CN114350739A

CN114350739A - High-throughput screening method for epsilon-caprolactone high-yield strains

Info

Publication number: CN114350739A
Application number: CN202111658178.1A
Authority: CN
Inventors: 李爽; 刘然; 陈和锋; 余皓
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-15

Abstract

The invention discloses a high-throughput screening method of an epsilon-caprolactone high-yield strain, belonging to the technical field of high-throughput screening. According to the invention, correlation analysis is carried out on the concentration of the epsilon-caprolactone which is a product of ADH and CHMO double-enzyme cascade reaction and the concentration of the cyclohexanone which is an intermediate product, so that after the strains with different catalytic performances are subjected to whole-cell catalytic reaction, the concentration of the cyclohexanone which is an intermediate product and the concentration of the epsilon-caprolactone which is a product are in a negative correlation relationship, and therefore, the cyclohexanone is used as a screening marker, and the strains with low cyclohexanone concentration are screened at high throughput to obtain the strains with high epsilon-caprolactone yield. The invention utilizes RBS engineering technology to construct a mutation library of the epsilon-caprolactone producing strain, seven mutant strains obtained by screening are subjected to shake flask experiment verification, and the epsilon-caprolactone yield of 70mM cyclohexanol is catalyzed, which is obviously improved compared with the original strain RCL 0. The method has simple operation steps, and can greatly reduce the experiment cost of the strain screening process and shorten the screening period.

Description

High-throughput screening method for epsilon-caprolactone high-yield strains

Technical Field

The invention relates to a high-throughput screening method of an epsilon-caprolactone high-yield strain, belonging to the technical field of high-throughput screening.

Background

The epsilon-caprolactone is colorless transparent oily liquid, has aromatic smell, is easily soluble in solvents such as water, ethanol, benzene and the like, is insoluble in petroleum ether, is an organic chemical intermediate with wide application, and can be used as a monomer to synthesize a high-performance polymer material such as polycaprolactone. Due to the unique biocompatibility, excellent biodegradability and good permeability of polycaprolactone, polycaprolactone is widely applied in the biomedical engineering field and the biodegradation field: can be used as various biodegradable medical materials, such as surgical suture, carrier for controlling drug release, bone splint, etc.; can be used for synthesizing environment-friendly materials, such as degradable plastics, fully degradable membrane materials and the like, and can be used for favorably reducing white pollution.

Currently, the synthesis method of epsilon-caprolactone is mainly divided into a chemical oxidation method and a biological conversion method, and the synthesis is based on the Baeyer-Villiger reaction discovered in 1899 by Adoff von Baeyer and VictorVilliger, which refers to a kind of organic synthesis reaction for oxidizing ketone into ester. According to different oxidants, the chemical oxidation method can be divided into a peroxyacid oxidation method, a hydrogen peroxide oxidation method and an oxygen oxidation method, and currently, the technology of oxidizing cyclohexanone by peroxyacid is mostly adopted in China to chemically produce epsilon-caprolactone, but the method has the defects of easy explosion, low safety, serious environmental pollution, complex operation and the like. In response to green chemistry calls, biocatalytic methods have gained attention in recent years.

In 1976, researchers identified an oxygen and NADHPH dependent cyclohexanone monooxygenase (CHMO) in Acinetobacter calcoaceticus sp. Since the participation of CHMO in enzymatic reactions in vitro requires the additional addition of the expensive cofactor NADPH, the in vitro biocatalytic method is not suitable for industrial large-scale applications. To solve this problem, a two-enzyme or multi-enzyme cascade is established in which whole cells catalyze the production of epsilon-caprolactone. Wherein, oxygen in the air is used as an oxidant, and the substrate cyclohexanol is catalyzed and oxidized by alcohol dehydrogenase ADH and cyclohexanone monooxygenase CHMO to further generate intermediate cyclohexanone and final product epsilon-caprolactone (Schmidt S, Scherkus C, Muschiol J, et al. an enzyme cascade synthesis of epsilon-caprolactone and its oligomers [ J ]. Angew Chem Int Ed Engl,2015,54(9): 2784-2787), and the cyclic utilization of the auxiliary factor NADPH can be realized by two-step oxidation reaction in the process. However, the reaction still has the problems of unbalanced NADPH, inhibited concentration of substrate and product, low oxygen transmission rate, and the like. In recent years, in order to further solve the problem of unbalanced NADPH in the cascade reaction process, related researches have been carried out on RBS modification of ADH and CHMO proteins in the cascade system, but due to the fact that the number of modified strains is small, the catalytic effect of mutant strains is only improved to a limited extent. The existing mutation library construction method is various and efficient, such as error-prone PCR, gene recombination, sequence saturation mutation (SESAM), gene editing and the like. To rapidly obtain the dominant mutant strain of interest from the mutant library, a rapid and accurate high-throughput screening method is essential.

The existing epsilon-caprolactone production strain screening is generally carried out by adopting shake flask catalysis and then using gas chromatography for detection, the process is complicated and time-consuming, and no one can screen epsilon-caprolactone high-yield strains by using a high-throughput screening technology at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a high-throughput screening method of an epsilon-caprolactone high-yield strain.

The invention aims to break through the bottleneck that the epsilon-caprolactone high-yield strain can not be obtained by high-throughput screening at present, and provides a simple and feasible high-throughput screening method for the epsilon-caprolactone high-yield strain, which has higher application value.

The purpose of the invention is realized by the following technical scheme:

according to the invention, correlation analysis is carried out on the concentration of the epsilon-caprolactone which is a product of ADH and CHMO double-enzyme cascade reaction and the concentration of the cyclohexanone which is an intermediate product, and the correlation relationship between the concentration of the cyclohexanone which is an intermediate product and the concentration of the epsilon-caprolactone which is a product is found out after the strains with different catalytic performances are subjected to whole-cell catalytic reaction, so that the intermediate product cyclohexanone can be used as a screening marker, and the strains with high yield of the epsilon-caprolactone can be obtained by screening the strains with low cyclohexanone concentration in a high throughput manner.

The invention provides a high-throughput screening method of an epsilon-caprolactone high-yield strain, which comprises the following steps:

(1) picking single colony of the mutation library strain for producing epsilon-caprolactone into a micropore plate filled with a liquid culture medium, and carrying out shake culture; transferring the seed solution into a new microplate liquid culture medium, and performing shake culture to OD₆₀₀Adding an inducer IPTG to induce protein expression when the expression level is between 0.5 and 0.7;

(2) centrifuging to remove a supernatant culture medium, using a buffer solution containing cyclohexanol with a certain concentration to resuspend thallus whole cells, and carrying out a reaction of catalyzing cyclohexanol to generate epsilon-caprolactone by the whole cells in a micropore plate;

(3) after the reaction is finished, adding a 2, 4-dinitrophenylhydrazine solution to ensure that an intermediate product cyclohexanone in a whole-cell catalytic system fully reacts with the 2, 4-dinitrophenylhydrazine to generate a corresponding phenylhydrazone compound; adding NaOH solution for color reaction to enable the corresponding phenylhydrazone compound to react with alkali liquor to generate a benzoquinone compound, centrifuging, taking supernate into a new micropore plate, measuring the light absorption value of a sample under a specific absorption wavelength by using an enzyme-labeling instrument, and measuring the concentration of cyclohexanone in a catalytic system through two-step reaction; taking an original strain RCL0 for producing epsilon-caprolactone as a negative control, reacting under the same condition, and taking a mutant strain with the ratio of the absorbance value under the characteristic absorption wavelength to the absorbance value of the negative control lower than 1 as a positive mutant strain; the lower the light absorption value ratio, the higher the yield of the epsilon-caprolactone of the corresponding strain;

(4) and carrying out shake flask catalysis experiment on the positive mutant strain obtained by screening through the color reaction of 2, 4-dinitrophenylhydrazine for yield verification.

Furthermore, the epsilon-caprolactone-producing mutation library strain in the step (1) is obtained by simultaneously mutating the original ribosome binding site sequences of ADH and CHMO genes in the original strain RCL0 by using RBS engineering technology, taking Escherichia coli BL21(DE3) as a host and pRSFDuet-1 as an expression vector.

The starting strain RCL0 is a recombinant strain BDR-3 and is disclosed in a document 'construction of a system for efficiently synthesizing epsilon-caprolactone by complete cells of Ursus Jingzu Escherichia coli and research on catalytic performance [ D ]. university of south China, 2020'.

Preferably, the micro-porous plate in the steps (1), (2) and (3) is a 96-hole or 48-hole micro-porous plate;

preferably, the shaking culture conditions in the step (1) are 36-38 ℃, 900-1100 rpm and 10-14 hours; further culturing at 37 deg.C and 1000rpm for 12 hr;

preferably, the liquid culture medium in the step (1) is LB liquid culture medium;

preferably, the condition for inducing protein expression in the step (1) is inducing for 6-8 h at 28-32 ℃ and 800-1000 rpm; further inducing at 30 deg.C and 900rpm for 7 h;

preferably, the final concentration of IPTG in the step (1) is 0.4-0.6 mM; further 0.5 mM;

preferably, the centrifugation in the step (2) is performed for 10-15 min at 1800-2000 g and 4-8 ℃; further centrifuging at 4 deg.C and 2000g for 10 min;

preferably, the final concentration of the cyclohexanol in the step (2) is 40-60 mM; further 60 mM;

preferably, the buffer solution in the step (2) is Tris-HCl buffer solution; further 20mM, pH7.5 Tris-HCl buffer;

preferably, the reaction conditions in the step (2) are 23-27 ℃ and 700-900 rpm for 14-18 h; further reacting for 16h at 25 ℃ and 800 rpm;

preferably, the concentration of the 2, 4-dinitrophenylhydrazine solution in the step (3) is 15 to 25mM (further 20 mM); dissolving with 95% ethanol (analytically pure) and 3% sulfuric acid solution, and storing in dark place;

preferably, the concentration of the NaOH solution in the step (3) is 1.2-1.8M; further 1.5M;

preferably, in the step (3), the cyclohexanone and the 2, 4-dinitrophenylhydrazine are reacted fully under the conditions of 28-32 ℃ and 700-900 rpm for 25-35 min; further reacting for 30min at the temperature of 30 ℃ and the rotating speed of 800 rpm;

preferably, in the step (3), the condition of the color reaction is that the mixture is kept still for reaction for 10-20 min at 28-32 ℃; further standing and reacting for 15min at 30 ℃;

preferably, in the step (3), the centrifugation is performed for 5-10 min at 1800-2000 g and 4-8 ℃; further centrifuging at 2000g for 5min at 4 deg.C;

preferably, the specific absorption wavelength in the step (3) is 530-550 nm; further 540 nm.

Preferably, the flow of the reaction between the intermediate product cyclohexanone and the 2, 4-dinitrophenylhydrazine in the catalytic system in the step (3) is as follows: after the whole cell catalytic reaction of a microporous plate (96 pore plate) of the mutant strain is finished, taking 20 mu L of catalytic reaction liquid into the microporous plate (96 pore plate), adding 20 mu L of 2, 4-dinitrophenylhydrazine solution into the microporous plate, uniformly mixing, and reacting for 30min at the temperature of 30 ℃ and the rotating speed of 800 rpm; then, 200 μ L of 1.5M NaOH solution is added into the mixture, and the mixture is evenly mixed and then stands for reaction for 15min at 30 ℃; subsequently, the mixture was centrifuged at 2000g for 5min at 4 ℃ and 100. mu.L of the supernatant was applied to a new microplate (96-well plate) and the absorbance at 540nm was measured using a microplate reader.

The high-throughput screening method of the epsilon-caprolactone high-yield strain is applied to screening of the epsilon-caprolactone high-yield strain.

The invention also provides an epsilon-caprolactone high-yield strain which is obtained by the high-throughput screening method.

The epsilon-caprolactone high-yield strain is RCL01, RCL02, RCL03, RCL04, RCL05, RCL06 and RCL 07; the method is characterized in that the original ribosome binding site sequences of ADH and CHMO genes in an original strain RCL0 are simultaneously mutated by an RBS engineering technology, escherichia coli BL21(DE3) is taken as a host, and pRSFDuet-1 is taken as an expression vector;

wherein, the ribosome binding site sequences of ADH in RCL 01-RCL 07 are respectively shown as SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO. 17;

the sequences of the ribosome binding sites of CHMO in RCL 01-RCL 07 are respectively shown in SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO. 18.

The application of the epsilon-caprolactone high-yield strain in fermentation production of epsilon-caprolactone.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a high-throughput screening method of an epsilon-caprolactone high-yield strain, which solves the bottleneck that the screening process of the epsilon-caprolactone production strain depends on gas chromatography in the prior art, innovatively takes a cascade reaction intermediate product as a screening mark, detects the concentration of intermediate product cyclohexanone in a whole-cell catalytic reaction post-system of different mutant strains through high throughput, and screens the strain with high yield of the product epsilon-caprolactone through high throughput. The method has simple operation steps, can greatly reduce the experiment cost in the strain screening process and shorten the screening period.

(2) The invention utilizes RBS engineering technology to construct a mutation library of the epsilon-caprolactone producing strain, seven mutant strains are obtained by screening and are verified by a shake flask experiment, and the epsilon-caprolactone yield of 70mM cyclohexanol is catalyzed, which is obviously improved compared with the original strain RCL 0; the optimal mutant strain RCL07 is high in catalytic efficiency and strong in long-term stability, and can achieve the epsilon-caprolactone space-time yield of 0.236mM/gcell/h within 54h in a fed-batch experiment of 40mM cyclohexanol, and the record is the highest record of the epsilon-caprolactone yield and the space-time yield in the existing fed-batch report.

Drawings

FIG. 1 is a diagram of the cascade reaction of Escherichia coli whole cell catalysis cyclohexanol to produce epsilon-caprolactone.

FIG. 2 is a graph of the correlation between the concentration of cyclohexanone as an intermediate product and the concentration of epsilon-caprolactone as a product after the different engineered strains of example 1 participated in the whole-cell catalytic reaction of 40mM (a) and 60mM (b) cyclohexanol.

FIG. 3 is a composition diagram of a combinatorial RBS mutation library of ADH and CHMO genes.

FIG. 4 is a schematic diagram of the reaction of cyclohexanone, an intermediate product of the cascade reaction, and 2, 4-dinitrophenylhydrazine.

FIG. 5 is a graph showing the results of the different engineered strains in example 4 catalyzing 70mM cyclohexanol.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

It is noted that the following processes, if not described in particular detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents, materials and equipment used, unless otherwise specified, are all considered to be conventional products commercially available.

The cascade reaction diagram of the invention for producing epsilon-caprolactone by using Escherichia coli whole cell to catalyze cyclohexanol is shown in figure 1.

The ADH (GenBank: AY267012.1) of the invention has a codon-optimized nucleotide sequence shown in SEQ ID NO. 1.

CHMO in the invention refers to CHMO (GenBank: BAA86293.1) mutant CHMO-QM, which comprises 4 mutation sites: C376L/M400I/T415C/A463C, and the nucleotide sequence after codon optimization is shown as SEQ ID NO. 2.

Example 1 correlation of intermediate product concentration to product concentration in a cascade reaction

A plurality of epsilon-caprolactone producing bacteria BDR-2 and BDR-5-BDR-13 are constructed in the early stage of a laboratory, and the 10 strains are respectively used for whole-cell catalytic reaction of 40mM cyclohexanol and 60mM cyclohexanol, so that the yield of the epsilon-caprolactone of each strain is found to be different (construction of a system for efficiently synthesizing epsilon-caprolactone from whole cells of escherichia coli of bear Jing plant and research on catalytic performance [ D ]. university of south China's Rich worker 2020.). Through further analysis of the whole-cell catalysis results of the series of strains, the inventor finds that after different strains catalyze substrate cyclohexanol with the same concentration, the concentration of the product epsilon-caprolactone in the reaction system and the concentration of intermediate product cyclohexanone are in a better negative correlation relationship (figure 2). Therefore, we speculate that the strain can be screened by detecting the concentration of the intermediate product cyclohexanone in the system after the catalytic reaction to characterize the yield of the caprolactone, namely the efficiency of the cascade reaction.

Example 2 construction of a library of epsilon-caprolactone-producing mutant strains based on RBS engineering

A strain containing recombinant vector pRSF-RBS is constructed in the early stage of a laboratory₁₀-ADH-RBS₂₀Construction of epsilon-caprolactone producing strain BDR-3 (bear Jing plant. Escherichia coli whole cell high-efficiency synthesis epsilon-caprolactone system and catalytic performance research [ D ] of (pRSF-A10C20) of (CHMO)]University of southern China, 2020). In the present invention, pRSF-RBS₁₀-ADH-RBS₂₀renamed-CHMO to pRSF-RBS_A0-ADH-RBS_C0-CHMO(RBS_A0And RBS_C0Respectively shown as SEQ ID NO.3 and SEQ ID NO. 4); BDR-3 was renamed to RCL 0. With the recombinant vector pRSF-RBS_A0-ADH-RBS_C0CHMO is an initial vector constructed by the Library, SD sequences in original ribosome binding site sequences of adh and CHMO genes are mutated, degenerate SD sequences of the two genes are designed by RBS Library Calculator webpage software and RedLibs algorithm, primers containing the degenerate SD sequences are designed, the initial vector is used as a template for amplification, and homologous recombination is carried out to construct a two-enzyme RBS combined mutation Library. The specific implementation steps are as follows:

acquisition of RBS degenerate sequences of ADH and CHMO

The SD sequence of 6bp of the adh and chmo genes on the starting carrier is set as a complete degenerated form (NNNNNN), the protein coding sequence (CDS) of the two genes and the RBS sequence after the complete degeneracy of the SD sequence are respectively input into RBS Library promoter v2.0(https:// salislab. net/software /) of a prediction mode, and 4096 RBS sequences of the two genes and corresponding initial translation rate values (TIR) data sets are obtained. The SD sequences with differences in RBS sequences were cut and the SD sequence-TIR data sets were input into the RedLibs algorithm (Jeschek M, Gerngross D, Panke S. Rationally reduced libraries for combinatorial procedure optimization experimental effort [ J]Nat Commun,2016,7: 11163), instructions see https:// www.bsse.ethz.ch/bpl/software/redlibs, library size was set to 24, library distribution was as close to uniform as possible, 6bp SD degenerate sequences of adh and chmo genes were obtained, respectively, corresponding to RBS degenerate sequences forming both genes, RBS degenerate sequences_A(Lib)And RBS_C(Lib)As shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

PCR amplification of fragments

Design and Synthesis of RBS-containing_A(Lib)Degenerate primers of the sequence ComRBS1-Deg-f and RBS-containing primers_C(Lib)Brief description of the sequencesAnd primer ComRBS2-Deg-r, and plasmid skeleton primer pairs BB-right-f and BB-left-r are designed and synthesized. And using a starting vector as a template, amplifying a segment containing degenerate SD sequences of adh and chmo and adh genes by using a ComRBS1-Deg-f primer pair and a ComRBS2-Deg-r primer pair, and amplifying a segment containing the chmo genes and pRSFDuet-1 plasmid skeleton by using a BB-right-f primer pair and a BB-left-r primer pair.

ComRBS1-Deg-f：5′-TAATTTTGTTTAACTTTAATARGRRHATATACCATGACGGATCGTCTG-3′；

ComRBS2-Deg-r：5′-GCTGCTGCCCATATGTATATDNCCYCCTTATACTTAACTAATATAC-3′；

BB-right-f：5′-ATATACATATGGGCAGCAGCCATCACCATCATC-3′；

BB-left-r：5′-ATTAAAGTTAAACAAAATTATTTCTACAG-3′。

3. Construction of a library of epsilon-caprolactone-producing RBS combinatorial mutants

Use of

II, recombining the two fragments containing the homologous arms to obtain a plasmid library pRSF-RBS containing adh and chmo gene RBS sequence mutation_A(Lib)-ADH-RBS_C(Lib)CHMO, library size 576, plasmid library DNA was transferred by chemical transformation into e.coli BL21(DE3) competent cells to obtain RBS combinatorial mutant strain library (fig. 3).

Example 3 high throughput screening of Epsilon-caprolactone producing strains

1. Orifice plate culture and whole cell catalysis of mutant library strains

1743 single colonies of the mutant strain library of example 2 were picked and inoculated into 150. mu.LLB medium (containing kanamycin) in 96-well plates, and after culturing at 37 ℃ and 1000rpm for 12 hours, the seed solution was mixed at 1: 50 were transferred to 200. mu.L of fresh LB medium (containing kanamycin) and cultured under the same conditions until OD₆₀₀To 0.5-0.7, the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5mM to induce protein expression. After inducing at 30 deg.C and 900rpm for 7h, the fermentation broth was centrifuged at 4 deg.C and 2000g for 10min to collect the Escherichia coli cells, and 200. mu.L of 6-containing solution was used to remove the supernatantThe cells were resuspended in Tris-HCl buffer (20mM, pH7.5) containing 0mM cyclohexanol, and whole-cell catalytic reaction was carried out at 25 ℃ and 800rpm for 16 hours.

2. High throughput screening of mutant library strains

After the catalytic reaction is carried out for 16 hours, the cyclohexanone concentration of the catalytic reaction liquid in the pore plate is detected in a high flux manner by using a 2, 4-dinitrophenylhydrazine solution. The first step of reaction: 20 mul of catalytic reaction solution is taken to be put into a 96-well plate containing 20 mul of 2, 4-dinitrophenylhydrazine solution (20mM), and the mixture is evenly mixed and put into a microplate constant temperature oscillator to react for 30min under the conditions of 30 ℃ and 800 rpm. The second step of reaction: adding 200 μ L NaOH solution (1.5M) into the mixture, mixing, and standing in 30 deg.C incubator for reaction for 15 min; after the two-step reaction (FIG. 4) was completed, the 96-well plate was centrifuged using a plate centrifuge at 4 ℃ for 5min at 2000g, and then 100. mu.L of the supernatant was put in a new 96-well plate, and its absorbance at 540nm was measured using a microplate reader to detect the concentration of cyclohexanone. Lower absorbance values indicate lower detected concentrations of cyclohexanone, higher epsilon-caprolactone production by the corresponding strain. Using original strain RCL0 producing epsilon-caprolactone as negative control, reacting under the same condition, using the mutant strain whose ratio of light absorption value measured by mutant strain to light absorption value of negative control is less than 1 as positive mutant strain, and respectively naming 7 positive mutant strains with lowest ratio as RCL01, RCL02, RCL03, RCL04, RCL05, RCL06 and RCL07, which respectively contain plasmid pRSF-RBS_A(01)-ADH-RBS_C(01)-CHMO、pRSF-RBS_A(02)-ADH-RBS_C(01)-CHMO、pRSF-RBS_A(03)-ADH-RBS_C(02)-CHMO、pRSF-RBS_A(04)-ADH-RBS_C(02)-CHMO、pRSF-RBS_A(05)-ADH-RBS_C(03)-CHMO、pRSF-RBS_A(06)-ADH-RBS_C(04)-CHMO and pRSF-RBS_A(07)-ADH-RBS_C(05)-CHMO. The RBS sequences of ADH and CHMO of the above 7 mutant strains were different: RBS_A(01)RBS as shown in SEQ ID NO.7_C(01)As shown in SEQ ID NO. 8; RBS_A(02)As shown in SEQ ID NO. 9; RBS_A(03)RBS as shown in SEQ ID NO.10_C(02)As shown in SEQ ID NO. 11; RBS_A(04)As shown in SEQ ID NO. 12; RBS_A(05)RBS as shown in SEQ ID NO.13_C(03)As shown in SEQ ID NO. 14; RBS_A(06)RBS as shown in SEQ ID NO.15_C(04)As shown in SEQ ID NO. 16; RBS_A(07)RBS as shown in SEQ ID NO.17_C(05)As shown in SEQ ID NO. 18.

EXAMPLE 4 Shake flask experimental validation of the yield of epsilon-caprolactone from seven mutant strains

The 7 positive mutants of example 3 were removed from a-80 ℃ freezer, activated on LB solid plates containing kanamycin, cultured in a 37 ℃ incubator for 12 hours, and single colonies were picked up and inoculated in LB liquid medium containing kanamycin in test tubes, and cultured overnight at 37 ℃ with shaking at 220rpm for 12 hours. Subsequently, the seed liquid was mixed in a ratio of 1: 50 proportion was transferred to LB medium containing kanamycin in shake flasks for scale-up and inducers were added during the logarithmic phase to induce ADH and CHMO expression in the strains. The method comprises the following specific steps:

1. shaking culture and protein induction expression of seven mutant strains

The seed solution after overnight culture was mixed according to the ratio of 1: 50 volume ratio of the culture medium is transferred into a 500mL shake flask containing 200mL LB liquid medium (containing kanamycin), and the mixture is placed in a 37 ℃ shaking table at 220rpm for 1.5-2.5 h, during which the OD is measured₆₀₀Value, until the thallus is in logarithmic growth phase (OD)₆₀₀Between 0.5 and 0.7), 100. mu.L of 1M isopropyl-beta-D-thiogalactoside (IPTG) stock was added to a final concentration of 0.5 mM. Placing the shake flask in a 30 ℃ shaking table, inducing for 7h at the rotating speed of 180rpm, collecting the bacterial liquid in a 50mL centrifuge tube, centrifuging at 1340g and 4 ℃ for 30min to collect thalli, removing the supernatant culture medium, and then using 20mM Tris-HCl (pH7.5) buffer solution to re-suspend the thalli until the thalli concentration is 100g_wcwL, preservation in a refrigerator at 4 ℃.

2. Shake flask yield validation of seven mutant strains

Using 7 mutant strains induced to express in example 4, whole cell catalytic reaction was carried out using cyclohexanol as a substrate. Mixing the raw materials in a ratio of 1: 100 to 100g_wcwAdding dimethyl sulfoxide (DMSO) into/L bacterial suspension, mixing, freezing for 30min, centrifuging to remove supernatant, resuspending with 20mM Tris-HCl (pH7.5) buffer solution to original volume, and storing in refrigerator at 4 deg.C for useThe reaction is catalyzed in whole cells.

The whole-cell catalytic reaction was carried out in a 50mL shake flask, and the reaction system is shown in Table 1 below.

TABLE 1

Reagent	Volume of
		Cyclohexanol	74.0μL(70mM)
100g_wcwCell suspension/L	1mL
		20mM Tris-HCl (pH7.5) buffer	9mL

Firstly, adding 9mL of buffer solution into a 50mL shake flask, then adding 74 mu L of cyclohexanol solution, uniformly blowing to fully dissolve cyclohexanol in the buffer solution, finally adding 1mL of whole-cell suspension, immediately sealing by using a sealing film after uniformly mixing, and placing the mixture in a shaking table at 25 ℃ and at 120rpm for reaction for 16 h. Three replicates were set up for each mutant strain. And (3) replacing 1mL of whole cell suspension with 1mL of 20mM Tris-HCl (pH7.5) buffer solution in the control group, uniformly mixing the components, freezing 800 mu L of sample in a 2mL centrifuge tube, and detecting the sample as an actual substrate input value before catalysis.

3. Extraction and detection of product epsilon-caprolactone

After the catalytic reaction is finished, uniformly mixing the reaction solution in the shake flask, putting 800 mu L of the reaction solution into a 2mL centrifuge tube, simultaneously taking out the reaction solution of the control group which is frozen, respectively adding 800 mu L of ethyl acetate solution (containing 2mM acetophenone) into the centrifuge tube, shaking and extracting for 10min by an oscillator, centrifuging for 5min at the rotating speed of 14000rpm, absorbing 500 mu L of the upper organic phase by a 1mL syringe, filtering by a 0.22 mu m sterile filter head, and detecting by using GC.

The organic phase content was measured by gas chromatography using Shimadzu GC-2014C, HP-5 column (30 m × 0.32mm × 0.25 μm), Flame Ionization Detector (FID), and injector temperatures of 280 deg.C and 250 deg.C, respectively. Nitrogen gas is used as carrier gas, and the ratio of 30: a split ratio of 1, flow rate of 3mL/min, 1 μ L aliquot was injected in split mode. The procedure set up was as follows: the initial temperature of the column box is 50 ℃, the temperature is increased to 100 ℃ at the speed of 10 ℃/min, and then the temperature is increased to 200 ℃ at the speed of 20 ℃/min, and the total time is 17 min.

The GC analysis results are shown in FIG. 5, and the concentrations of epsilon-caprolactone, which is the product obtained by catalyzing 70mM cyclohexanol by 7 mutant strains, are different and are higher than those of the original strain RCL 0. Among them, the yield of the mutant strain RCL07 was the highest, and reached 47.0 mM. This shows that the catalytic ability of 7 positive mutant strains obtained by high-throughput screening is verified to be stronger than that of the original strain, and the conjecture that the product epsilon-caprolactone concentration and the intermediate product cyclohexanone concentration are in a better negative correlation in example 1 is verified to be feasible, which also shows that the high-throughput screening method for epsilon-caprolactone high-yield strains provided by the invention is effective.

Example 5 fed-batch catalytic application of mutant Strain RCL07

The RCL07 mutant strain with the highest epsilon-caprolactone yield in example 4 was used in fed-batch whole-cell catalysis experiments to further increase the epsilon-caprolactone yield. The strain culture and protein expression steps are the same as in example 4, and the fed-batch catalysis step is as follows:

1. fed-batch catalyzed system composition

Using 100g of DMSO-treated sample in example 4 with 40mM cyclohexanol as a substrate_wcwThe RCL07 cell suspension at/L was subjected to fed-batch whole-cell catalytic reaction. The reaction was carried out in a 250mL shake flask, and the system is shown in Table 2 below.

TABLE 2

Reagent	Volume of
		Cyclohexanol	211μL(40mM)
100g_wcwCell suspension/L	5mL
		20mM Tris-HCl (pH7.5) buffer	45mL

2. Procedure and results of fed-batch catalysis

Firstly, adding 45mL of buffer solution into a 250mL shake flask, then adding 211 mu L of cyclohexanol solution, uniformly mixing to fully dissolve cyclohexanol in the buffer solution, finally adding 5mL of cell suspension, immediately sealing by using a sealing film after uniformly mixing, placing on a shaking table at 25 ℃ at 120rpm, and sampling once every several hours. Three are arranged in parallel. And (3) replacing 5mL of cell suspension with 5mL of 20mM Tris-HCl (pH7.5) buffer solution in the control group, uniformly mixing the components, freezing 800 mu L of sample in a 2mL centrifuge tube, and detecting the sample as an actual substrate input value before catalysis. And after the single batch reaction is finished, collecting a catalytic system, centrifuging for 30min at a low temperature of 4 ℃ by using a centrifuge to recover the thallus, preparing the heavy suspension thallus of the catalytic system containing cyclohexanol with the same concentration again to start the next batch fed-batch experiment, and carrying out 4 batches of reactions in total, wherein the first two batches of reactions are carried out for 11h, the second two batches of reactions are carried out for 16h, and the total input amount of the substrate is 160 mM. At intervals of several hours, 800. mu.L of the reaction solution was sampled and assayed for changes in the concentrations of the substrate and the product by the procedure described in example 4.

The mutant strain RCL07 has strong catalytic activity and long-term stability, and fed-batch results (Table 3) show that the mutant strain can catalyze 4 batches of substrates within 54h to realize the epsilon-caprolactone space-time yield of 0.236mM/g/h, which is the highest record of the epsilon-caprolactone yield and the space-time yield in the existing fed-batch report.

TABLE 3

a space-time yield refers to the concentration of epsilon-caprolactone produced by bacterial catalysis per unit biomass per unit time.

Reference documents:

[1]Silva AL P,Batista P K,Filho A D,et al.Rapid conversion of cyclohexenone,cyclohexanone and cyclohexanol toε-caprolactone by whole cells of Geotrichumcandidum CCT1205[J].Biocatalysis and Biotransformation,2017,35(3):185-190.

[2]Schmidt S,Scherkus C,Muschiol J,et al.An enzyme cascade synthesis of epsilon-caprolactone and its oligomers[J].Angew Chem Int Ed Engl,2015,54(9):2784-2787.

[3]Kohl A,Srinivasamurthy V,Bottcher D,et al.Co-expression of an alcohol dehydrogenase and a cyclohexanone monooxygenase for cascade reactions facilitates the regeneration of the NADPH cofactor[J].Enzyme Microb Technol,2018,108:53-58.

[4]Xiong J,Chen H,Liu R,et al.Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration forε-caprolactone production[J].Bioresources and Bioprocessing,2021,8(1).

the above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Claims

1.A high-throughput screening method of epsilon-caprolactone high-yield strains is characterized in that: the method comprises the following steps:

correlation analysis is carried out on the concentration of the product epsilon-caprolactone of the ADH and CHMO double-enzyme cascade reaction and the concentration of the intermediate product cyclohexanone, and the correlation relationship between the concentration of the intermediate product cyclohexanone and the concentration of the product epsilon-caprolactone is found out after the strains with different catalytic performances are subjected to whole-cell catalytic reaction, so that the intermediate product cyclohexanone is used as a screening marker, and the strains with low cyclohexanone concentration are screened in high throughput to obtain the strains with high yield of epsilon-caprolactone.

2. The high-throughput screening method for epsilon-caprolactone high-producing strains according to claim 1, which is characterized in that: the method specifically comprises the following steps:

3. The high-throughput screening method for epsilon-caprolactone high-producing strains according to claim 2, which is characterized in that:

the epsilon-caprolactone-producing mutation library strain in the step (1) is obtained by simultaneously mutating the original ribosome binding site sequences of ADH and CHMO genes in an original strain RCL0 by using RBS engineering technology, taking Escherichia coli BL21(DE3) as a host and pRSFDuet-1 as an expression vector;

the starting strain RCL0 is a recombinant strain BDR-3.

4. The method for high-throughput screening of epsilon-caprolactone high-producing strains according to claim 2 or 3, wherein:

the microporous plate in the steps (1), (2) and (3) is a 96-hole or 48-hole microporous plate;

the shaking culture conditions in the step (1) are 36-38 ℃, 900-1100 rpm and 10-14 hours;

inducing protein expression in the step (1) for 6-8 h at 28-32 ℃ and 800-1000 rpm; the final concentration of the IPTG in the step (1) is 0.4-0.6 mM.

5. The method for high-throughput screening of epsilon-caprolactone high-producing strains according to claim 2 or 3, wherein:

centrifuging for 10-15 min under the conditions of 4-8 ℃ and 1800-2000 g in the step (2);

the final concentration of the cyclohexanol in the step (2) is 40-60 mM;

the buffer solution in the step (2) is Tris-HCl buffer solution;

the reaction conditions in the step (2) are 23-27 ℃ and 700-900 rpm for 14-18 h.

6. The method for high-throughput screening of epsilon-caprolactone high-producing strains according to claim 2 or 3, wherein:

the concentration of the 2, 4-dinitrophenylhydrazine solution in the step (3) is 15-25 mM;

the concentration of the NaOH solution in the step (3) is 1.2-1.8M;

in the step (3), the cyclohexanone and the 2, 4-dinitrophenylhydrazine are fully reacted for 25-35 min under the conditions of 28-32 ℃ and 700-900 rpm;

in the step (3), the condition of the color reaction is that the mixture is kept still for reaction for 10-20 min at the temperature of 28-32 ℃;

in the step (3), the centrifugation is carried out for 5-10 min at the temperature of 4-8 ℃ and the speed of 1800-2000 g;

the specific absorption wavelength in the step (3) is 530-550 nm.

7. The high-throughput screening method of the epsilon-caprolactone high-producing strain disclosed by any one of claims 1-6 is applied to screening of the epsilon-caprolactone high-producing strain.

8. An epsilon-caprolactone high-yield strain obtained by the high-throughput screening method of any one of claims 1 to 6.

9. The epsilon-caprolactone high-producing strain of claim 8, wherein:

the epsilon-caprolactone high-yield strain is RCL01, RCL02, RCL03, RCL04, RCL05, RCL06 or RCL 07; the method is characterized in that the original ribosome binding site sequences of ADH and CHMO genes in an original strain RCL0 are simultaneously mutated by an RBS engineering technology, escherichia coli BL21(DE3) is taken as a host, and pRSFDuet-1 is taken as an expression vector;

10. Use of the epsilon caprolactone-producing strain of claim 8 or 9 in the fermentative production of epsilon caprolactone.