CN115851692A - Beta-caryophyllene synthase mutant, coding gene and application thereof in beta-caryophyllene synthesis - Google Patents

Abstract

The invention discloses a beta-caryophyllene synthase mutant, a coding gene and application thereof in beta-caryophyllene synthesis. The invention relates to the field of biotechnology, in particular to a beta-caryophyllene synthase mutant, a coding gene and application thereof in beta-caryophyllene synthesis. The amino acid sequence of the beta-caryophyllene synthase mutant obtained by error-prone PCR is the sequence 2 in the sequence table. Through directional modification, the catalytic efficiency of the beta-caryophyllene synthase is improved, the synthesis of the beta-caryophyllene with the saccharomyces cerevisiae strain SQ3-6-ECPS11 as the chassis bacteria can generate 600-800mg/L beta-caryophyllene after 96h of fermentation, and the yield index has important industrial application potential.

Description

Beta-caryophyllene synthase mutant, coding gene and application thereof in beta-caryophyllene synthesis

Technical Field

The invention relates to the technical field of biology, in particular to a beta-caryophyllene synthase mutant, a coding gene and application thereof in beta-caryophyllene synthesis.

Background

Terpene compounds are compounds with isoprene as a basic structural unit, have various physiological and pharmacological functions such as oxidation resistance, virus resistance, parasite resistance, tumor resistance, immunoregulation and the like, and are widely applied to the fields of nutrition, health care, medicine and the like. The extraction of animals and plants is the main source of terpene compounds, but the problems of long growth period of animals and plants, low ratio of effective components, complex raw material pretreatment, animal resource protection and the like cause the direct extraction method to face severe high cost and industrial scale limitation, and the market demand for rapid promotion is difficult to meet.

The terpenoid synthesized by the microbial fermentation method has the obvious characteristics of short period, easy treatment of raw materials, controllable process and the like, not only has green natural property, but also has economical efficiency and environmental friendliness, and is a production way with obvious competitive advantages. Yeasts are microorganisms which are widely applied in the field of industrial biotechnology, have a natural terpene compound synthesis precursor pathway, namely a mevalonate pathway, and are therefore preferred underpan cells for synthesizing terpene compounds. Many terpene compounds having extremely high added value are unique to animals and plants. Thus, the synthesis of terpene compounds of animal and plant origin using yeast as the underpan cells requires first the expression of a heterologous terpene synthase. The activity level of terpene synthase is a key rate-limiting factor affecting the efficiency of terpene compound synthesis. The directed evolution and semi-rational design and modification of the enzyme provide an effective technical means for solving the problem of low activity of the natural terpene synthase, but because the terpene compounds have complex structures, the analysis and detection are usually carried out by means of chromatography and mass spectrometry, so that the analysis flux is very low, and the detection cost is also very high. Therefore, establishing an effective high-throughput screening method for screening terpene synthase mutants is a key technology to be solved for terpene synthase directed evolution.

beta-Caryophyllene (beta-caryophylelene) is a bicyclic sesquiterpene, which is a plant volatile compound commonly found in plants such as artemisia annua, ocimum basilicum, cinnamomum cassia, etc., and is often used as a cosmetic and food additive due to its strong wood odor. Meanwhile, the beta-caryophyllene also has various physiological activities including anti-inflammatory action, anticancer action, antibacterial action, antioxidant action, analgesic action and the like. The direct extraction from plants such as sweet wormwood herb and the like is the main production mode of the beta-caryophyllene at present, and the yield is low and the cost is high due to the limitations of long plant growth cycle, low beta-caryophyllene content, complex extraction process and the like. The realization of the efficient synthesis of the beta-caryophyllene by taking the saccharomycetes as the underpan cells is an urgent need and an important direction for the development of the industry. beta-Caryophyllene is synthesized by dephosphorylation and cyclization reaction by taking farnesyl diphosphate synthesized by a mevalonate pathway as a substrate under the catalysis of beta-Caryophyllene synthase (CPS). The low activity of natural beta-caryophyllene synthase is a key factor for limiting the synthesis efficiency of beta-caryophyllene.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the activity of the microbial beta-caryophyllene synthase.

In order to solve the above technical problems, the present invention provides a protein, which may be any one of the following:

a1 Protein with an amino acid sequence of SEQ ID No. 2;

a2 A fusion protein having the same function obtained by attaching a tag to the N-terminus and/or C-terminus of A1).

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

The nucleotide sequence encoding the protein of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution or point mutation. Those nucleotides which are artificially modified to have 75% or more than 75% identity to the nucleotide sequence of the protein isolated according to the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the protein and have the same protein function.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, per response Gap cost, and Lambda ratio to 11,1 and 0.85 (default values), respectively, and performing a calculation to search for identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

The proteins described above are derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae).

The invention also provides a biological material related to the protein, wherein the biological material can be any one of the following materials:

b1 Nucleic acid molecules encoding the proteins described above;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1);

b4 A recombinant vector containing the expression cassette of B2);

b5 A recombinant microorganism containing the nucleic acid molecule according to B1);

b6 A recombinant microorganism containing the expression cassette described in B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism containing the recombinant vector of B4).

In the above biological material, the nucleic acid molecule of B1) may be a DNA molecule represented by any one of the following:

d1 ) the nucleotide sequence is the DNA molecule shown in SEQ ID NO. 3;

d2 The sequence of the coding region is a DNA molecule shown as SEQ ID NO.3 in the sequence table;

d3 A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined under d 1) or d 2) and codes for the protein.

The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be an RNA, such as a gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

Vectors described herein are well known to those skilled in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. In particular, the vector is YEp-GMZC.

In the above biomaterial, the recombinant microorganism is a recombinant yeast.

In the biological material, the recombinant yeast is recombinant saccharomyces cerevisiae.

In the biological material, the recombinant saccharomyces cerevisiae also contains ABC transporter mutant gene STE6 ^T1025N And does not contain a saccharomyces cerevisiae alcohol dehydrogenase gene ADH5.

In the biological material, the recombinant saccharomyces cerevisiae is constructed according to the following method.

The invention also provides a method for constructing the recombinant saccharomyces cerevisiae.

The method for constructing the recombinant saccharomyces cerevisiae comprises the steps of knocking out the ethanol dehydrogenase gene in the recipient saccharomyces cerevisiae, and encoding the protein encoding gene and the ABC transporter encoding gene STE6 ^T1025N The recipient saccharomyces cerevisiae is introduced.

In the above method, the method further comprises adding P _IRA1 Introducing the promoter into the receptor Saccharomyces cerevisiae to make P _IRA1 The promoter drives transcription of the ABC transporter encoding gene.

Herein, the introduction may be to transform the vector carrying the DNA molecule of the present invention into a host bacterium by any known transformation method such as chemical transformation or electroporation. The introduced DNA molecule may be in single or multiple copies. The introduction may be the integration of the foreign gene into the host chromosome or the extrachromosomal expression from a plasmid.

The invention also provides a method for producing the beta-caryophyllene, which comprises the steps of culturing the recombinant saccharomyces cerevisiae to obtain a fermentation product, and obtaining the beta-caryophyllene from the fermentation product.

The invention also provides the method for constructing the recombinant saccharomyces cerevisiae and the application of the recombinant saccharomyces cerevisiae in any one of the following applications:

p1, application in producing beta-caryophyllene;

p2 and improving the yield of the beta-caryophyllene.

The invention adopts an error-prone PCR-based directed evolution strategy to transform beta-caryophyllene synthase, thereby improving the catalytic efficiency of the enzyme. Will carry the selection markers GMZ, STE6 ^T1025N The expression cassette fragments are transferred into a saccharomyces cerevisiae strain SQ3-4 through an electrical transformation method, a recombinant strain SQ3-6 is obtained through forward screening of bleomycin resistance and reverse screening of galactose induction, a plasmid pYE-ECPS11 with a beta-caryophyllene synthase E353D mutant coding gene expression cassette is converted into the strain SQ3-6, the yield of beta-caryophyllene of the obtained recombinant strain SQ3-6-ECPS11 reaches 62.44mg/L, compared with the strain SQ3-6-CPS, the yield is improved by 78.2%, the strain SQ3-6-ECPS11 is cultured in a fermentation tank, 600-800mg/L beta-caryophyllene can be produced after 96h of fermentation, and the synthesis of beta-caryophyllene with saccharomyces cerevisiae as a chassis is greatly improved.

Drawings

FIG. 1 is a comparison of the tolerance of different Saccharomyces cerevisiae strains to salt stress.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative experiments in the following examples, unless otherwise specified, were carried out in triplicate.

PrimeSTAR Max Premix (2 x) is a product of TaKaRa, inc. under the product designation R045A.

The beta-caryophyllene standard product is an Allantin reagent (Shanghai) Limited company product, and the product number is 87-44-5.

The Error-prone PCR Kit InstantError-prone PCR Kit is a product of Tiannze company, and the product number is 101005-100.

The plasmid pYE-CPS is a plasmid which is constructed in the earlier stage of the research group and is provided with a Zeocin (bleomycin) resistance selection marker and a beta-caryophyllene synthase coding gene QHS1 expression cassette, and the construction method is described in the following documents: lu S, zhou C, guo X, du Z, chen Y, wang Z, he X. (2022) Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and beta-alkane.

The plasmid YEp-GMZC is a plasmid which is constructed in the earlier stage of the research group and is provided with a Zeocin resistance screening marker and a galactose-induced mazF expression cassette is a reverse screening marker, and the construction method is described in paragraphs 0167-0183 of the Chinese invention patent application with the publication number CN 113151262B (the patent number is ZL202110188875.9, the invention name is yeast promoter with weakened regulation strength and the application thereof in metabolic flux regulation).

Saccharomyces cerevisiae SQ3-4 is described in the following references: lu S, zhou C, guo X, du Z, chen Y, wang Z, he X. (2022) Enhancing fluxes through the potent pathway in Saccharomyces cerevisiae by engineering the HMGR and β -alkane microorganism, microb Biotechnol,15 (8): 2292-2306, hereinafter referred to as Saccharomyces cerevisiae SQ3-4, publicly available from the institute of microbiology, a national academy of sciences, only for the purpose of repeating the present invention.

Saccharomyces cerevisiae SQ3-4-CPS is obtained by transferring multiple copies of Saccharomyces cerevisiae-Escherichia coli shuttle plasmid pYE-CPS into Saccharomyces cerevisiae SQ3-4, and the construction method is described in the literature: lu S, zhou C, guo X, du Z, chen Y, wang Z, he X. (2022) Enhancing fluxes through the potent pathway in Saccharomyces cerevisiae by engineering the HMGR and β -alkane microorganism, microb Biotechnol,15 (8): 2292-2306, hereinafter referred to as Saccharomyces cerevisiae SQ3-4-CPS, publicly available from the institute of microbiology, is used only for the repetition of the present invention.

YPD medium: the culture medium consists of solute and solvent; the solutes are yeast powder, peptone and glucose, and the solvent is water; the concentrations of solutes were as follows: 10g/L yeast powder, 20g/L peptone and 20g/L glucose; natural pH; bleomycin at 100. Mu.g/mL was added as required.

The solid culture medium of the above culture medium is prepared by adding 20g/L agar powder, the rest components and concentration are the same as the liquid culture medium, and 200. Mu.g/mL bleomycin is added according to the need.

Example 1 establishment of high throughput screening method for terpene synthases

1. Effect of salt stress on growth of Saccharomyces cerevisiae

(1) Inoculating Saccharomyces cerevisiae SQ3-4 into 3mL YPD liquid culture medium, and culturing at 30 deg.C and 200rpm for 20h; inoculating to 3mL YPD liquid medium at 1% (volume ratio), culturing at 30 deg.C and 200rpm to OD ₆₀₀ Up to 1.0. OD conversion Using sterile Water ₆₀₀ And (3) carrying out gradient dilution on the bacterial liquid of 1.0 time by 10 times, and fully and uniformly mixing for later use.

(2) NaCl was added to the YPD solid medium to give final concentrations of 0g/L, 20g/L and 40g/L, respectively, to obtain YPD solid medium plates having different NaCl concentration gradients. Aspirate 20. Mu.L of dilution 10 ^-5 And (3) uniformly coating the SQ3-4 bacterial liquid on a NaCl concentration gradient culture medium plate, performing static culture in an incubator at the temperature of 30 ℃ for 48 hours, counting the number of colonies on different plates, and calculating the survival rate.

As shown in Table 1, the addition of NaCl produced a significant inhibition of the growth of Saccharomyces cerevisiae cells, whereas the addition of 20g/L and 40g/L NaCl reduced the cell viability by 57.5% and 97.3%, respectively, compared to the absence of NaCl, and yeast strain SQ3-4 had hardly grown on the medium plate containing 40g/L NaCl (FIG. 1).

TABLE 1 Effect of NaCl on cell survival for different yeasts

2. Effect of salt stress on growth of beta-caryophyllene synthetic strains

(1) And (2) inoculating saccharomyces cerevisiae SQ3-4-CPS with a plasmid-form expression cassette of the beta-caryophyllene synthase coding gene QHS1 into 3mL YPD liquid culture medium, and preparing bacterial liquids with different dilutions according to the method in the step one (1).

(2) Preparing NaCl concentration gradient plate by the method described in the previous step (2), and sucking 20. Mu.L of dilution with 10 ^-5 And uniformly coating the SQ3-4-CPS bacterial liquid on a NaCl concentration gradient culture medium plate, performing static culture in an incubator at the temperature of 30 ℃ for 48 hours, counting the colony number on different plates, and calculating the survival rate.

As shown in Table 1, the addition of NaCl also produced a significant inhibition of the growth of the SQ3-4-CPS cells, and the addition of 20g/L and 40g/L NaCl decreased the cell viability by 29.4% and 88.7%, respectively, compared to the absence of NaCl. However, SQ3-4-CPS has better salt stress tolerance than the strain SQ3-4 (FIG. 1). The increase of the content of active oxygen in the cell is a general biological process in the response of salt stress injury of the yeast cell, and the beta-caryophyllene has the physiological activity of resisting oxidation and can effectively neutralize and eliminate the active oxygen in the cell so as to provide antioxidant protection for the yeast cell. Therefore, the growth of the beta-caryophyllene synthetic strain SQ3-4-CPS expressing the beta-caryophyllene synthase is less influenced than that of the developed strain SQ3-4 under the condition of salt stress.

The results show that the synthesis of the beta-caryophyllene in the saccharomyces cerevisiae is beneficial to the improvement of the salt stress tolerance of the strain, and the high-activity beta-caryophyllene synthase can catalyze and synthesize more beta-caryophyllene so as to generate stronger salt stress tolerance, so that the activity of the beta-caryophyllene synthase can be screened through salt stress.

Example 2 directed evolution of beta-Carophyllene synthase and screening of highly active enzymes

1. Directed evolution of beta-caryophyllene synthase

1.1 error-prone PCR of beta-caryophyllene synthase encoding gene QHS1

(1) The following primers were designed and synthesized based on the nucleotide sequence of the beta-caryophyllene synthase encoding gene QHS1 in plasmid pYE-CPS (sequence 1 in the sequence Listing):

ECPS-F:5’-ATGGACATGCCAGCTAAAG-3’

ECPS-R:5’-TCAAATTGGGATAGGGTGAAC-3’

(2) Error-prone PCR amplification was performed using the Instant Error-pro PCR Kit with plasmid pYE-CPS as a template.

Error-prone PCR reaction system: 10ng of DNA template, 3 mu L of error-prone PCR Mix, 3 mu L of error-prone PCR-dedicated dNTP and MnCl for error-prone PCR ₂ 3 μ L, error-prone PCR-specific Taq DNA polymerase (0.5U/. Mu.L) 0.5 μ L. The concentration of each primer in the reaction system is 0.3 mu mol/L, the system is supplemented to 30 mu L by deionized water, and the mixture is uniformly mixed.

Error-prone PCR reaction conditions: circulating for 1 time at 94 ℃/3 min; 94 ℃/1min,45 ℃/1min,72 ℃/1min,45 cycles; no extension process is required.

The error-prone PCR amplified to about 1.5kb DNA fragment from plasmid pYE-CPS using primers ECPS-F and ECPS-R was designated ECPS.

1.2 construction of QHS1 mutant library

(1) And carrying out PCR amplification on the plasmid pYE-CPS by taking the plasmid pYE-CPS as a template and the ECPS as a long primer to obtain a linearized plasmid fragment with the ECPS connected to two ends.

PCR amplification System: primeSTAR Max Premix (2X) 25. Mu.L, plasmid pYE-CPS 40ng, ECPS 400ng, make up to 50. Mu.L with deionized water and mix well.

And (3) PCR reaction conditions: cycling for 1 time at 98 ℃/3 min; 98 ℃/10sec,55 ℃/10sec,72 ℃/30sec,32 cycles; 72 ℃/5min.

(2) The PCR product obtained in (1) was digested with a restriction enzyme DpnI (NEW ENGLAND Biolabs, inc., cat. R0176L), and the PCR template was removed therefrom.

Enzyme digestion system: PCR product 20. Mu.L, cutsmart 5. Mu.L, dpnI 2. Mu.L, deionized water 23. Mu.L.

The enzyme digestion reaction conditions are as follows: the reaction is carried out for 2h at 37 ℃.

(3) And (3) cyclizing the linear plasmid by using an escherichia coli homologous recombination mechanism to construct a QHS1 mutant library.

Adding the PCR product obtained in the step (2) and removing the template into 200 mu L of escherichia coli competent cells, gently mixing uniformly, and carrying out ice bath for 30min; performing water bath heat shock at 42 ℃ for 90sec, taking out, and immediately performing ice bath for 2min; after adding 800. Mu.L of LB liquid medium and performing static culture at 37 ℃ for 1 hour, the bacterial solution was spread on an LB medium plate containing 100. Mu.g/mL ampicillin and subjected to static culture at 37 ℃ for 16 hours.

Scraping all the Escherichia coli transformants on the plate into a 1.5mL centrifuge tube filled with 1mL sterile water, and centrifuging at 12000rpm for 2min to collect thalli; washing thallus with 500 μ L STE solution, centrifuging at 12000rpm for 2min, collecting thallus, extracting plasmid by conventional alkali extraction method to obtain QHS1 mutant library, named pYE-ECPS, and storing in refrigerator at-20 deg.C for use.

2. Screening of high-activity beta-caryophyllene synthase

2.1 construction of Saccharomyces cerevisiae strains expressing QHS1 mutant pools

The QHS1 mutant library pYE-ECPS is introduced into saccharomyces cerevisiae SQ3-4 by an electric transformation method (electric transformation conditions: 1.5kV,50 muF, 200 omega, 3 mSec), 100 muL of transformed bacterial liquid is taken and coated on a YPD medium plate containing 200 mug/mL antibiotic Zeocin and 20g/L NaCl, and the bacterial liquid is statically cultured at 30 ℃ until obvious single colony is formed. Single colonies of recombinant yeast having Zeocin resistance and salt stress tolerance were obtained and named SQ3-4-ECPS.

2.2 screening of beta-Caryophyllene synthase mutants

(1) Single colony screening based on salt stress: selecting a single colony SQ3-4-ECPS with a good growth state and a large colony to 1mL of sterile water, diluting by 1000 times, and standing for 3h at room temperature; vortex, shake and mix evenly, take 5 mul spot plate to YPD medium plate containing 200 mug/mL antibiotic Zeocin and 40g/L NaCl, after static culture 72h at 30 deg.C observe growth state, named SQ 3-4-ECPS-M1-SQ 3-4-ECPS-M42 colony with best growth state, carry on sequencing and follow-up analysis.

(2) Sequence analysis and comparison: PCR amplification was carried out using yeast cell lysate as a template and upstream and downstream primers S091 (5-. Sequence analysis finds that: 15 mutation hotspots were identified from QHS1 of the 42 salt stress resistant single colonies screened compared to the coding sequence of native QHS1 (table 2).

TABLE 2 analysis of mutational hot spots in salt stress resistant single colonies

Hot spot of nucleotide mutation	Amino acid changes
		A26G	K9R
T62C	V21A
		G133A	E45K
T376C	F126L
		C404A	S135Y
G478T	G160C
		C617A	T206N
G793A	A265T
		A935T	Y312F
T1031	V344D
		A1059C	E353D
C1087T	H363Y
		C1100T	A367V
G1534T	V512F
		G1583A	G528D

2.3 Effect of beta-Carophyllene synthase mutant on Yeast beta-Carophyllene Synthesis ability

(1) The single colonies selected to have only the single point mutation in the QHS1 coding region were renamed to SQ3-4-ECPS1 to SQ3-4-ECPS15. Respectively inoculating the strain and the strain SQ3-4-CPS into a 3mLYPD culture medium, and performing shake culture at 30 ℃ and 200rpm for 24h to obtain an activated bacterial liquid; transferring the strain to 3mL YPD medium with 10% (volume ratio), and shake culturing at 30 deg.C and 200rpm for 24h to obtain seed bacteria liquid; the cells were inoculated in 5mL of YPD medium in an inoculum size of 10% (by volume), followed by addition of 1mL of dodecane and shake culture at 30 ℃ and 200rpm for 48 hours to obtain a fermentation broth.

(2) The fermentation broth was centrifuged at 5000rpm for 5min, and the bacterial cells and the dodecane organic phase were collected separately. The thalli is dried in a 65 ℃ oven to constant weight and weighed, and the cell biomass is gram (g/L) of dry thalli in each liter of fermentation liquor. And detecting the content of the beta-caryophyllene by using a dodecane organic phase through GC-MS.

The results are shown in Table 3, with minor differences between the cell biomass of the different strains.

(3) Preparing a beta-caryophyllene standard curve: accurately weighing beta-caryophyllene standard substances, dissolving the beta-caryophyllene standard substances in dodecane to prepare dodecane solutions of the beta-caryophyllene with the concentrations of 14.5mg/L, 29mg/L, 58mg/L, 116mg/L and 232mg/L respectively, and setting three times for each concentration. Performing GC-MS analysis by using a SHIMADZU GCMS-QP2010 Ultra infiit gas chromatography-mass spectrometry system, wherein a chromatographic analysis column is DB-5MS; the carrier gas is helium, and the flow rate is 3mL/min; the injection port temperature is 240 ℃, the FID is 280 ℃, and the detection temperature is respectively 150 ℃ and is kept for 1min, and the speed of 20 ℃/min is increased to 280 ℃; the flow splitting ratio is 50; the sample size was 1. Mu.L. Fitting a standard curve by taking the concentration of the beta-caryophyllene solution as a horizontal coordinate and the measured peak area as a vertical coordinate to obtain a function formula between the concentration of the beta-caryophyllene and the peak area:

concentration of beta-caryophyllene (mg/L) = (peak area + 165152)/73335

(4) And (4) carrying out GC-MS detection on a dodecane organic phase obtained by fermentation by the method in the step (3), and calculating the content of beta-caryophyllene in different fermentation liquids according to peak areas, wherein the yield of the beta-caryophyllene is milligrams (mg/L) of the beta-caryophyllene in each liter of the fermentation liquid.

As shown in Table 3, compared with the strain SQ3-4-CPS expressing wild-type beta-caryophyllene synthase, the beta-caryophyllene yield of the strains SQ3-4-ECPS5, SQ3-4-ECPS9, SQ3-4-ECPS11 and SQ3-4-ECPS14 is respectively improved by 27.1%,25.6%,73.3% and 43.8%; compared with the wild type, the beta-caryophyllene synthase of the strains has the following amino acid site mutations respectively: Y312F, V344D, E353D, V21A. Yeast plasmids were extracted from the strains SQ3-4-ECPS5, SQ3-4-ECPS9, SQ3-4-ECPS11 and SQ3-4-ECPS14, respectively, and designated pYE-ECPS5, pYE-ECPS9, pYE-ECPS11 and pYE-ECPS14, respectively.

TABLE 3 cell growth and beta-caryophyllene Synthesis by expression of mutant beta-caryophyllene synthase strains

2.4 analysis of enzymatic Activity of beta-Caryophyllene synthase and mutants thereof

(1) Construction of expression plasmids and strains: the following primers were designed and synthesized based on the nucleotide sequence of pYE-CPS:

S213：5’-CTTTAGCTGGCATGTCCATCTGTTTTTTTAGAAAGAGCC-3’

S214：5’-TTTTTTTGTTTTTTATGTCTAAGCTTG-3’

S215：5’-GGCTCTTTCTAAAAAAACAGATGGACATGCCAGCTAAAG-3’

S216：5’-cagtcacgacgttgtaaaacgacggccagtgccaagcttAGACATAAAAAACAAAAAA

ATTACTTTTCGAACTG-3’

PCR amplification was carried out using the plasmids pYE-CPS, pYE-ECPS5, pYE-ECPS9, pYE-ECPS11 and pYE-ECPS14 as templates, respectively, and the PCR reaction system and reaction conditions were as shown in 1.2 in this example.

6802bp of plasmid skeleton is amplified by primers S213 and S214, 1756bp of CPS and ECPS coding sequences with plasmid skeleton homology arms and Strep labels are amplified by primers S215 and S216. Plasmid backbone and CPS coding sequence or ECPS coding sequence were expressed as 1:1, then introducing the mixture into Saccharomyces cerevisiae SQ3-4 by an electric conversion method (electric conversion condition: 1.5kV,50 muF, 200 omega, 3 mSec), connecting by utilizing a homologous recombination mechanism of the Saccharomyces cerevisiae, and screening a converted strain by bleomycin resistance; obtaining recombinant strains SQ3-4-CPS-Strep for expressing Strep-labeled beta-caryophyllene synthase and recombinant strains SQ3-4-ECPS5-Strep, SQ3-4-ECPS9-Strep, SQ3-4-ECPS11-Strep and SQ3-4-ECPS14-Strep for expressing Strep-labeled beta-caryophyllene synthase mutants.

(2) Protein purification of beta-caryophyllene synthase and beta-caryophyllene synthase mutants: culturing the constructed saccharomyces cerevisiae recombinant strain in 20mL YPD medium at 30 ℃ and 200rpm for 24h; then transferring the culture medium to 200mL YPD medium, and culturing at 30 ℃ and 200rpm for 24h; then transferring the mixture into 2000mL YPD culture medium, and culturing for 48h at 30 ℃ and 200 rpm; the cells were collected by centrifugation at 5000rpm for 5min and washed 3 times with distilled water.

The washed cells were resuspended in 50mL Buffer W (100 mmol/L Tris-HCl (pH 8.0), 150mmol/L NaCl,1mmol/L EDTA), and disrupted using a sonicator at 350Hz with a 10sec pause per 20sec run for 120 cycles. And (3) centrifuging the crushed bacterial suspension for 30min at 4 ℃ and 12000rpm, and collecting supernatant, namely the total protein extraction solution.

Passing the total protein solution through a filter column filled with Strep-Tactin XT filler, washing the column with 5mL of Buffer W, and removing impurity proteins; and eluting the target protein which is combined with the column and is provided with a Strep label by taking 3mL of Buffer BXT (100 mmol/L Tris-HCl (pH 8.0), 150mmol/L NaCl,1mmol/L EDTA and 5mmol/L biotin) to obtain purified beta-caryophyllene synthase and beta-caryophyllene synthase mutant protein.

(3) Determination and calculation of enzymatic Activity: a250. Mu.L enzyme reaction system was prepared containing 4. Mu.g of the enzyme protein, 5mM 4-hydroxyethylpiperazineethanesulfonic acid (pH 7.0), 1mM MgCl ₂ 1mM dithiothreitol and 8. Mu.M farnesyl pyrophosphate FPP. The reaction was carried out at 30 ℃ for 15min. The reaction was stopped by adding 250. Mu.L of ethanol. Shaking and extracting with 250 μ L dodecane for 15min. And (3) determining the content of the beta-caryophyllene in the dodecane organic phase by GC-MS analysis. The enzyme kinetic parameters such as the maximum reaction rate Vmax, the Michaelis constant Km and the catalytic constant Kcat are calculated by nonlinear fitting through originPro. The catalytic constant (Kcat) of the enzyme is defined as the number of molecules of FPP that can be converted per second per molecule of β -caryophyllene synthase at 30 ℃, pH 7.0.

The result shows that compared with wild beta-caryophyllene synthase, only E353D mutation leads to remarkable improvement of enzyme activity, and the maximum reaction rate is improved by 34.8%; enzyme catalysis constant Kcat of 0.0077s ^-1 Compared with wild beta-caryophyllene synthase, the beta-caryophyllene synthase is improved by 30.5 percent; the ratio of Kcat/Km is increased by 35.5% compared with wild enzyme. Therefore, the mutant of the beta-caryophyllene synthase E353D is a mutant enzyme with improved activity of the beta-caryophyllene synthase. Beta-caryophylleneThe nucleotide sequence of the synthase E353D mutant is a sequence 3 in a sequence table.

Example 3 construction of efficient Synthesis of beta-Caryophyllophora

1. Increasing transmembrane transporter level and increasing synthesis of beta-caryophyllene

(1) Expression of ABC transporter mutant Ste6 ^T1025N Construction of the Saccharomyces cerevisiae Strain

The following primers were designed and synthesized based on the nucleotide sequences of the NCBI reported Saccharomyces cerevisiae alcohol dehydrogenase gene ADH5 (GenBank No. NC-001134.8), GTPase activator gene IRA1 (GenBank No. CP046082.1), plasma membrane ABC transporter encoding gene STE6 (GenBank No. NM-001179774.1), and plasmid YEp-GMZC:

S181：5’-ATGCCTTCGCAAGTCATTCC-3’

S182：5’-TTCAACGCCTTATAAACAGTGATACCTG-3’

S183：5’-caatcttgctgaagttgccccaatcttgtgtgcaggtatcactgtttataaggcgttgaaGCATGCCCGCGGTGCTC-3’

S184：5’-cgtcatgaagacactacttgtaaaattaGCAAATTAAAGCCTTCGAG-3’

S185：5’-ctcgaaggctttaatttgcTAATTTTACAAGTAGTGTCTTCATGACG-3’

S186：5’-gtgtcggctcgcttagagGATGCTTTGATTTTGTAGATATGTAGTT-3’

S187：5’-aactacatatctacaaaatcaaagcatcCTCTAAGCGAGCCGACAC-3’

S188：5’-gtagtcttaaaacttaaaaagttcatCTGTTTTTTTAGAAAGAGCC-3’

S189：5’-ggctctttctaaaaaaacagATGAACTTTTTAAGTTTTAAGACTAC-3’

S190：5’-CATTATTGTTTTCAACCTCTAGGTTATTATGCTTTTCATCAAGA ATCCTA-3’

S191：5’-TAGGATTCTTGATGAAAAGCATAATAACCTAGAGGTTGAAAA CAATAATG-3’

S192：5’-atagcttatataaaaagtaaaaatatattcatcaaattcgttacaaaagaTTAACTGCTTTGGTTGGAAAC-3’

S193：5’-TCTTTTGTAACGAATTTGATGAATATATTTTTACTTTTTATAT-3’

S194：5’-TTCTCATAGGGGCAGGAGC-3’

the PCR reaction system and reaction conditions were as shown in 1.2 of example 2.

Amplifying a 5' end homology arm ADH5-uparm of ADH5 with the length of 500bp by taking the genome of the saccharomyces cerevisiae strain SQ3-4 as a template and taking S181 and S182 as primers; amplifying an upstream sequence ADH5-5up of the 5' end of ADH5 with the length of 537bp by taking S185 and S186 as primers; amplifying a 3' end homologous arm ADH5-downarm of ADH5 with the length of 500bp by taking S193 and S194 as primers; amplification of 463bp IRA1 promoter P with S187 and S188 as primers _IRA1 . The 5 'terminal sequence (3117 bp) and the 3' terminal sequence (876 bp) of the ABC transporter encoding gene STE6 of Saccharomyces cerevisiae were amplified using S189, S190, S191 and S192 as primers, respectively.

The DNA fragment STE6 with the length of 3943bp is obtained by fusion PCR ^T1025N ，STE6 ^T1025N The nucleotide sequence is a sequence 4 in a sequence table, wherein the 1 st to 20 th sites of the sequence 4 are related to a promoter P _IRA1 Overlapping sequence, STE6 at positions 21 to 3893 ^T1025N 3894 to 3943 are sequences overlapping ADH 5-downlarm. The amino acid sequence of the encoded protein is shown as sequence 5 in the sequence table, wherein the base C at position 3074 of the STE6 encoding sequence is replaced by the base A, so that the amino acid at position 1025 of the encoded protein is changed from threonine T to asparagine N.

A screening marker expression cassette GMZ with the length of 2478bp is amplified by taking a plasmid YEp-GMZC as a template and S183 and S184 as primers, the nucleotide sequence is a sequence 6 in a sequence table, wherein the 1 st to 60 th sites of the sequence 6 are sequences overlapped with ADH5-uparm, the 61 st to 2450 th sites are screening marker GMZ sequences, and the 2451 st to 2478 th sites are sequences overlapped with ADH5-5 up.

ADH5-uparm, GMZ, ADH5-5up, P in equal molar number _IRA1 、STE6 ^T1025N And ADH 5-downearm as template, and performing fusion PCR with primer pair S181/S194 to obtain 8172bp DNA with screening markers GMZ and STE6 ^T1025N DNA fragments of the expression cassette and ADH5 homology arm (nucleotide sequence is shown as sequence 7 in the sequence table), wherein the 1 st to 500 th sites of the sequence 7 are ADH5-uparm, the 501 th to 2890 th sites are GMZ, the 2891 th to 3390 th sites are ADH5-5up, and the 3391 th to 3799 th sites are P _IRA1 STE6 at bits 3800-7672 ^T1025N Positions 7673 to 8172 are ADH 5-downloams.

Transferring the DNA fragment into a saccharomyces cerevisiae strain SQ3-4 by an electric transformation method, and obtaining the endogenous ADH5 gene in the genome of the strain SQ3-4 by forward screening of bleomycin resistance and reverse screening of galactose induction to replace with STE6 ^T1025N A recombinant strain of the expression cassette was designated SQ3-6.

(2) Expression of Ste6 ^T1025N Influence on the Synthesis of Saccharomyces cerevisiae beta-caryophyllene

The plasmid YEp-CPS was transformed into the strain SQ3-6 to obtain a recombinant strain SQ3-6-CPS. The yeasts SQ3-4-CPS and SQ3-6-CPS were cultured according to the method described in step 2.3 of example 2, and the content of beta-caryophyllene was determined. As shown in Table 4, the yield of beta-caryophyllene of the strain SQ3-6-CPS reaches 35.04mg/L, which is improved by 45.9% compared with the strain SQ 3-4-CPS.

2. Expression of beta-caryophyllene synthase E353D mutant for improving synthesis of saccharomyces cerevisiae beta-caryophyllene

And transforming the plasmid pYE-ECPS11 with the beta-caryophyllene synthase E353D mutant encoding gene expression cassette into a strain SQ3-6 to obtain a recombinant strain SQ3-6-ECPS11.

The yeasts SQ3-6-CPS and SQ3-6-ECPS11 were cultured as described in (1) of step 2.3 of example 2. As shown in Table 4, the yield of beta-caryophyllene of the strain SQ3-6-ECPS11 reaches 62.44mg/L, which is improved by 78.2% compared with the strain SQ3-6-CPS.

TABLE 4 comparison of beta-caryophyllene yields for different strains

Example 4 production of beta-Carophyllene by fermentation of Yeast

1. Seed liquid culture

(1) Activating strains: inoculating an inoculating loop strain SQ3-6-ECPS11 into 5mL YPD liquid culture medium, and shake culturing at 30 deg.C and 200rpm for 18h to obtain activated bacteria liquid.

(2) First-stage seed liquid: the activated strain was inoculated into 20mL YPD medium at an inoculum size of 2% (by volume), and shake-cultured at 30 ℃ and 200rpm for 18 hours to obtain a first-order seed strain.

(3) Secondary seed liquid: the primary seed solution was transferred to 200mL YPD medium at an inoculum size of 5% (by volume), and shake-cultured at 30 ℃ and 200rpm for 18 hours to obtain a secondary seed solution.

2. Fermentation culture

(1) Transferring the secondary seed liquid into a 5-L fermentation tank filled with 2000mL of fermentation medium according to the inoculation amount of 10% (volume ratio), wherein the sugar content of the medium is 4%, and the medium comprises the following components: 80g of carbon source (glucose, sucrose, cane molasses and corn starch hydrolysate), 30g of nitrogen source (ammonium sulfate and ammonia water), 20g of corn steep liquor, 12g of monopotassium phosphate, 6g of magnesium sulfate, 100mg of zinc sulfate, 10mg of copper sulfate and 2000mL of water. Controlling pH at 5.0-5.5 with fed-batch ammonia water, fermenting at 30 deg.C for 12h, and regulating aeration and stirring speed to maintain dissolved oxygen at above 40%.

(2) After 12h of culture, feeding and fermenting, wherein the feeding liquid comprises the following components: 500g/L glucose, 10g/L potassium dihydrogen phosphate, 5g/L magnesium phosphate, 1g/L zinc sulfate, 1g/L copper sulfate and the balance of water. The feeding rate is gradually increased from 15-30mL/h, so that the glucose content in the fermentation liquor is maintained at about 1 g/L. Controlling pH at 5.0-5.5 with fed-batch ammonia water, fermenting at 30 deg.C for 84h, and regulating aeration and stirring speed to maintain dissolved oxygen at above 20%.

(3) After 24h of culture, 20% (volume ratio) of dodecane was added, and the fermentation culture was continued under the conditions described in step (2).

3. Analysis of the product

Samples were taken at intervals during the fermentation process and the concentration of beta-caryophyllene in the fermentation broth was determined by GC-MS as described in step 2.3 of example 2. The strain SQ3-6-ECPS11 can produce 600-800mg/L beta-caryophyllene after being fermented for 96 h. The yield index has important industrial application potential.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims (10)

1. A protein, which is any one of the following:

a1 Protein of which the amino acid sequence is SEQ ID No. 2;

a2 A fusion protein having the same function obtained by attaching a tag to the N-terminus and/or C-terminus of A1).

2. A biomaterial related to the protein of claim 1, said biomaterial being any one of the following:

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette containing the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1);

b4 A recombinant vector containing the expression cassette of B2);

b5 A recombinant microorganism containing the nucleic acid molecule according to B1);

b6 A recombinant microorganism containing the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism containing the recombinant vector of B4).

3. The biomaterial of claim 2, wherein: the recombinant microorganism is recombinant yeast.

4. The biomaterial of claim 2, wherein: the recombinant yeast is recombinant saccharomyces cerevisiae.

5.The biomaterial of claim 4, wherein: the recombinant saccharomyces cerevisiae also contains ABC transporter mutant gene STE6 ^T1025N And does not contain a saccharomyces cerevisiae alcohol dehydrogenase gene ADH5.

6. The recombinant saccharomyces cerevisiae yeast of any of claims 4-5, wherein: the recombinant saccharomyces cerevisiae yeast is constructed according to the method of claim 7 or 8.

7. The method for constructing the recombinant saccharomyces cerevisiae is characterized by comprising the following steps: the method comprises knocking out the alcohol dehydrogenase gene ADH5 of claim 5, the gene encoding the protein of any one of claims 1 to 5 and the ABC transporter encoding gene STE6 from the recipient Saccharomyces cerevisiae ^T1025N The recipient saccharomyces cerevisiae is introduced.

8. The method of claim 7, wherein: the method also includes coupling P _IRA1 Introducing the promoter into the receptor Saccharomyces cerevisiae to make P _IRA1 The promoter drives transcription of the ABC transporter encoding gene.

9. A method for producing β -caryophyllene, comprising culturing the recombinant saccharomyces cerevisiae of any one of claims 4-6 to obtain a fermentation product, and obtaining β -caryophyllene from the fermentation product.

10. The use of the recombinant saccharomyces cerevisiae yeast of claim 7 or 8 or any of claims 4-6 in any of the following applications:

p1, application in producing beta-caryophyllene;

p2 and improving the yield of the beta-caryophyllene.

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