Novel method for producing polyester through biological catalysis

Abstract

The invention relates to the field of biosynthesis high polymer materials, in particular to a biosynthesis method and application of omega-hydroxy fatty acid polyester (omega-PHFA) based on a whole-cell catalytic system. The method comprises the following steps: constructing a recombinant microbial whole-cell catalysis system containing P450 monooxygenase, CoA ligase and acyltransferase, and synthesizing omega-PHFA with a C12-C18 long carbon chain skeleton by taking renewable fatty acid as a substrate. Compared with a traditional chemical / enzymatic synthesis method, the technology is greener, and the performance defects of existing biosynthetic PHA materials are overcome. The invention provides related genetically engineered bacteria and a method for producing omega-PHFA by using the genetically engineered bacteria, and the genetically engineered bacteria have a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biosynthetic polymer materials, and specifically relates to a method for biosynthesis of ω-hydroxy fatty acid polyester (ω-PHFA) based on a whole-cell catalytic system and its application. In particular, the present invention realizes the green synthesis of ω-PHFA with a C12-C18 long carbon chain skeleton by constructing a recombinant microbial whole-cell catalytic system comprising P450 monooxygenase, fatty acid CoA ligase and acyltransferase, using renewable fatty acids as substrates. This technology is greener than traditional chemical / enzymatic synthesis, and at the same time provides a new optimization strategy for problems such as mechanical property defects of polyhydroxyalkanoates (PHA) that can be produced by microbial metabolism. The present invention has broad application prospects, especially in the fields of plastics, fibers, medical materials, etc., and can realize the production of low-cost and green biodegradable polymer materials, which is helpful to promote the sustainable development of the polymer industry. Technical Background

[0002] Under the background of the carbon neutrality strategy, the green synthesis technology of bio-based degradable polyester has become the core development direction in the field of polymer materials. PHA is currently the only biodegradable bio-based polyester material that has been reported to be synthesized through biological pathways and applied to industrial production. However, because this biosynthetic pathway is limited by the catalytic mechanism of the PHA synthase PhaC (which can only catalyze C4-C8 short-chain monomers), the main chain ester bond density of its product is too high and the methylene segment length is insufficient, resulting in the material having defects such as low crystallinity (<40%), insufficient tensile strength (<30MPa) and poor thermal stability (Tm<160℃). This results in its mechanical properties and processing properties being restricted, making it difficult to meet the actual application needs of high-performance polyester materials.

[0003] Comparative studies have shown that extending the length of the methylene segment in the polyester main chain and reducing the proportion of ester bonds in the molecule can help improve the crystallinity and mechanical properties of polyester materials, thereby enhancing their application potential in the field of polymer materials. Specifically, materials such as polyω-hydroxynonanoate, polyω-hydroxydecanoate and polyω-hydroxytetradecanoate have been shown to have excellent thermal stability and hydrolytic stability. Through comparative studies of the physical and chemical properties of different ω-PHFAs, it was found that as the number of methylene units in the polymerized monomer increases, the melting temperature (Tm) and glass transition temperature (Tg) of the polyester gradually increase. This shows that by increasing the number of methylene units in the main chain, the thermal stability and mechanical properties of polyester materials can be effectively improved.

[0004] Although the existing chemical synthesis method can prepare long-chain ω-PHFA, it relies on heavy metal catalysts and high temperature and high pressure conditions (>150℃, 3-5MPa), with high production costs and heavy environmental load. In addition, heavy metal catalysts are difficult to separate from the product, the product purification cost is high, and the application in biomedical materials also faces problems such as toxic residues; and the traditional enzyme catalysis method faces technical bottlenecks such as poor enzyme stability, low substrate conversion rate, and excessive use of organic reagents. In comparison, the whole-cell catalytic synthesis of polyesters using cell metabolic pathways has obvious advantages. By modifying or constructing cell metabolic pathways, a low-cost and green sustainable production model can be more effectively achieved. This method is more in line with the current concept of green chemistry and biomanufacturing. The esterification reaction in the cell is usually achieved by two consecutive reactions. First, the carboxyl group of the substrate consumes ATP to form a high-energy acyl-CoA compound under the action of CoA ligase; then, the acyl-CoA compound completes the acyl transfer reaction with the hydroxyl compound under the action of acyltransferase, releasing the CoA molecule and forming an ester bond. At present, based on the synthesis pathway of intracellular ester compounds, CoA ligase and acyltransferase are often used as key nodes, and by introducing the ethanol metabolic pathway, the cell's fatty acid synthesis pathway is connected with the ethanol synthesis pathway to directly generate fatty acid ethyl esters. However, there is no work report on the application of CoA ligase and acyltransferase in polyester synthesis.

[0005] The innovation of the present invention is that it catalyzes the synthesis of long-chain ω-PHFA through cellular metabolic pathways, opening up a new biocatalyst. This not only overcomes the problem of insufficient polyester performance in the prior art, but also makes the production method in the form of whole-cell catalysis more green and sustainable. The proposal of this new production pathway not only provides a new green synthesis route for the polymer field, but also provides a theoretical basis and practical guidance for the development of bio-based polyester materials with better performance. Summary of the invention

[0006] The present invention proposes an innovative biocatalytic strategy to solve the problems of material mechanical properties defects caused by the short main chain methylene chain in the existing biosynthetic PHA technology, as well as environmental pollution in the traditional chemical synthesis method. By systematically transforming the genome of chassis microorganisms and introducing exogenous gene modules, a highly efficient ω-hydroxy fatty acid polyester synthesis cell catalyst was successfully constructed, realizing the green biomanufacturing of long carbon chain ω-hydroxy fatty acid polyester (C12-C18). The technical core of the present invention is to establish a cascade catalytic system of P450 monooxygenase, CoA ligase and acyltransferase for the first time, breaking through the limitations of the traditional intracellular ester compound synthesis path. The present invention constructs a cascade reaction system containing three key enzymes through heterologous expression: 1) P450 monooxygenase catalyzes the hydroxylation reaction of fatty acid terminals to generate ω-hydroxy fatty acids with bifunctional groups; 2) CoA ligase catalyzes the activation of carboxyl groups to acyl-CoA intermediates under ATP energy supply conditions; 3) Acyltransferase mediates acyl transfer reaction, and forms high molecular weight polyester chains through continuous polycondensation of hydroxyl groups and acyl-CoA. Compared with the prior art, the PHA synthesis pathway cannot achieve the synthesis of polyhydroxy fatty acids with longer carbon chain skeletons; for the three key enzymes involved, fatty acid P450 monooxygenase is only used for the hydroxylation of alkanes or fatty acids in current research reports to produce hydroxy fatty acids and diacids; the research on CoA ligase and acyltransferase is mainly combined with the intracellular alcohol metabolism pathway, and is mostly used to prepare monoesters or structural esters. Although the chemical method can also achieve the synthesis of long-chain ω-PHFA, in addition to environmental pollution and harsh reaction conditions, heavy metal catalysts are difficult to effectively remove during the product purification process, and the high cost also brings potential risks to its application in biomedical materials. Enzyme catalysis needs to face the high price of enzyme preparations and the unavoidable use of a large amount of organic reagents. In the present invention, the whole cell catalysis process is mild and only requires the use of a small amount of organic solvents. At the same time, the non-use of heavy metal catalysts also avoids the difficulty of subsequent purification, and the product safety is more reliable.

[0007] The breakthrough of the present invention is that the hydroxyl functional group is introduced at the end of the fatty acid by P450 enzyme, and after activation by CoA ligase, each monomer carries both hydroxyl and acyl activation sites, thereby achieving monomer iterative polycondensation. Based on this, the number of methylene groups in polyester monomers can be effectively expanded to the C12-C18 range, creating a new model for the biosynthesis of ω-hydroxy fatty acid polyesters, and providing a new platform and strategy for the development of high-performance bio-based polyester materials.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a genetically engineered bacterium for synthesizing long-chain ω-hydroxy fatty acid polyesters, wherein the genetically engineered bacterium can express P450 monooxygenase, CoA ligase and acyltransferase.

[0010] In some embodiments of the present invention, the starting strain of the bottom plate bacteria is Escherichia coli K-12 or its derivative strains, preferably Escherichia coli BL21 or its derivative strains, and more preferably Escherichia coli BL21 (DE3).

[0011] Preferably, the fadD (NCBI accession number: CP081489.1, SEQ ID NO.1) and fadE (NCBI accession number: CP081489.1, SEQ ID NO.2) genes in the Escherichia coli base strain are knocked out, and the expression of the fadL gene (NCBI accession number: CP081489.1, SEQ ID NO.3) is enhanced.

[0012] In some embodiments of the present invention, the expression of the P450 monooxygenase, CoA ligase and acyltransferase can be achieved by introducing an expression vector carrying the encoding gene into the chassis bacteria or directly inserting the encoding gene into the genome.

[0013] Preferably, the expression of the P450 monooxygenase, CoA ligase and acyltransferase is achieved by introducing an expression vector comprising genes encoding the P450 monooxygenase, CoA ligase and acyltransferase.

[0014] The P450 monooxygenase described above is preferably derived from P450BM3 (CYP102A1) of Bacilus megaterium (NCBI accession number: P14779.2, SEQ ID NO.4). Or derived from CYP153A of Marinobacter (NCBI accession number: WP_011784142.1, SEQ ID NO.5)

[0015] Preferably, the P450 monooxygenase has an amino acid sequence as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0016] The CoA ligase described above is preferably derived from ACOS5 of Arabidopsis thalian (NCBI accession number: NP_176482.1, SEQ ID NO.6)

[0017] Preferably, the CoA ligase has an amino acid sequence as shown in SEQ ID NO.6.

[0018] The acyltransferase described above is preferably derived from MhWS2 of Marinobacter hydrocarbonoclasticus (NCBI accession number: AVN88338.1, SEQ ID NO.7)

[0019] Preferably, the acyltransferase has an amino acid sequence as shown in SEQ ID NO.7.

[0020] Optional expression vectors include expression vectors suitable for chassis microorganisms such as Escherichia coli, yeast, Bacillus subtilis, including but not limited to common Escherichia coli pET, pRSFDuet and other series expression vectors, yeast pGAP, pRS and other expression systems, and Bacillus subtilis pHY300PLK, amyE and other expression systems.

[0021] In some embodiments of the present invention, when E. coli is preferably used as the chassis, the preferred expression vector is pET-22b(+).

[0022] In a second aspect, the present invention provides the use of the genetically engineered bacteria described above in the synthesis of ω-hydroxy fatty acid polyesters.

[0023] In a third aspect, the present invention provides a method for preparing ω-hydroxy fatty acid polyester, the method comprising: synthesizing ω-hydroxy fatty acid polyester using medium-chain fatty acids in the genetically engineered bacteria described above.

[0024] In some embodiments of the present invention, it mainly includes constructing an expression vector or an integration vector, transforming into a gene knockout chassis cell, and realizing controllable gene expression. After obtaining the mature protein, fatty acids are added as substrates, a whole-cell catalytic reaction is carried out, ω-PHFA is accumulated in the cell, and the changes in the substrate and intracellular products during the catalytic process are detected. After the reaction is completed, the intracellular product is extracted, and the product is obtained by alcohol precipitation using cold ethanol, and the properties are characterized after drying.

[0025] Preferably, the method comprises: mixing a whole-cell biocatalyst with a fatty acid to synthesize ω-hydroxy fatty acid polyesters in the cell.

[0026] Preferably, the preparation method comprises the preparation of a whole-cell biocatalyst and the use of the whole-cell biocatalyst to catalyze the synthesis of ω-hydroxy fatty acid polyesters from fatty acids.

[0027] Among them, the preparation of whole-cell biocatalysts includes: vector construction, codon optimization of the three core enzymes for chassis cell species, design and use of homologous recombination to connect them into expression vectors or integration vectors; cell transformation and protein expression, introducing the recombinant vector into chassis cells by electroporation / chemical transformation, and culturing the expression of core enzymes to obtain whole-cell biocatalysts.

[0028] Preferably, the chassis cell used is Escherichia coli BL21 DE3, and more preferably, the fadD and fadE genes in the Escherichia coli chassis bacteria are knocked out, and the expression of the fadL gene is enhanced.

[0029] In some embodiments of the present invention, the expression vectors of P450 monooxygenase, CoA ligase and acyltransferase are transferred into the above-mentioned E. coli chassis bacteria, cultured and grown to the mid-logarithmic phase, and then IPTG is used to induce protein expression. The induced cells are collected by centrifugation to obtain the whole-cell biocatalyst.

[0030] The method for synthesizing ω-hydroxy fatty acid polyester from fatty acid using the whole-cell biocatalyst comprises: washing the whole-cell biocatalyst prepared above with a buffer solution, resuspending it in a fresh simple culture medium, adding 5-20 mM straight-chain fatty acid to start cell catalysis, and performing the reaction under constant temperature oscillation. After the reaction is completed, the product is separated, purified and characterized.

[0031] Preferably, a surfactant may be additionally added to the above simple culture medium to enhance the efficiency of substrate transmembrane transport.

[0032] Preferably, the above-mentioned product separation and purification method includes, after the whole-cell catalytic process is completed, collecting the bacteria, drying and grinding, using solvent graded extraction, and then using cold ethanol to separate the products, and obtaining the polyester product after drying.

[0033] Preferably, the above-mentioned product property characterization includes that the molecular weight and PDI of ω-PHFA polyester are determined by gel exclusion chromatography (GPC). The structure of the polyester product is characterized by Fourier transform infrared spectroscopy (FTIR) combined with nuclear magnetic resonance (NMR) data. The thermal stability of the polyester is detected by a thermogravimetric analyzer (TGA), and the thermal stability of the product is determined by the pyrolysis temperature of the product; the crystallization performance of the polyester is determined by the dynamic non-isothermal crystallization performance of the polyester by a rotational rheometer; and the crystallization temperature (Tc) and the melting temperature (Tp) of the product are determined by a differential scanning calorimeter (DSC).

[0034] The beneficial effects of the present invention include at least:

[0035] The present invention provides a method for biosynthesis of ω-hydroxy fatty acid polyester (ω-PHFA) based on whole-cell catalysis, and successfully synthesizes high-performance polyester with C12-C18 long carbon chain by introducing a multi-enzyme cascade catalytic system of P450 monooxygenase, CoA ligase and acyltransferase. The method adopts a green and sustainable biocatalytic method, avoids high temperature and high pressure and heavy metal catalysts in traditional chemical synthesis, and can effectively improve the crystallinity, thermal stability and mechanical properties of polyester. In addition, the entire synthesis process is simple and efficient, suitable for low-cost production, and has broad application prospects, especially in the fields of plastics, fibers and medical materials, which meets the current needs of environmental protection and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 This is a schematic diagram of the whole-cell catalytic intracellular metabolic process pathway of ω-hydroxy fatty acid polyester (ω-PHFA);

[0038] Figure 2 After the polyester is precipitated with cold ethanol, it is allowed to stand for phase separation and the product is washed and purified and then collected by centrifugation;

[0039] Figure 3 This is the H NMR spectrum result of polyω-hydroxylauric acid;

[0040] Figure 4 GPC results for polyω-hydroxylauric acid DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The present invention provides a genetically engineered bacterium that expresses P450 monooxygenase, CoA ligase and acyltransferase. Based on the above recombinant bacteria, the present invention constructs a whole-cell catalyst that can prepare ω-hydroxy fatty acid polyesters from fatty acid substrate sources. Specifically, it includes:

[0043] This embodiment provides an Escherichia coli chassis strain, and the construction method thereof is as follows:

[0044] Example 1 Preparation of whole cell catalyst

[0045] S1: The fadD and fadE genes in the Escherichia coli BL21 (DE3) genome were knocked out using CRISPR-Cas9 gene editing technology to obtain E. coli BL21 (DE3) (△fadD, △fadE).

[0046] S2: Construct the expression vector pCDFDuet-FadL+P450BM3, pETDuet-ACOS5 / MhWS2, as shown in Figure XX, to enhance the expression of fatty acid transport protein FadL, and introduce three core enzymes P450BM3, ACOS5, and MhWS2. Transfer the plasmid pCDFDuet-FadL+P450BM3, pETDuet-ACOS5 / MhWS2 into E.coliBL21(DE3)(△fadD, △fadE) to obtain BL21(DE3)(ω-PHFA), which is the genetically engineered E. coli bacteria.

[0047] S3: Pick a single colony of the recombinant Escherichia coli and place it in 4 mL of LB medium containing 100 μg / mL ampicillin sodium and 50 μg / mL kanamycin sulfate, and culture at 37°C, 220 rpm for 12-16 h to obtain a seed solution.

[0048] S4: The seed solution prepared in S3 was transferred to a fermentation medium (LB medium) containing 100 μg / mL ampicillin sodium and 50 μg / mL kanamycin sulfate, and cultured at 37°C until OD600 reached 0.6-0.8, then the inducer was added, and the induction culture was carried out at 18°C ​​for 16 hours.

[0049] S5: Collect the bacteria by centrifugation to obtain the biocatalyst.

[0050] Example 2 Synthesis of ω-PHFA from fatty acids (FFA) using biocatalysts

[0051] This embodiment provides a method for synthesizing ω-PHFA using FFA catalyzed by a biocatalyst, the steps are as follows:

[0052] S1: Add 20 g / L (wet weight of bacteria) of the biocatalyst (prepared by the method of Example 1), 5 mM lauric acid, 1% DMSO, and 1 g / L glucose to a fresh simple culture medium, and carry out oxygen catalysis for 36 h at 30° C. while maintaining pH at 6.5-7.0.

[0053] S2: During the catalytic process of S1, samples were taken and centrifuged every hour. After derivatization, the supernatant was quantified by GC-FID. Parallel samples were quickly frozen and crushed by liquid nitrogen, concentrated after extraction, and the products were separated by alcohol precipitation using cold ethanol. After alkaline hydrolysis, the composition and cumulative amount of the polyester products were analyzed by GC.

[0054] S3: After the catalysis of S1 was completed, the cells were collected by centrifugation and milled using a ball mill (zirconia beads, ) was finely ground under low temperature of liquid nitrogen to a particle size of <50μm; then solvent fractionation extraction was performed: the first round of extraction: chloroform-methanol mixed solvent (3:1, v / v), ultrasonic assisted extraction for 30min (40kHz, 25℃), two additional fresh extraction solvents were added, the organic phases were combined and concentrated by rotary evaporation to 10% of the original volume; 10 times the volume of pre-cooled ethanol (-20℃) was slowly added to the concentrate, and the phases were allowed to stand at 4℃ for 12h for phase separation (see Appendix Figure 2 The precipitate was washed with cold ethanol 3 times (10 mL / g each time) and vacuum dried to obtain a white flocculent polyester product. The yield in this example was calculated to be 25 mg / g (cell dry weight).

[0055] S4: Product Characterization, Utilization 1 The structure of the polyester product was further characterized by H-NMR (see Appendix Figure 3 ), confirming that the product is ω-PHFA. The number of hydrogen atoms on the α-carbon and ω-carbon of the ω-PHFA monomer was calculated based on the peak area ratio of the proton signal, and its degree of polymerization was estimated to be about 39. Figure 4 ) The polyester products of ω-hydroxylauric acid and ω-hydroxypalmitic acid were characterized respectively. The obtained polyω-hydroxylauric acid ester had an Mn of about 7000, an Mw of about 9800, and a PDI of 1.4.

[0056] The difference between Example 3 and Example 2 is that by adding fatty acids of different chain lengths such as lauric acid, myristic acid, and palmitic acid as substrates for whole-cell catalysis, corresponding polyester products can be obtained. The specific molecular weight and PDI are shown in the table below.

[0057] Substrate Yield (mg / L) Degree of Polymerization Molecular weight Mn(Da) PDI C12:0 20±5.3 20-42 4000-9000 1.4 C14:0 16±4.4 17-35 4000-8500 1.3 C16:0 15±7.2 10-33 2700-7300 1.4

Claims

1. A genetically engineered bacterium for synthesizing long-chain ω-hydroxy fatty acid polyesters, wherein the genetically engineered bacterium needs to express P450 monooxygenase, CoA ligase and acyltransferase, characterized in that: 1) P450 monooxygenase catalyzes the hydroxylation reaction of the fatty acid terminal to generate ω-hydroxy fatty acids with bifunctional groups; 2) CoA ligase catalyzes the activation of the carboxyl group to an acyl-CoA intermediate under ATP energy supply conditions; 3) Acyltransferase mediates the acyl transfer reaction to form a high molecular weight polyester chain through the continuous condensation of the hydroxyl group and acyl-CoA. The expression of the fatty acid P450 monooxygenase, CoA ligase and acyltransferase is achieved by introducing an expression vector carrying the coding gene into the chassis bacteria or directly inserting it into the genome.

2. The genetically engineered bacterium according to claim 1, wherein The P450 monooxygenase (catalyzing fatty acid hydroxylation) is preferably derived from P450BM3 (CYP102A1) of Bacilus megaterium (NCBI accession number: P14779.2) and CYP153A from Marinobacter (NCBI accession number: WP_011784142.1), and the P450 monooxygenase has an amino acid sequence as shown in SEQ ID NO.4 and SEQ ID NO.

5.

3. The genetically engineered bacterium according to claim 1, wherein The CoA ligase is preferably derived from ACOS5 of Arabidopsisthaliana (NCBI accession number: NP_176482.1), and the CoA ligase has an amino acid sequence as shown in SEQ ID NO.

6.

4. The genetically engineered bacterium according to claim 1, wherein The acyltransferase is preferably derived from MhWS2 (AVN88338.1) of Marinobacterhydrocarbonoclasticus, and the acyltransferase has an amino acid sequence as shown in SEQ ID NO.

6.

5. The genetically engineered bacterium according to claim 1, wherein The expression vector is selected from expression vectors suitable for chassis microorganisms such as Escherichia coli, yeast, Bacillus subtilis, etc.

6. A method for preparing ω-hydroxy fatty acid polyester, characterized in that: In the genetically engineered bacteria according to claims 1-5, medium- and long-chain fatty acids are used to synthesize ω-hydroxy fatty acid polyesters.

7. The preparation method according to claim 6, wherein: The method comprises the preparation of a whole-cell biocatalyst and the use of the whole-cell biocatalyst to catalyze the synthesis of ω-hydroxy fatty acid polyester from fatty acids. The preparation method of the whole-cell biocatalyst comprises: codon-optimizing the coding genes of P450 monooxygenase, CoA ligase and acyltransferase for chassis cell species, and inserting them into an expression vector or an integration vector by homologous recombination; introducing the recombinant vector into chassis cells by electroporation or chemical transformation, and achieving the expression of P450 monooxygenase, CoA ligase and acyltransferase to obtain the whole-cell biocatalyst; The method for synthesizing ω-hydroxy fatty acid polyesters by catalyzing fatty acids with whole-cell biocatalysts is as follows: the whole-cell biocatalyst prepared above is washed with a buffer solution and then resuspended in a fresh simple culture medium, and 5-20 mM straight-chain fatty acids are added to start cell catalysis. The reaction is carried out under constant temperature oscillation. After the reaction is completed, the product is separated, purified and characterized.

8. The preparation method according to claim 7, wherein: The conditions of the catalytic reaction include: a reaction time of 24 to 72 hours, a reaction temperature controlled at 25 to 37° C., and a pH value adjusted to 6.5 to 8.0 to optimize enzyme activity and catalytic efficiency.

9. According to the preparation methods of claims 6, 7, and 8, ω-hydroxy fatty acid polyesters with a molecular weight Mn ranging from 1000 to 50000 Da and a PDI ranging from 1.1 to 1.8 can be finally obtained.

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