A carboxylate reductase mutant and its application in biosynthesis of (z)-13-octadecenal

Abstract

This invention provides a carboxylic acid reductase mutant and its application in biosynthesis ( Z The application of 1,3-octadecenal belongs to the field of biocatalysis and synthetic biotechnology. The carboxylic acid reductase mutant is based on wild-type carboxylic acid reductase. Mc The wild-type carboxylic acid reductase was obtained by CAR-based mutation in one of G274A, M298L, or G274A / M298L. Mc The amino acid sequence of CAR is shown in SEQ ID NO: 1. The biosynthesis provided by this invention ( Z The method of )-13-octadecenal, using pheromone precursors ( Z Using 13-octadecenoic acid as a substrate and catalyzed by a carboxylic acid reductase mutant, the sex pheromone of the rice stem borer was synthesized. Z The enzymatic preparation of 13-octadecenal is a green and environmentally friendly preparation route with mild reaction conditions and high yield of the target product.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis and synthetic biotechnology, and particularly relates to a carboxylic acid reductase mutant and its application in biosynthesis. Z Applications of )-13-octadecenal. Background Technology

[0002] In the field of pest control, insect sex pheromones have become an important alternative or supplement to chemical pesticides due to their environmental friendliness, high specificity, and low likelihood of developing resistance. Sex pheromones are key chemical signals for mating communication between insect sexes. By releasing specific components to interfere with pest mating behavior, effective monitoring and green control of target populations can be achieved. In recent years, numerous studies and practical applications have demonstrated the enormous potential and broad application prospects of sex pheromones in integrated pest management.

[0003] ( Z )-13-octadecenal is a major pest of rice, specifically the rice stem borer ( ). Chilo suppressalis This compound is one of the core components of sex pheromones. Sex pheromone decoys constructed based on this compound have been widely studied and applied in population monitoring, mass trapping, and mating interference of the rice stem borer (Experimental study on the whole process of prevention and control of Chilo suppressalis sex pheromone [J]. Modern Agricultural Science and Technology , 2016, 18: 98. The application technology test of insect sex pheromone mixiang method to control the Chilo suppressalis[J]. Friends of Farmers to Riches , 2018, 10: 117.). Therefore, green and efficient synthesis ( Z )-13-octadecenal is of great practical significance for supporting the green control of rice stem borer.

[0004] at present,( Z The synthesis of 13-octadecenal mainly relies on traditional chemical methods, including the Wittig reaction and alkyne-selective reduction. However, these methods generally suffer from harsh reaction conditions, the use of toxic and hazardous reagents, and numerous byproducts (such as triphenylphosphine, which is difficult to separate in the Wittig route), limiting the feasibility of its large-scale green manufacturing. Therefore, developing a novel environmentally friendly synthetic route is crucial. Z13-Octadecanal has become a research hotspot in both academia and industry.

[0005] In recent years, the rapid development of synthetic biology has provided new technological pathways for the green manufacturing of complex natural products. For example, Honglei Wang et al. have genetically engineered flaxseed to produce a precursor to moth sex pheromones (…). Z In vivo synthesis of 1,1-hexadecenoic acid, followed by in vitro chemical transformation to obtain the target pheromone component (Insect pest management with sex pheromone precursors from engineered oilseed plants [J]. Nature Sustainability , 2022, 5(11): 981-90.). This study provides an important paradigm for the synthesis of insect sex pheromones based on plant chassis, and also suggests that similar strategies can be used for synthesis ( Z )-13-octadecenoic acid, and then through a key conversion step to obtain ( Z )-13-octadecenal.

[0006] In this context, ( Z )-13-octadecenoic acid is efficiently converted into ( Z 13-Octadecanal is one of the key bottlenecks in constructing a complete biosynthetic route. Therefore, developing a highly active biocatalyst that can specifically catalyze this reaction is the core task for realizing this process. This invention successfully constructed a catalytic (13-octadecenal) biocatalyst by screening carboxylic acid reductases. Z A novel biocatalytic pathway for the formation of aldehydes from 13-octadecenoic acid was discovered. By combining this pathway with structure-guided protein engineering, the screened enzyme elements were modified to obtain mutants with enhanced catalytic activity, providing important components for the biosynthesis of insect sex pheromones. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a carboxylic acid reductase mutant and its application in biosynthesis ( Z Applications of )-13-octadecenal.

[0008] This invention provides a carboxylic acid reductase mutant, wherein the carboxylic acid reductase mutant is based on wild-type carboxylic acid reductase. Mc The wild-type carboxylic acid reductase was obtained by CAR-based mutation in one of G274A, M298L, or G274A / M298L. Mc The amino acid sequence of CAR is shown in SEQ ID NO: 1.

[0009] This invention provides a gene encoding the aforementioned carboxylic acid reductase mutant.

[0010] The present invention provides a recombinant vector expressing the carboxylic acid reductase mutant, comprising an initial vector and the gene, wherein the initial vector is the pRSFDuet-1 plasmid.

[0011] Preferably, the gene and the phosphate pantothenic acid thioethylamine transferase (Sfp) gene are co-expressed on the initial vector.

[0012] This invention provides a genetically engineered bacterial strain, obtained by transferring the recombinant vector into a host bacterium; the host bacterium is *Escherichia coli*. E. coli BL21 (DE3).

[0013] This invention provides the aforementioned carboxylic acid reductase mutant, the aforementioned gene, the aforementioned recombinant vector, and the aforementioned genetically engineered bacteria in biosynthesis (…). Z Applications of )-13-octadecenal.

[0014] This invention provides a biosynthesis ( Z A method for 1,3-octadecenal includes the following steps: by( Z Using 1,3-octadecenoic acid as a substrate, a reduction reaction is carried out by the carboxylic acid reductase mutant or the genetically engineered bacteria in the presence of a cofactor. The cofactors include NADPH, ATP, and MgCl2; The reduction reaction is carried out in a water-heptane two-phase system; The reduction reaction is accompanied by oscillation; The reduction reaction is carried out at a temperature of 28~32℃.

[0015] Preferably, after the reduction reaction is completed, 2-4 times the volume of heptane is added for extraction, the organic phase is collected, dried, and concentrated to obtain (…). Z )-13-octadecenal.

[0016] Preferably, the concentration of the substrate is 0.8~1.2mM, and the concentration of the carboxylic acid reductase mutant is 0.1~0.3mg / ml.

[0017] Preferably, the concentration of NADPH is 4-6 mM, the concentration of ATP is 4-6 mM, and the concentration of MgCl2 is 8-12 mM.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The carboxylic acid reductase mutant provided by the present invention, with wild-type carboxylic acid reductase McBased on CAR, the carboxylic acid reductase mutant is obtained by mutation in one of G274A, M298L, or G274A / M298L. The carboxylic acid reductase mutant described in this invention is obtained by modifying the wild-type carboxylic acid reductase using a structure-guided semi-rational design molecular modification method, thus solving the problem of wild-type carboxylic acid reductase. Mc CAR addresses the issue of low enzyme activity in non-natural substrates, with the optimal mutant G274A / M298L exhibiting catalytic activity 0.36 times higher than the wild type.

[0019] The biosynthesis provided by this invention ( Z The method of )-13-octadecenal, using pheromone precursors ( Z Using 1,3-octadecenoic acid as a substrate, a carboxylic acid reductase mutant was used for catalysis. Z Preparation by reduction of 1,3-octadecenoic acid ( Z )-13-octadecenal, achieving the dimorphothiocarbamate sex pheromone ( Z The enzymatic preparation of 1,3-octadecenal provides a green and environmentally friendly preparation route.

[0020] Using the method described in this invention, the carboxylic acid reductase mutant catalyzed the reaction for 24 h, achieving a target product concentration of 0.59 mM, a molar yield of 59%, and a total yield of 76%, providing a sustainable alternative method for the biosynthesis of insect sex pheromones. Attached Figure Description

[0021] Figure 1 This invention utilizes a carboxylic acid reductase mutant to synthesize a pure enzyme. Z The reaction route of )-13-octadecenal.

[0022] Figure 2 This invention utilizes the crude enzyme solution of a carboxylic acid reductase mutant for biosynthesis ( Z The reaction route of )-13-octadecenal.

[0023] Figure 3 The results of screening for saturation mutations at the G274 and M298 sites.

[0024] Figure 4 For commercial standard products ( Z )-13-octadecenal and ( Z The GC-FID spectrum of 1,3-octadecenol (0.5 mM) showed a retention time of 17.064 min. Z )-13-octadecenal, retention time 17.834 min ( Z )-13-octadecenol.

[0025] Figure 5 Wild type in Example 4 McGC-FID chromatogram of a sample taken 2 h after the CAR WT catalytic reaction.

[0026] Figure 6 The mutant in Example 4 Mc GC-FID chromatogram of a sample taken 2 h after CAR M298L catalytic reaction.

[0027] Figure 7 The mutant in Example 4 Mc GC-FID chromatogram of a sample taken after 2 h of CAR G274A / M298L catalytic reaction.

[0028] Figure 8 The mutant in Example 5 Mc GC-FID chromatogram of a sample taken 24 h after the CAR G274A / M298L catalytic reaction. Detailed Implementation

[0029] This invention provides a carboxylic acid reductase mutant, wherein the carboxylic acid reductase mutant is based on wild-type carboxylic acid reductase. Mc The wild-type carboxylic acid reductase was obtained by CAR-based mutation in one of G274A, M298L, or G274A / M298L. Mc The amino acid sequence of CAR is shown in SEQ ID NO: 1, and is as follows: MPTETRDERLARRIADLHATDPEFAAATPDDAISETIDQPGVRLPQIMATVLDGYADRPALGQRAVRFVIDPQTGRTSADLLPRFETITYAELSARVHAVMNTLTDVAPGDRVALLGFTSVDYTVIDMALALSGAV SVPLQTSAPAATLRPIIAETEPVIIASAVDHLADAVELAREADTVRRVIVFDHRAEVDDHRDAVADARTRLTEGGRAIEVLTLAEVLEHGATLPAAQPFSSPEQDPLTLLIYTSGSTGAPKGAMYPERLVAGAWLRS G RSTWYGEHATPSITLNFLPMSHM M.

[0030] The sites in bold and underlined in the above sequence are mutation sites.

[0031] The present invention also provides a gene encoding the aforementioned carboxylic acid reductase mutant, wherein the gene is preferably codon-optimized and synthesized by a biotechnology company.

[0032] The gene sequence of the G274A mutation is as follows (SEQ ID NO: 2): ATGCCTACAGAGACACGTGATGAACGTCTGGCTCGTAGAATTGCCGATCTGCATGCAACCGATCCTGAATTTGCCGCAGCTACACCTGATGATGCAATTAGCGAAACCATCGACCAGCCGGGTGTTCGTTTACCTCAAATTATGGCAACCGTGCTGGACGGTTATGCAGATAGACCTGCACTGGGTCAGCGTGCAGTGCGTTTCGTGATCGACCCTCAGACCGGTCGTACCAGCGCAGACCTGCTGCCTCGTTTCGAGACCATCACCTATGCAGAACTGAGTGCCCGTGTTCATGCAGTTATGAATACCCTGACCGACGTTGCACCGGGTGACCGTGTGGCACTGCTGGGTTTCACCAGCGTGGACTACACCGTGATTGATATGGCCCTGGCACTGAGTGGTGCAGTGAGCGTGCCTCTGCAGACCAGCGCACCTGCAGCAACCCTGCGTCCTATCATCGCAGAGACCGAGCCTGTGATTATTGCAAGCGCAGTTGATCACCTGGCCGATGCAGTGGAGTTAGCACGTGAGGCAGATACAGTTCGCCGTGTGATTGTTTTCGACCATCGTGCTGAAGTGGACGATCATCGTGATGCAGTGGCAGATGCACGTACAAGACTGACAGAAGGCGGTAGAGCAATTGAAGTTCTGACCCTGGCCGAAGTGCTGGAGCACGGTGCAACCCTGCCTGCAGCACAGCCTTTCAGCAGCCCTGAGCAGGACCCTCTGACCCTGCTGATCTACACCAGCGGTAGCACCGGTGCACCTAAGGGTGCAATGTATCCTGAACGTCTGGTGGCAGGTGCTTGGCTGCGTAGC GCA The gene sequence of the M298L mutation is as follows (SEQ ID NO: 3): ATGCCTACAGAGACACGTGATGAACGTCTGGCTCGTAGAATTGCCGATCTGCATGCAACCGATCCTGAATTTGCCGCAGCTACACCTGATGATGCAATTAGCGAAACCATCGACCAGCCGGGTGTTCGTTTACCTCAAATTATGGCAACCGTGCTGGACGGTTATGCAGATAGACCTGCACTGGGTCAGCGTGCAGTGCGTTTCGTGATCGACCCTCAGACCGGTCGTACCAGCGCAGACCTGCTGCCTCGTTTCGAGACCATCACCTATGCAGAACTGAGTGCCCGTGTTCATGCAGTTATGAATACCCTGACCGACGTTGCACCGGGTGACCGTGTGGCACTGCTGGGTTTCACCAGCGTGGACTACACCGTGATTGATATGGCCCTGGCACTGAGTGGTGCAGTGAGCGTGCCTCTGCAGACCAGCGCACCTGCAGCAACCCTGCGTCCTATCATCGCAGAGACCGAGCCTGTGATTATTGCAAGCGCAGTTGATCACCTGGCCGATGCAGTGGAGTTAGCACGTGAGGCAGATACAGTTCGCCGTGTGATTGTTTTCGACCATCGTGCTGAAGTGGACGATCATCGTGATGCAGTGGCAGATGCACGTACAAGACTGACAGAAGGCGGTAGAGCAATTGAAGTTCTGACCCTGGCCGAAGTGCTGGAGCACGGTGCAACCCTGCCTGCAGCACAGCCTTTCAGCAGCCCTGAGCAGGACCCTCTGACCCTGCTGATCTACACCAGCGGTAGCACCGGTGCACCTAAGGGTGCAATGTATCCTGAACGTCTGGTGGCAGGTGCTTGGCTGCGTAGCGGTCGTAGCACCTGGTACGGTGAGCACGCAACCCCTAGCATCACCCTGAACTTTCTGCCTATGAGCCACATG CTG Gene sequence of G274A / M298L mutation (SEQ ID NO: 4): ATGCCTACAGAGACACGTGATGAACGTCTGGCTCGTAGAATTGCCGATCTGCATGCAACCGATCCTGAATTTGCCGCAGCTACACCTGATGATGCAATTAGCGAAACCATCGACCAGCCGGGTGTTCGTTTACCTCAAATTATGGCAACCGTGCTGGACGGTTATGCAGATAGACCTGCACTGGGTCAGCGTGCAGTGCGTTTCGTGATCGACCCTCAGACCGGTCGTACCAGCGCAGACCTGCTGCCTCGTTTCGAGACCATCACCTATGCAGAACTGAGTGCCCGTGTTCATGCAGTTATGAATACCCTGACCGACGTTGCACCGGGTGACCGTGTGGCACTGCTGGGTTTCACCAGCGTGGACTACACCGTGATTGATATGGCCCTGGCACTGAGTGGTGCAGTGAGCGTGCCTCTGCAGACCAGCGCACCTGCAGCAACCCTGCGTCCTATCATCGCAGAGACCGAGCCTGTGATTATTGCAAGCGCAGTTGATCACCTGGCCGATGCAGTGGAGTTAGCACGTGAGGCAGATACAGTTCGCCGTGTGATTGTTTTCGACCATCGTGCTGAAGTGGACGATCATCGTGATGCAGTGGCAGATGCACGTACAAGACTGACAGAAGGCGGTAGAGCAATTGAAGTTCTGACCCTGGCCGAAGTGCTGGAGCACGGTGCAACCCTGCCTGCAGCACAGCCTTTCAGCAGCCCTGAGCAGGACCCTCTGACCCTGCTGATCTACACCAGCGGTAGCACCGGTGCACCTAAGGGTGCAATGTATCCTGAACGTCTGGTGGCAGGTGCTTGGCTGCGTAGC GCA CGTAGCACCTGGTACGGTGAGCACGCAACCCCTAGCATCACCCTGAACTTTCTGCCTATGAGCCACATG CTG This invention provides a recombinant vector for expressing the carboxylate reductase mutant, comprising an initial vector and the gene, wherein the initial vector is the pRSFDuet-1 plasmid. This invention does not specifically limit the source of the pRSFDuet-1 plasmid; any commercially available product in the art is acceptable. In this invention, the gene is preferably co-expressed with the phosphoproteopancreatoylthioethylamine transferase (Sfp) gene in the initial vector. This invention does not specifically limit the specific sequence of the phosphoproteopancreatoylthioethylamine transferase (Sfp) gene; any known phosphoproteopancreatoylthioethylamine transferase (Sfp) gene sequence is acceptable. The function of expressing the phosphoproteopancreatoylthioethylamine transferase (Sfp) gene is to modify the carboxylate reductase, thereby endowing it with catalytic activity. This invention does not specifically limit the preparation method of the recombinant vector; any method known in the art is acceptable, and it can be prepared in-house or by a biotechnology company.

[0033] This invention provides a genetically engineered bacterial strain, obtained by transferring the recombinant vector into a host bacterium; the host bacterium is *Escherichia coli*. E. coli BL21 (DE3). This invention does not impose any particular limitation on the preparation method of the genetically engineered strain; any preparation method commonly used in the field may be employed.

[0034] This invention provides the aforementioned carboxylic acid reductase mutant, the aforementioned gene, the aforementioned recombinant vector, and the aforementioned genetically engineered bacteria in biosynthesis (…). Z Applications of )-13-octadecenal.

[0035] This invention provides a biosynthesis ( Z A method for using 13-octadecenal includes the following steps: using ( Z Using 1,3-octadecenoic acid as a substrate, a reduction reaction is carried out by the carboxylic acid reductase mutant or the genetically engineered bacteria in the presence of a cofactor.

[0036] In this invention, the concentration of the substrate is preferably 0.8-1.2 mM, more preferably 0.9-1.1 mM, and most preferably 1.0 mM; the concentration of the carboxylic acid reductase mutant is preferably 0.1-0.3 mg / ml, more preferably 0.15-0.25 mg / ml, and most preferably 0.2 mg / ml. In this invention, the cofactors include NADPH, ATP, and MgCl2; the concentration of NADPH is preferably 4-6 mM, more preferably 5 mM; the concentration of ATP is preferably 4-6 mM, more preferably 5 mM; and the concentration of MgCl2 is preferably 8-12 mM, more preferably 9-11 mM. The reduction reaction of this invention is carried out in a water-heptane two-phase system; in this invention, the aqueous medium of the reduction reaction is a Na2HPO4-NaH2PO4 buffer solution, and heptane is added during the reaction to promptly extract the aldehyde product; the volume of heptane added during the reaction is equal to the volume of the aqueous phase. The reduction reaction described in this invention is accompanied by oscillation, preferably at a rotation speed of 800-1200 rpm, more preferably 900-1100 rpm, and most preferably 1000 rpm. The temperature of the reduction reaction is preferably 28-32°C, more preferably 29-32°C, and most preferably 30°C; the duration of the reduction reaction is 100-140 min, more preferably 110-130 min, and most preferably 120 min.

[0037] In this invention, after the reduction reaction is completed, 2-4 times the volume of heptane is added for extraction, the organic phase is collected, dried, and concentrated to obtain (…). Z )-13-Octadecanal; In this invention, the organic phase is preferably dried using anhydrous Na2SO4, and then the organic layer is transferred for nitrogen blowing concentration to obtain the product.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0040] Reagents used in upstream genetic engineering: DPN I used in the embodiments of this invention was purchased from TaKaRa, Takara Bio Engineering (Dalian) Co., Ltd.; plasmid extraction kit and DNA recovery and purification kit were purchased from Axygen Hangzhou Co., Ltd. E. coliBL21(DE3) and plasmid pRSFDuet-1 were purchased from Novagen; DNA markers, low molecular weight standard proteins, and agarose gel electrophoresis reagents were purchased from Beijing TransGen Biotech Co., Ltd.; primer synthesis and sequencing were performed by Qingke Biotechnology Co., Ltd. Refer to the product instructions for the usage of the above reagents.

[0041] Reagents used in downstream catalytic processes: ( Z 13-Octadecanoic acid was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., NADPH was purchased from Bangtai Biotechnology (Shenzhen) Co., Ltd., and ATP, anhydrous MgCl2 and dimethyl sulfoxide (DMSO) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0042] Carboxylic acid reductase activity screening system (GC-FID method): Appropriate amount of crude enzyme solution, 1 mM substrate (dissolved in DMSO, final DMSO reaction volume 0.5% v / v), 5 mM NAPDH, 5 mM ATP, 10 mM MgCl2, total reaction volume 1 mL, reaction medium 50 mM pH 7.5 Na2HPO4-NaH2PO4 buffer and 0.5 mL heptane. The reaction was carried out at 30 ℃ for 2 h. After the reaction, the sample was centrifuged at 12000 rpm for 2 min, and the upper organic layer was collected and dried with anhydrous sodium sulfate. Quantitative analysis of each substance in the sample was performed using GC-FID.

[0043] Pure enzyme catalytic preparation ( Z 13-Octadecenal system (GC-FID method): Appropriate amount of purified enzyme, 1 mM substrate (dissolved in DMSO, final reaction volume of DMSO: 0.5% v / v), 5 mM NAPDH, 5 mM ATP, 10 mM MgCl2, total reaction volume 1 mL, reaction medium: 50 mM pH 7.5 Na2HPO4-NaH2PO4 buffer and 0.5 mL heptane. The reaction was carried out at 30 ℃ for 2 h. After the reaction, the mixture was centrifuged at 12000 rpm for 2 min, and the upper organic layer was collected and dried with anhydrous sodium sulfate. Quantitative analysis of each substance in the sample was performed using GC-FID.

[0044] Example 1

[0045] Construction of genetically engineered bacteria

[0046] In Example 1, enzymes from different sources that can catalyze the reduction of carboxylic acid were screened, and genetically engineered bacteria expressing different enzymes were constructed.

[0047] 1.1 Screening of carboxylic acid reductases

[0048] According to literature reports, carboxyl reductases from different sources were screened, specifically from Nocardia otitis media in guinea pigs (…).Nocardia otitidiscaviarum Carboxylic acid reductase ( No CAR), Chlorpheniramine maleate ( Mycolicibacterium chlorophenolicum Carboxylic acid reductase ( Mc CAR), Mycobacterium smegmatis ( Mycolicibacterium smegmatis Carboxylic acid reductase ( Ms CAR1 Ms CAR2 Ms CAR3), Mycobacteria ( Mycobacterium sp.JLS Carboxylic acid reductase ( Msp CAR), Marine Mycobacterium ( Mycobacterium marinum Carboxylic acid reductase ( Mm CAR), Mycobacterium virginianum ( Mycolicibacterium neoaurum Carboxylic acid reductase ( Mn CAR) and E. awanocardiosis ( Nocardia iowensis Carboxylic acid reductase ( Ni CAR). They were numbered E1, E2, E3, E4, E5, E6, E7, E8, and E9 respectively, and then compared with those derived from Bacillus subtilis ( ). Bacillus subtilis The Sfp gene of phosphopanyl thioethylamine transferase was integrated into the pRSFDuet-1 plasmid and transformed into competent E. coli cells.

[0049] In summary, a total of 9 candidate enzymes were selected, and the details of the 9 candidate enzymes are shown in Table 1.

[0050] Table 1. Carboxyl reductases from different sources

[0051] The gene sequences of carboxylic acid reductase in Table 1, after codon optimization, were sent to General Biosystems (Anhui) Co., Ltd. for whole-genome synthesis, and were respectively synthesized with those derived from Bacillus subtilis (…). Bacillus subtilis The Sfp gene (GenBank: YCU06296.1) of the phosphopantoylthioethylamine transferase was integrated into the pRSFDuet-1 plasmid to obtain recombinant expression plasmids, which were then used to form nine recombinant expression vectors. These nine recombinant expression vectors were then transformed into expression hosts. E.coli In BL21(DE3), after sequencing verification, glycerol with a final concentration of 25% was added to the bacterial culture of each engineered bacterium and stored at -80 ℃ for later use.

[0052] Example 2

[0053] catalytic( Z Preparation of 1,3-octadecenoic acid substrate ( Z )-13-Octadecanal

[0054] In this Example 2, the nine engineered bacteria obtained in Example 1 were used to catalyze ( Z )-13-octadecenoic acid to prepare ( Z )-13-Octadecanal. Specifically, it includes the following: 2.1 Microbial Culture Liquid LB medium consists of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl. Dissolve in deionized water and bring to a final volume. Sterilize at 121 °C for 20 min. For solid LB medium, add 15 g / L agar.

[0055] 1) The nine engineered bacteria containing alcohol oxidase or alcohol dehydrogenase genes obtained in Example 1 were inoculated into 5 mL of liquid LB medium (containing 50 μg / mL kanamycin) and cultured at 37 °C with shaking at 200 rpm for 8 h.

[0056] 2) Inoculate at a rate of 1% (v / v) into 10 mL of liquid LB medium (containing 50 μg / mL kanamycin) and culture. OD 600 Once the concentration reaches 0.6-0.8, add the inducing agent IPTG (final concentration 0.01 mM) and induce at 18 ℃ for 16 h.

[0057] 3) After the culture is completed, take 8 mL of culture medium, pour it into a 10 mL centrifuge tube, centrifuge at 4000 rpm for 10 min, discard the supernatant, collect the bacterial cells, wash the cells twice with 50 mM phosphate buffer (pH 8.0), and then store them in an ultra-low temperature freezer at -80 ℃ for later use.

[0058] 2.2 Preparation of crude enzyme solution

[0059] The cryopreserved bacterial cells obtained in step 2.1 were resuspended in 2 mL of 50 mM phosphate buffer (pH 7.5), the bacterial cell suspension was sonicated and the supernatant obtained after centrifugation was the target crude enzyme solution.

[0060] 2.3. Crude enzyme solution catalysis ( Z )-13-octadecenoic acid

[0061] Reaction system (0.5 mL): Take 0.45 mL of crude enzyme solution obtained in step 2.2, 1 mM substrate (dissolved in DMSO, DMSO final reaction volume is 0.5% v / v), 5 mM NAPDH, 5 mM ATP, and 10 mM MgCl2, the total reaction volume is 0.5 mL, and the reaction medium is 50 mM pH 7.5 Na2HPO4-NaH2PO4 buffer. The reaction was carried out at 30 ℃ and 1000 rpm in a metal bath with shaking for 2 h. After the reaction, 0.5 mL of heptane was added and mixed, centrifuged at 12000 rpm for 2 min, and the upper organic layer was collected and dried with anhydrous sodium sulfate. The substances in the sample were quantitatively analyzed by GC-FID. Because the crude enzyme solution of *E. coli* contains multiple reductases, it can reduce the aldehyde product to an alcohol (…). Figure 2 Therefore, in the enzyme screening process, the concentration of cis-13-octadecenol generated in the reaction system was used as an indirect indicator of carboxylic acid reductase activity. The results are shown in Table 2.

[0062] Table 2. Concentration of cis-13-octadecenol produced from crude enzyme solutions obtained by different engineered bacteria

[0063] a ND: Not detected

[0064] As shown in Table 2, E1, E2, E5, and E6 can all catalyze ( Z The reduction reaction of 1,3-octadecenoic acid showed that the concentration of the final product cis-1,3-octadecenool, produced by E2 catalysis, was 0.256 mM, which was the highest value.

[0065] Example 3

[0066] Construction and screening of single-point mutation libraries

[0067] In this Example 3, the E2 engineered bacteria obtained in Example 2 were molecularly modified. Specifically, the following steps were taken: 3.1 Construction of the alanine scanning library 1. Whole plasmid PCR: pRSFDuet-sfp- Mc Using CAR plasmids as templates, upstream and downstream primers covering the mutation points were designed (Table 3) for whole plasmid PCR.

[0068] Table 3 Primers used for the alanine scanning mutant library

[0069] PCR amplification system: DNA polymerase 25 µL; Upstream primer (10 pmol / µL) 2.5 µL; Downstream primer (10 pmol / µL) 2.5 µL; Template 0.5 µL; ddH2O 19.5 µL.

[0070] PCR amplification conditions: 1) Pre-denaturation: 95 ℃ for 5 min; 2) Denaturation: 98 ℃ for 10 s; Annealing: 58 ℃ for 15 s; Extension: 72 ℃ for 60 s; 30 cycles in total; 3) Post-extension: 72 ℃ for 10 min; 4) Store at 4℃.

[0071] 2. Template digestion: The PCR product was subjected to agarose gel electrophoresis and then recovered. The plasmid template was digested with DpnI enzyme. The digestion system consisted of 1 μL DpnI enzyme, 17 μL PCR product, and 2 μL buffer. Digestion was completed at 37 °C for 2 hours.

[0072] 3. Transformation and Validation: After the digestion products were verified by nucleic acid agarose gel electrophoresis, they were transformed into Escherichia coli using a 42 °C heat shock method. E.coli BL21 (DE3) competent cells. The specific process is as follows: (1) Thaw competent cells on ice for 15 min; (2) Add 10 μL of DNA to 100 μL of competent cells under sterile conditions and mix gently. Place on ice for 30 min. (3) The EP tube was placed in a 42 ℃ metal bath for 90 s heat shock, and then placed on ice to cool for 2 min after the heat shock. (4) Add 800 μL of LB medium to the EP tube and mix well with the pipette tip. Incubate at 37 °C for 40-60 min in a shaker at 200 rpm. (5) After concentration, apply the corresponding resistance plates and incubate in a 37 ℃ incubator for 12-16 h to produce colonies.

[0073] (6) Select a single colony for culture and sequencing. If the sequencing is correct, preserve the strain.

[0074] 3.2 Screening of alanine scanning libraries

[0075] The mutant obtained in step 3.1 was cultured to prepare a crude enzyme solution (the culture method and crude enzyme solution preparation method are the same as in steps 2.1-2.2). Then, the crude enzyme solution was catalyzed according to the method in step 2.3 of Example 2. Z Catalytic activity screening of 13-octadecenoic acid.

[0076] The screening results are shown in Table 4. After screening, mutants G274A and M298A with catalytic activity of crude enzyme solution increased by 18% and 2% respectively compared with wild type.

[0077] Table 4. Results of mutant screening

[0078] b ND: Not detected

[0079] 3.3 Construction and Screening of Site-Directed Saturation Mutant Libraries for G274 and M298

[0080] pRSFDuet-sfp- Mc Using the CAR plasmid as a template, upstream and downstream primers covering the mutation point were designed for whole plasmid PCR. The construction and screening of mutants were similar to steps 3.1 and 3.2.

[0081] The filtering results are as follows Figure 3 As shown, after screening, among the saturation mutations at the G274 site, G274A exhibited relatively high crude enzyme catalytic activity, which was 18% higher than that of the wild type; while among the saturation mutations at the M298 site, M298L exhibited relatively high crude enzyme catalytic activity, which was 14% higher than that of the wild type.

[0082] 3.4, G274A and M298L combined mutations

[0083] By combining two advantageous mutants, a two-point mutant G274A / M298L with 50% higher catalytic activity in crude enzyme solution than wild-type McCAR was finally obtained through screening.

[0084] Example 4

[0085] Carboxylic acid reductase Mc Purification and activity assay of CAR and its mutants

[0086] 4.1. Wild type Mc CAR and purification of mutants

[0087] The bacterial cells were resuspended in a pre-cooled 20 mM imidazole solution (containing 20 mM imidazole, 0.5 M NaCl and 20 mM pH 7.0 Na2HPO4-NaH2PO4 buffer); after sonication, the cells were centrifuged at 4000 rpm for 30 min at 4 ℃. The crude enzyme supernatant was filtered through a 0.22 μm filter membrane and placed in an ice-water bath for later use. First, the ethanol solution in the Ni-NTA-Sefinose pre-packed gravity column (5 mL) was replaced with deionized water, then the column was equilibrated with 10 column volumes of 20 mM imidazole solution at a flow rate of 1 mL / min. After column equilibration, the filtered crude enzyme solution was added to the column, and the sample was loaded at a flow rate of 1 mL / min. After loading, 10 column volumes of 20 mM imidazole solution (containing 20 mM imidazole, 0.5 M NaCl, and 20 mM pH 7.0 Na2HPO4-NaH2PO4 buffer) were used to elute contaminating proteins. Further elution with 10 column volumes of 50 mM imidazole solution (containing 50 mM imidazole, 0.5 M NaCl, and 20 mM pH 7.0 Na2HPO4-NaH2PO4 buffer) was performed. Finally, 10 column volumes of 100 mM imidazole solution were used to elute contaminating proteins. Elute contaminating proteins with 100 mM imidazole solution (containing 100 mM imidazole, 0.5 M NaCl, and 20 mM pH 7.0 Na2HPO4-NaH2PO4 buffer); then elute the target protein with 250 mM imidazole solution (containing 250 mM imidazole, 0.5 M NaCl, and 20 mM pH 7.0 Na2HPO4-NaH2PO4 buffer), collecting approximately 50 mL; place the collected protein solution in an ultrafiltration tube, centrifuge at 4000 rpm for 40 min at 4 °C to concentrate the protein sample, then wash twice with 50 mM pH 7.0 Na2HPO4-NaH2PO4 buffer, finally add glycerol (final concentration 20%), mix well, and store at -80 °C.

[0088] 4.2. Wild type Mc CAR and mutant activity assay

[0089] Carboxylic acid reductase McCAR enzyme activity standard detection system (GC-FID method): 0.2 mg / mL pure enzyme, 1 mM substrate (dissolved in DMSO, final DMSO reaction volume 0.5% v / v), 5 mM NAPDH, 5 mM ATP, 10 mM MgCl2, total aqueous reaction volume 0.5 mL, reaction medium 50 mM pH 7.5 Na2HPO4-NaH2PO4 buffer, with an additional 0.5 mL heptane added for timely extraction of the aldehyde product. The reaction was carried out in a metal bath at 30 ℃ and 1000 rpm for 120 min. After the reaction, the sample was extracted by centrifugation at 12000 rpm and dried with anhydrous sodium sulfate. Quantitative analysis of each substance in the sample was performed using GC-FID. Enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol (Z)-13-octadecenal product per minute under standard reaction conditions.

[0090] In the final measured enzyme activity data, the optimal mutant was... Mc The specific enzyme activity of CAR G274A / M298L is 6.45 × 10⁻⁶. -4 U / mg, compared to wild type Mc CAR enzyme activity (4.75×10) -4 The U / mg concentration increased by 0.36 times, while the mutant... Mc The specific enzyme activity of CAR M298L (4.68×10⁻⁶) -4 The concentration (U / mg) is similar to that of the wild type.

[0091] Example 5

[0092] Carboxylic acid reductase Mc CAR mutant catalysis ( Z Preparation of 1,3-octadecenoic acid ( Z )-13-Octadecanal

[0093] In this Example 5, the reaction time is extended based on Example 4.2. Specifically: Reaction system (0.5 mL): mutant McCAR G274A / M298L purified enzyme (0.2 mg / mL), 1 mM substrate (dissolved in DMSO, final DMSO volume 0.5% v / v), 5 mM NAPDH, 5 mM ATP, and 10 mM MgCl2 were used in a total aqueous reaction volume of 0.5 mL. The reaction medium was 50 mM pH 7.5 Na2HPO4-NaH2PO4 buffer. An additional 0.5 mL of heptane was added during the reaction for timely extraction of the aldehyde product. The reaction was carried out in a metal bath at 30 °C and 1000 rpm for 120 min. After the reaction, the sample was extracted by centrifugation at 12000 rpm and dried with anhydrous sodium sulfate. Quantitative analysis of the substances in the sample was performed by GC-FID.

[0094] Finally, after 24 hours of reaction, the reaction system was measured to contain ( Z The concentration of 1,3-octadecenal reached 0.59 mM, and the yield of the target compound reached 59%. Furthermore, 0.17 mM of ( Z The formation of 13-octadecenol is presumably a further reduction product of the aldehyde in the presence of excess NADPH. Therefore, the overall (aldehyde + alcohol) yield of the reaction catalyzed by this mutant can reach 76%.

[0095] As can be seen from the above embodiments, the carboxylate reductase mutant provided by the present invention is obtained by modifying the wild type through a structure-guided semi-rational design molecular modification method, thus solving the problem of wild-type carboxylate reductase. Mc CAR addresses the issue of low enzyme activity in non-natural substrates, with the optimal mutant G274A / M298L exhibiting catalytic activity 0.36 times higher than the wild type.

[0096] The method provided by this invention has mild reaction conditions, high yield of the target product, and achieves the realization of the dicarboxylic acid pheromone (…). Z The enzymatic preparation of 13-octadecenal provides a green and environmentally friendly preparation route, offering a sustainable alternative for the biosynthesis of insect sex pheromones.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A carboxylic acid reductase mutant, characterized in that, The carboxylic acid reductase mutant is a wild-type carboxylic acid reductase. Mc The wild-type carboxylic acid reductase was obtained by CAR-based mutation in one of G274A, M298L, or G274A / M298L. Mc The amino acid sequence of CAR is shown in SEQ ID NO:

1.

2. A gene encoding the carboxylic acid reductase mutant of claim 1.

3. A recombinant vector expressing the carboxylic acid reductase mutant of claim 1, characterized in that, It includes an initial vector and the gene as described in claim 2, wherein the initial vector is the pRSFDuet-1 plasmid.

4. The recombinant vector according to claim 3, characterized in that, The gene and the phosphate pantothenic acid thioethylamine transferase (Sfp) gene are co-expressed on the initial vector.

5. A genetically engineered strain, characterized in that, The recombinant vector of claim 3 or 4 is obtained by transferring it into a host bacterium; the host bacterium is Escherichia coli. E. coli BL21 (DE3).

6. The carboxylic acid reductase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, and the genetically engineered bacteria of claim 5 in biosynthesis ( Z Applications of )-13-octadecenal.

7. A biosynthesis ( Z The method for 1,3-octadecenal is characterized by, Includes the following steps: by( Z Using 1,3-octadecenoic acid as a substrate, and in the presence of a cofactor, a reduction reaction is carried out by the carboxylic acid reductase mutant of claim 1 or the genetically engineered bacteria of claim 5 catalyzed by the reaction. The cofactors include NADPH, ATP, and MgCl2; The reduction reaction is carried out in a water-heptane two-phase system; The reduction reaction is accompanied by oscillation; The reduction reaction is carried out at a temperature of 28~32℃.

8. The method according to claim 7, characterized in that, After the reduction reaction is completed, 2-4 times the volume of heptane is added for extraction, the organic phase is collected, dried, and concentrated to obtain ( Z )-13-octadecenal.

9. The method according to claim 7, characterized in that, The concentration of the substrate is 0.8~1.2mM, and the concentration of the carboxylic acid reductase mutant is 0.1~0.3mg / ml.

10. The method according to claim 7, characterized in that, The concentration of NADPH is 4-6 mM, the concentration of ATP is 4-6 mM, and the concentration of MgCl2 is 8-12 mM.

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