 pSEVA341 HEO , plasmid map such as Figure 6 The above three plasmids were used as templates to obtain xylE, xylFGH, HEO Sequence, verification results such as Figure 7 As shown, xylE, xylFGH, HEOThe lengths were 1476 bp, 1407 bp, and 3771 bp, respectively. The results were in line with expectations. Part of the product was sent to a biological company for sequencing, and single clones with the correct sequence were selected for amplification and joined to MDF-09-4sor-1 (for specific steps, refer to Example 1) to obtain strains MDF-09-4sor-2, MDF-09-4sor-3, and MDF-09-4sor-4.

[0109] Amplification xylE, xylFGH, HEO The primer sequences (5'-3') of the pSEVA341 backbone are as follows:

[0110] xy -F: see SEQ ID No.27;

[0111] xy -R: see SEQ ID No.28;

[0112] xyF -F: see SEQ ID No.29;

[0113] xyF -R: see SEQ ID No.30;

[0114] HEO -F: see SEQ ID No.31;

[0115] HEO -R: see SEQ ID No.32;

[0116] pSEVA341-F: see SEQ ID No. 33;

[0117] pSEVA341-R: see SEQ ID No.34.

[0118] Example 4 Transporter Screening Fermentation Test

[0119] (1) Culture medium:

[0120] 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.

[0121] (2) Seed solution preparation

[0122] ① Bacteria activation

[0123] 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.

[0124] ② Primary seed culture

[0125] 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.

[0126] ③Secondary seed cultivation

[0127] 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.

[0128] (3) Preparation of fermentation medium

[0129] Fermentation medium (50 MM): xylose 20 g / L, glucose 20 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, H2BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, NaMoO4·2H2O 0.03 g / L

[0130] (4) Fermentation culture

[0131] Inoculate 5% of the seed solution (1 mL) into a 150 mL conical flask and culture it in a shaker at 37°C and 220 rpm for 48 h.

[0132] (5) Determination of cell dry weight and PHA content

[0133] Cell dry weight (CDW): Place 10-35 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).

[0134] Determination of P34HB content: Weigh 0.05 g of dry bacteria obtained by fermentation, grind them, and place them in a well-sealed esterification tube. Treat 3-hydroxybutyric acid, a 3HB detection standard, and 4-hydroxybutyric acid, a 4HB detection standard, in the same way as the samples, 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). High-purity nitrogen is used as the carrier gas. The program temperature is set as follows:

[0135] Table 5 Program temperature settings

[0136]

[0137] The injection volume was 1 μL, and the external standard method was used for quantitative analysis of PHA. The yield of PHA and the contents of 3HB and 4HB were calculated based on the peak area.

[0138] The fermentation results are shown in Table 6:

[0139] Table 6 Fermentation results of transporter proteins

[0140]

[0141] The results showed that the addition of glucose into the TCA cycle generated succinyl-CoA and α-ketoglutarate, and then 4hbd-sucd-ogdA-orfZ Gene cluster produces 4HB. By introducing xylose degradation pathway gene sequences xyB - xy - xyD - xy After that, the 4HB content in P34HB was increased to 7.63 mol%. 、 xyF 、The data of engineered bacteria fermentation after HEO showed that the strain MDF-9-4sor-4 expressing HEO had the highest dry weight, PHA and 4HB contents, which were 10.78 g / L, 80.11% and 8.63 mol%, respectively.

[0142] Example 5 Mutation of the HEO site of xylose transporter

[0143] In order to express the transport protein HEO more efficiently, HEO was randomly mutated (chemical method) and more efficient HEO was screened out from the mutants. The specific construction method steps are shown in Example 1.

[0144] Xylose concentration determination and xylose conversion rate calculation:

[0145] The xylose content in each sample fermentation broth was detected by high performance liquid chromatography. Take 1mL of fermentation broth, centrifuge at 14000rpm for 10min, and aspirate the supernatant to pass through a membrane (0.22μm) to complete the sample preparation. At the same time, prepare 0, 0.6, 1.2, 1.8, 2.4, and 3.0g / L xylose detection standards. The mobile phase is 0.005M dilute sulfuric acid prepared with ultrapure water. The analysis conditions are: RI: 55℃, flow rate: 0.6mL / min, column temperature: 85℃, injection volume 10μL. Calculate the xylose content based on the peak area.

[0146] Calculation of xylose conversion rate: cell dry weight*PHA (wt%)*4HB (mol%) / [(initial sugar addition amount-residual sugar amount)*100].

[0147] (1) Amplification of HEO primer sequence (5′-3′):

[0148] HEO -F: see SEQ ID No.31;

[0149] HEO -R: see SEQ ID No.32.

[0150] (2) The amplification system is shown in Table 7:

[0151] Table 7 Amplification system

[0152]

[0153] The Tris-HCl mix described in Table 7 comprises 10 mM Tris-HCl, 50 mM KCl and 2 mM MgCl2.

[0154] (3) After screening and sequencing, the forward mutants of the HEO transporter were obtained, namely: glutamate at position 105 and leucine at position 112 were both mutated to aspartic acid, named HEO-1 (E105D, L112D); serine at position 210 and aspartic acid at position 235 were both mutated to alanine, named HEO-2 (S210A, D235A); valine at position 65 was mutated to alanine, and glycine at position 96 was mutated to cysteine, named HEO-3 (V65A, G96C); glycine at position 326 was mutated to cysteine, named HEO-4 (G326C); alanine at position 348 was mutated to aspartic acid and threonine at position 351 was mutated to lysine, named HEO-5 (A348D, T351K).

[0155] The strain MDF-9-4sor-4 introduced with five HEO transporter forward mutants was named MDF-9-HEO-1 (E105D, L112D), MDF-9-HEO-2 (S210A, D235A), MDF-9-HEO-3 (V65A, G96C), MDF-9-HEO-4 (G326C), and MDF-9-HEO-5 (A348D, T351K), and fermentation tests were performed. The specific fermentation steps refer to Example 4, and the fermentation results are shown in Table 8:

[0156] Table 8 Fermentation results of HEO mutants

[0157]

[0158] The results showed that the 4HB content and xylose utilization rate of HEO were increased after HEO was mutated on the basis of MDF-9-4sor-4 strain. After the 105th glutamic acid and 112th leucine of HEO were mutated to aspartic acid, the cell dry weight, P34HB, 4HB content and xylose conversion rate were increased to 11.65 g / L, 84.09%, 13.86% and 34.99%, respectively.

[0159] Example 6 Optimization of the ratio of mixed carbon sources

[0160] The carbon source ratio of the fermentation medium of MDF-9-HEO-1 was optimized, and a total of 5 carbon source ratios were set for testing, namely glucose (g / L): xylose (g / L) = 0:30, 10:20, 20:20, 20:10, 30:0. The specific fermentation steps refer to Example 4, and the fermentation results are shown in Table 9:

[0161] Table 9 Fermentation test results of mixed carbon source ratio optimization

[0162]

[0163] The results in Table 9 show that after optimizing the carbon source ratio of the fermentation medium of the MDF-9-HEO-1 strain, the content of 4HB and the utilization rate of xylose by the strain were increased. Among them, when the ratio of Glu:xylose was 20:10, the cell dry weight, P34HB, 4HB content and xylose conversion rate increased to 11.84 g / L, 85.35% and 14.29%, 39.15%, respectively.

[0164] 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.

[0165] SEQ ID No.1 is the amino acid sequence of HEO; SEQ ID No.2 is HEO Nucleotide sequence; SEQ ID No.3 is xy Nucleotide sequence; SEQ ID No.4 is xyF Nucleotide sequence; SEQ ID No.5 is 4hbd - sucd - oGD - f Nucleotide sequence, of which 1~1116 bp are 4hbd , 1181~2542 bp sucd , 2570~4207 bp oGD , 4234~5523 bp f ; SEQ ID No.6 is xyB - xy - xyD - xy Nucleotide sequence, of which 1~747 bp is xyB , 796~1665 bp xy , 1711~3486 bp xyD , 3533~4687 bp xy .

Claims

1. Use of xylose transporter in the production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate), characterized in that: The xylose transporter is a mutant of the HEO protein, and the amino acid sequence of the HEO protein is shown in SEQ ID No. 1; the difference between the mutant of the HEO protein and the HEO protein is any one of the following: (i) Glutamic acid at position 105 and Leucine at position 112 were both mutated to aspartic acid; (ii) Serine 210 and aspartic acid 235 were both mutated to alanine.

2. A DNA molecule, characterized in that The DNA molecule is a nucleotide sequence encoding the mutant of the HEO protein according to claim 1.

3. A recombinant vector, characterized in that: The recombinant vector contains the DNA molecule according to claim 2.

4. An engineered bacterium for producing poly(3-hydroxybutyrate-co-4-hydroxybutyrate), characterized in that: The engineered bacteria express the 4hbd-sucd-ogdA-orfZ gene and the xylB-xylC-xylD-xylX gene and any one of the following: a. expressing the xylose transporter according to claim 1; b. comprising the DNA molecule according to claim 2; c. comprising the recombinant vector according to claim 3; The engineered bacteria is Halomonas lutescens MDF-9, and the preservation number is GDMCC NO.61850.

5. A method for constructing the engineered bacteria according to claim 4, characterized in that: The steps include: S1: amplify the 4hbd-sucd-ogdA-orfZ gene sequence and xylB-xylC-xylD-xylX gene sequence in vitro and introduce them into the strain; S2: Amplify the DNA molecule described in claim 2 in vitro, and introduce it into the strain described in S1 to obtain an engineered bacterium.

6. The construction method according to claim 5, characterized in that: The 4hbd-sucd-ogdA-orfZ gene sequence was introduced using the pRE112 vector; and / or the xylB-xylC-xylD-xylX gene sequence was introduced using the pSEVA321 vector.

7. A method for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) using the engineered bacteria according to claim 4, characterized in that: The steps include: (1) Seed solution preparation; (2) Shake flask seed culture; (3) Fermentation culture; The culture medium for fermentation culture in step (3) includes a carbon source, and the carbon source is glucose and xylose; the addition ratio of glucose to xylose is (1-2):1.

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