CN113604470A

CN113604470A - Recombinant yarrowia lipolytica T30pED for high yield of campesterol, construction method and application thereof

Info

Publication number: CN113604470A
Application number: CN202110938622.9A
Authority: CN
Inventors: 孟永宏; 苟元元; 郭建琦; 牛永洁; 杨璐
Original assignee: Xi'an Healthful Biotechnology Co ltd
Current assignee: Shaanxi Healthful Biological Engineering Co ltd
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-11-05
Anticipated expiration: 2041-08-16
Also published as: CN113604470B

Abstract

The invention relates to a recombinant yarrowia lipolytica T30pED for high yield of campesterol, a construction method and application thereof. The strong promoter TEF30 provided by the invention can be used for constructing a yarrowia lipolytica recombinant strain, and the maximum yield of campesterol of the obtained recombinant yarrowia lipolytica T30pED in shake flask fermentation reaches 664.1mg/L (33.21mg/g DCW), the maximum yield of the campesterol in a 5L fermentation tank reaches 1305.2mg/L (68.73mg/g DCW), and the yield is obviously higher than that of a wild promoter strain, so that the strong promoter TEF30 has prominent industrial application significance.

Description

Recombinant yarrowia lipolytica T30pED for high yield of campesterol, construction method and application thereof

[ technical field ] A method for producing a semiconductor device

The invention relates to recombinant Yarrowia lipolytica, in particular to a strong promoter TEF30 for improving the expression level of DHCR7 gene in Yarrowia lipolytica, a construction method of recombinant Yarrowia lipolytica T30pED containing the strong promoter and capable of producing campesterol in high yield, and application of the recombinant Yarrowia lipolytica.

[ background of the invention ]

The steroid medicine is a special polycyclic terpene compound widely existing in natural animals and plants, has various physiological activities and uses, is an important clinical medicine particularly in the medical field, and has remarkable anti-tumor, anti-inflammatory, anti-allergy, antibacterial and antiviral effects. At present, steroid drugs are produced mainly by chemical synthesis, plant extraction and biosynthesis. The chemical synthesis method has the advantages of complex production process, low product yield and serious environmental pollution, so that the method has no corresponding economic value. Most of the plant extraction methods obtain a mixture of phytosterol, and the phytosterol is difficult to separate into monomers with high purity and high yield for scientific research or production. Compared with animal and plant extraction and chemical synthesis methods, the biological synthesis method has more and more obvious advantages. The growth period of the microorganism is short, the growth and the propagation of the microorganism are not easily influenced by environmental factors, and the microorganism can be modified by means of genetic engineering to obtain a target product by fermenting the engineering strains.

Campesterol is an important synthetic precursor of steroid drugs and is also one of the main sterols of plant origin. The preparation of campesterol mostly adopts a plant extraction mode. The method has complicated operation process and low yield, and cannot be used for production. Campesterol differs from the main sterol of microbial origin (ergosterol) in that the C7-8 and C22-23 positions are saturated single bonds, and this similarity provides a theoretical basis for the feasibility of the microbiological process for the production of campesterol. In recent years, more and more researches are carried out to produce campesterol by a microbiological method, but the yield still does not reach the ideal level. Dogwooxin et al knocked out C-22 desaturase encoding gene ERG5 in Yarrowia lipolytica host, expressed gene DHCR7 encoded by 7-dehydrocholesterol reductase from Xenopus laevis, and obtained Campesterol with a yield of up to 106mg/L (Hao-Xing Du et al, engineering Yarrowia lipolytica for Campesterol over reduction [ J ]. PLoS ONE,2017,11 (1)). In Tanshina paper, yarrowia lipolytica is also taken as a host, the ERG5 gene is knocked out, and the DHCR7 gene from Xenopus laevis is freely expressed at the same time to obtain the highest yield of campesterol, which is 480ug/g DCW, and when the copy number of the DHCR7 gene is 2, the highest yield reaches 560ug/g DCW (Shinshina. research on the synthesis of campesterol by recombinant yarrowia lipolytica [ D ]. university of Shanxi Shimao, 2018.). Chinese patent application CN 107083338A discloses that when DHCR7 gene from zebra fish is expressed and ERG5 gene is knocked out, the yield of campesterol is 362.9mg/L (19.3mg/g DCW), and when acetyl CoA metabolism related gene POX2 is overexpressed on the basis, the yield of the campesterol obtained by fermentation reaches 942mg/L (36.5mg/g DCW). The prior art finds that the aim of high yield cannot be achieved only by means of expressing DHCR7 gene and knocking out ERG5 gene in the process of producing the campesterol by using yeast cells, and in addition, the yield of the campesterol is limited because the activity of a key enzyme, namely, 7-dehydrocholesterol reductase DHCR7, in the synthetic route is not high. Therefore, the synthesis process of campesterol needs to be continuously explored and innovated.

[ summary of the invention ]

The invention aims to overcome the defect of low yield of campesterol, the yield of the campesterol is improved by modifying an ergosterol synthesis way in yarrowia lipolytica, a promoter mutation library is constructed on the basis of the existing research of knocking out an ERG5 gene, a strong promoter is screened to realize the overexpression of a DHCR7 gene, the activity of the DHCR7 enzyme is improved, and the yield of the campesterol of yarrowia lipolytica is improved.

In order to achieve the above object, the present invention provides a strong promoter TEF30, the nucleic acid sequence of which is shown in SEQ ID No. 2.

The strong promoter TEF30 is obtained by screening and verifying a wild-type promoter TEF after mutation by an error-prone PCR technology, and the nucleic acid sequence of the wild-type promoter TEF is shown as SEQ ID No. 1.

The invention also provides application of the strong promoter TEF30 in improving the expression level of DHCR7 gene in Yarrowia lipolytica.

The invention also provides application of the strong promoter TEF30 in improving the yield of campesterol of Yarrowia lipolytica.

Based on the above, the invention provides a construction method of recombinant yarrowia lipolytica T30pED for high yield of campesterol, which comprises the following steps:

(1) construction of EGFP Gene expression vector pJN44-EGFP

An EGFP sequence is connected to a general plasmid pUC57 after being synthesized, an EGFP fragment is obtained by amplification through a primer EGFP-F-Hind III/EGFP-R-Sal I with an enzyme cutting site, a synthesized plasmid pUC57 is used as a template, HindIII/Sal I is respectively used as an enzyme cutting site for double enzyme cutting of a pJN44 vector and the EGFP fragment are connected to obtain a recombinant plasmid pJN44-EGFP, and the obtained recombinant plasmid pJN44-EGFP is correctly identified through the primer EGFP-F-Hind III/EGFP-R-Sal I;

(2) construction of recombinant yarrowia lipolytica polf-ERG5 containing EGFP gene expression vector pJN44-EGFP^--EGFP⁺

The recombinant plasmid pJN44-EGFP in the step (1) is linearized by restriction enzyme SmaI, and is transferred into yarrowia lipolytica engineering bacteria polf-ERG5 by a yeast transformation kit method^-Obtaining the recombinant yarrowia lipolytica polf-ERG5^--EGFP⁺The obtained recombinant yarrowia lipolytica polf-ERG5^--EGFP⁺The PCR identification is correct;

(3) construction of EGFP gene recombinant plasmid T30pJN44-EGFP containing strong promoter TEF30

The strong promoter TEF30 of claim 1 is subjected to double digestion by restriction enzymes BamHI/HindIII and then is connected with an expression vector pJN44-EGFP subjected to double digestion by BamHI and HindIII to obtain an EGFP gene recombinant plasmid T30pJN44-EGFP containing the strong promoter TEF 30;

(4) construction of recombinant yarrowia lipolytica TpEE containing recombinant plasmid T30pJN44-EGFP

The recombinant plasmid T30pJN44-EGFP in the step (3) is linearized by restriction enzyme Sma I, and is transferred into lipolytic subunit by a yeast transformation kit methodEngineering bacteria of rhodotorula rolfsii polf-ERG5^-The obtained recombinant yarrowia lipolytica TpEE is correctly identified by PCR;

(5) construction of lacZ Gene recombinant plasmid T30pJN44-lacZ containing Strong promoter TEF30

The lacZ gene is subjected to double enzyme digestion by restriction endonuclease Hind III/SalI, and then is connected with an expression vector T30pJN44-EGFP subjected to double enzyme digestion by Hind III/SalI to obtain lacZ gene recombinant plasmid T30pJN44-lacZ containing a strong promoter TEF 30;

(6) construction of recombinant yarrowia lipolytica TpEZ containing recombinant plasmid T30pJN44-lacZ

The recombinant plasmid T30pJN44-lacZ obtained in the step (5) is linearized by restriction enzyme Sma I, and is transferred into yarrowia lipolytica engineering bacterium polf-ERG5 by a yeast transformation kit method^-The obtained recombinant yarrowia lipolytica TpEZ is correctly identified by PCR;

(7) construction of DHCR7 Gene recombinant plasmid T30pJN44-DHCR7 containing Strong promoter TEF30

Connecting the DHCR7 fragment subjected to double enzyme digestion by Xma I and SalI with the recombinant plasmid T30pJN44-lacZ obtained in the step (5) to obtain a recombinant plasmid T30pJN44-DHCR7 containing a strong promoter TEF 30;

(8) construction of recombinant Strain T30pED containing recombinant plasmid T30pJN44-DHCR7

Transferring the recombinant plasmid T30pJN44-DHCR7 obtained in the step (7) into yarrowia lipolytica engineering bacteria polf-ERG5 by a yeast transformation kit method^-In the method, the recombinant yarrowia lipolytica T30pED is obtained, and the obtained recombinant yarrowia lipolytica T30pED is correctly identified by PCR.

In the invention, the DHCR7 gene is from zebra fish, and the sequence number is GenBank: BC 055631.1; the EGFP sequence of the gene comes from a cloning vector p15A-HNS-GFP, and the sequence number is GenBank: MN623123.1 (part gFP); the lacZ sequence is derived from Escherichia coli BL21, and the sequence number is GenBank: CP 035822.1.

In the invention, yarrowia lipolytica engineering bacteria polf-ERG5^-Reference can be made to the technical scheme disclosed in research on the synthesis of campesterol by recombinant yarrowia lipolytica (Tan Tu Miyuan); pJN44 the vector can be found in "research on the synthesis of microbial oil by recombinant yarrowia lipolytica YeastRecord in Dougui Ru.

The invention also provides a method for producing campesterol by the recombinant yarrowia lipolytica T30pED obtained by the construction method, which is characterized in that after the recombinant yarrowia lipolytica T30pED strain is activated at 30 ℃ and 200r/min, a single colony is selected to be inoculated into a small bottle liquid YPD seed culture medium of 10mL/50mL, and shake-flask culture is carried out for 24 hours at 30 ℃ and 200 r/min; then transferring the cells into 50mL/250mL liquid YPD culture medium with the inoculation amount of 2%, fermenting for 48h under the conditions of 30 ℃ and 200r/min, collecting the cells, transferring the cells into 50mL/250mL liquid YPD culture medium, and further fermenting for 96h under the conditions of 30 ℃ and 200 r/min; collecting cells, and extracting campesterol.

The invention also provides a method for producing the campesterol by the recombinant yarrowia lipolytica T30pED obtained by the construction method, which is characterized in that the recombinant yarrowia lipolytica T30pED is subjected to high cell density fermentation in a 5L fermentation tank, the sugar content in the fermentation tank is maintained to be 30g/L, thalli are collected after fermentation is carried out for 6 days, bacterial liquid is treated, and the yield of the campesterol in the samples is determined;

the medium composition in the fermentor was: glucose 4%, (NH)₄)₂SO₄ 1.51％，YNB0.85％，KH₂PO₄1.25％，MgSO₄·7H₂0.25% of O, 1% of uracil and 0.2% of yeast powder;

the fermentation conditions were: the culture temperature is maintained at 28 ℃, the initial rotating speed is set to be 200r/min, when the thalli grow into the logarithmic phase, the rotating speed is set to be 400r/min, the rotating speed and the dissolved oxygen amount are connected after the thalli grow into the stationary phase, the rotating speed is set to be 800r/min, the dissolved oxygen amount is maintained to be 20%, and the pH value is maintained to be 5.5 by using 10mol/L KOH solution until the fermentation is finished.

According to the invention, a TEF promoter mutation library is constructed, a strong promoter TEF30 is screened, a yarrowia lipolytica recombinant strain is constructed, and the activity of 7-dehydrocholesterol reductase is improved, so that the highest yield of campesterol of the recombinant strain in shake flask fermentation reaches 664.1mg/L (33.21mg/g DCW), the highest yield of the campesterol in a 5L fermentation tank reaches 1305.2mg/L (68.73mg/g DCW), the yield of the wild type promoter strain is improved by 2.34 times on the basis of 372.5mg/L (20.6mg/g DCW), and the yield is obviously superior to the highest yield 942mg/L (36.5mg/g DCW) in the prior art.

[ description of the drawings ]

FIG. 1 shows the result of PCR identification of recombinant yarrowia lipolytica engineering bacteria containing mutant promoter;

FIG. 2 shows the result of PCR identification of a recombinant strain containing a second reporter gene lacZ;

FIG. 3 shows the result of PCR identification of recombinant strain expressing DHCR7 gene;

FIG. 4 is a comparison of relative fluorescence intensity of mutant promoter and beta-galactosidase activity.

[ detailed description ] embodiments

The following examples serve to illustrate the technical solution of the present invention without limiting it.

In the present invention, "%" used for specifying concentrations is, unless otherwise specified, "%" used for specifying the ratio of amounts ": all the terms "are mass ratios.

YPD medium: 2% of glucose, 1% of yeast powder, 2% of peptone and 1.7% of agar powder, and sterilizing the mixture for 20min by high-pressure steam at 121 ℃.

YPD liquid medium: 2% of glucose, 1% of yeast powder and 2% of peptone, and sterilizing for 20min by high-pressure steam at 121 ℃.

Culture medium in fermenter: glucose 4%, (NH)₄)₂SO₄ 1.51％，YNB 0.85％，KH₂PO₄ 1.25％，MgSO₄·7H₂0.25% of O, 1% of uracil and 0.2% of yeast powder.

The invention relates to the following primers:

TABLE 1 primer information

Example 1 construction and screening of TEF promoter mutation library

1. Construction of first reporter gene EGFP recombinant strain and detection of relative fluorescence intensity

1.1 construction of first reporter Gene EGFP expression vector

Downloading an EGFP sequence (GenBank: MN623123.1) from NCBI, carrying out biological synthesis, connecting the EGFP sequence to a universal plasmid pUC57, designing a primer EGFP-F-Hind III/EGFP-R-SalI with a restriction enzyme site, amplifying to obtain an EGFP fragment by taking a synthetic plasmid pUC57 as a template, and respectively taking Hind III/SalI as the restriction enzyme site, carrying out double restriction enzyme pJN44 on the vector and the EGFP fragment, and connecting to obtain a recombinant plasmid pJN 44-EGFP. The recombinant plasmid is identified by using a primer EGFP-F-Hind III/EGFP-R-SalI, the size of the target segment EGFP is 732bp, and is consistent with that of the synthetic segment, which shows that the recombinant plasmid is successfully constructed and can be used for transforming engineering bacteria polf-ERG5 of yarrowia lipolytica^-(also written as recombinant yarrowia lipolytica pE).

1.2 construction and verification of the first reporter Gene recombinant Strain pEE

The constructed recombinant plasmid pJN44-EGFP is linearized by restriction enzyme SmaI, and is transferred into yarrowia lipolytica engineering bacteria polf-ERG5 by a yeast transformation kit method^-Constructing recombinant bacteria polf-ERG5^--EGFP⁺(also written as recombinant yarrowia lipolytica pEE). In order to detect whether the fragment is integrated on the host chromosome after gene linearization, a plurality of pEE single colonies are respectively picked as templates to carry out colony PCR.

Yeast Transformation the Yeast Transformation Kit from Epigentics (Frozen-EZ Yeast Transformation II Kit from Epigenetics) was used, and the procedure was as follows:

(1) activating the host bacteria to be transformed on YPD medium, and culturing at 28 deg.C for 1 day;

(2) picking single colony to inoculate in 2mL YPD liquid medium, culturing cell growth to logarithmic phase (OD is 0.8-1.0) at 28 ℃;

(3) centrifuging at 4000 Xg for 4min to collect thallus, and discarding supernatant;

(4) adding 500uL solution I to wash and clear the cells, centrifuging for 2min, and removing the supernatant;

(5) adding 50uL of solution II for resuspending the thalli, adding 5 mu L of plasmid recovered after linearization, and mixing uniformly;

(6) adding 500uL solution III, and performing vortex oscillation, culturing in a shaking table at 28 ℃ and 160r/min for 4h, and performing vortex oscillation for several seconds every other hour;

(7) and (3) coating a proper amount of bacterial liquid on an SD-LEU culture medium, culturing for 2-4 days in an incubator at 28 ℃, and obtaining a single bacterial colony to be subjected to PCR verification.

The colony PCR process was as follows:

and (3) PCR reaction system:

PCR procedure:

1.3 detection of relative fluorescence intensity of the first reporter recombinant Strain pEE

After YPD culture of the recombinant strain pEE and the original strain pE, collecting thalli, washing once with deionized water, resuspending with PBS, sampling 250uL to a 96-well plate, detecting EGFP fluorescence intensity (excitation wavelength: 488nm, emission wavelength: 505nm) by using a multifunctional microplate reader, and detecting OD₆₀₀When the fluorescence intensity of EGFP is detected, the original strain pE not expressing EGFP gene is used as a control to remove background interference. Specific fluorescence intensity F/OD₆₀₀(RFU/OD₆₀₀) Is the intensity value of the fluorescence intensity compared to the corresponding cell density OD₆₀₀. And calculating the relative fluorescence intensity of the strain according to the measurement result, and detecting whether the TEF promoter can be successfully expressed in the yarrowia lipolytica engineering bacterium pE and whether the EGFP gene can be used as a reporter gene for constructing a TEF promoter library by using the relative fluorescence intensity of the strain.

Mutation of the TEF promoter

Plasmid pJN44 is used as a template, TEF-F-BamHI/TEF-R-Hind III is used as a primer, the TEF promoter is mutated by using an error-prone PCR technology to obtain a TEF promoter mutation fragment TEFp, and the mutation TEF promoter mixed fragment is connected with a vector pJN44-EGFP which is subjected to double enzyme digestion by BamHI and Hind III through restriction enzymes BamHI/Hind III to obtain a recombinant mixed plasmid. The recombinant mixed plasmid is linearized by restriction enzyme Sma I, and a yeast transformation kit method is adoptedTransferred into yarrowia lipolytica engineering bacterium polf-ERG5^-And when single colonies grow out, randomly selecting 30 single colonies to carry out colony PCR to identify transformants, wherein the theoretical size of a PCR band of the positive clone is about 732 bp.

Error-prone PCR reaction system

Error-prone PCR reaction procedure

As shown in FIG. 1, the result shows that the recombinant yarrowia lipolytica engineering bacterium TpEE containing the mutant promoter is successfully constructed.

Construction and screening of TEF promoter mutation library

3.1 first screening of TEF promoter mutant library strains

And detecting the fluorescence intensity (500) of all the recombinant strain TpEE transformants by using a multifunctional microplate reader, comparing with the wild promoter recombinant strain pEE, removing the transformants with unchanged fluorescence intensity, and only keeping 150 transformants with obviously changed fluorescence intensity. Then selecting 1 transformant from the transformants with the same fluorescence intensity to construct a TEF promoter mutation library, displaying the variation range and trend of the promoter intensity, selecting 30 transformants in total to construct the TEF promoter mutation library, and performing fluorescence intensity and OD (optical Density)₆₀₀Detection and relative fluorescence intensity calculation.

In the TEF promoter mutation library, the activity of the TEF mutant No.1 is the lowest, which is 10% of the wild-type TEF strength; the activity of the TEF mutant No. 30 was the highest, 160% of the wild-type TEF intensity. And respectively selecting mutations No. 5,7,12,18,22,27,29 and 30 from the TEF promoter mutation library for subsequent experimental analysis. The intensities of the mutant TEF promoters with the numbers of 5,7,12 and 18 are lower than that of the wild-type TEF promoter, and are respectively 64RFU,115RFU,133RFU and 165RFU, which are respectively reduced by 72.17%, 50.00%, 42.17% and 28.26% compared with the wild-type TEF promoter. The mutant TEF promoters with numbers 22,27,29 and 30 are all stronger than the wild type TEF promoter, 287RFU,345RFU,398RFU and 421RFU respectively, and are respectively improved by 1.25 times, 1.50 times, 1.73 times and 1.83 times compared with the wild type TEF promoter, as shown in FIG. 1.

3.2 second screening for TEF mutant promoter Strength

In order to ensure the accuracy of detecting the strength of the TEF promoter, the strength of the mutant No. 5,7,12,18,22,27,29 and 30 relative to the wild TEF promoter is further determined by using lacZ gene in Escherichia coli BL21 as a second reporter gene. The lacZ gene is a common reporter gene for coding beta galactosidase, the lacZ gene is expressed by utilizing mutation seeds with numbers of 5,7,12,18,22,27,29 and 30 to construct a recombinant strain, and the enzyme activity of the beta galactosidase of the recombinant strain is detected, so that the strength of the mutation seeds can be determined again.

3.2.1 construction of second reporter Gene lacZ expression vector

Taking Escherichia coli BL21 genome as a template and lacZ-F-Hind III/lacZ-R-Sal I as a primer, amplifying lacZ gene of coded beta galactosidase to obtain lacZ gene fragment with the length of 3087bp, and respectively taking Hind III/Sal I as enzyme cutting sites to carry out double enzyme cutting pJN44 and lacZ fragment and connecting to obtain recombinant plasmid pJN 44-lacZ. The recombinant plasmid is identified by using the primers lacZ-F-Hind III/lacZ-R-Sal I, and the size of the target fragment lacZ is consistent with that of the synthesized fragment, so that the successful construction of the recombinant plasmid pJN44-lacZ is shown, and the recombinant plasmid can be used for transforming the yarrowia lipolytica engineering bacterium pE.

The 9 mutant strain genomes screened above are taken as templates, primers TEF-F-BamH I and TEF-R-Hind III containing enzyme cutting sites are used for amplifying mutant promoters TEF5, TEF7, TEF12, … and TEF30, after BamH I and Hind III are respectively cut by enzyme and are connected with recombinant plasmids pJN44-lacZ cut by BamH I and Hind III, recombinant plasmids T5pJN44-lacZ, T7pJN44-lacZ, … and T30pJN44-lacZ containing the mutant promoters are obtained, the recombinant plasmids are identified by PCR, the sizes of the fragments are consistent, the construction success of the recombinant plasmids is shown, and the recombinant plasmids can be used for transforming engineering bacteria pE of yarrowia lipolytica.

3.2.2 construction and validation of the second reporter Gene lacZ recombinant Strain

The constructed recombinant plasmid pJN44-lacZ, T5pJN44-lacZ, T7pJN44-lacZ, … and T30pJN44-lacZ are respectively linearized by restriction enzyme SmaI, and transformed into yarrowia lipolytica engineering bacteria polf-ERG5 by a yeast transformation kit method^-And constructing recombinant bacteria pEZ, T5pEZ, T7pEZ, … and T30 pEZ. A plurality of single colonies were picked up and subjected to colony PCR identification, and the results are shown in FIG. 2. .

3.2.3 beta-galactosidase enzyme Activity assay

The beta-galactosidase enzyme activities of the recombinant strain pEZ and the mutant promoter-containing recombinant strains T5pEZ, T7pEZ, …, T30pEZ were examined as follows. Comparing the activity of beta-galactosidase of each recombinant strain, and verifying whether the activity is consistent with the trend of fluorescence intensity:

(1) accurately subpackaging 5mL YPD medium in a test tube, inoculating yeast strain for one ring, and performing shake culture in a shaking table at 30 ℃ for 12h at 180 r/min;

(2) completely pouring 5mL of the bacterial liquid into a 150mL conical flask which is subpackaged with 45mL of YPD medium, and carrying out shake culture in a shaking table at 30 ℃ for 4 h;

(3) subpackaging the bacteria liquid into centrifuge tube, cleaning thallus with sterile deionized water for 2 times, and detecting OD of bacteria liquid to be detected₆₀₀A value;

(4) 100uL of bacterial liquid, 900uL of Z-Buffer,100uL of chloroform and 50uL of 0.1% SDS solution are sequentially added into a 2mL centrifuge tube, and are subjected to high-speed vortex oscillation lOs by a vortex oscillator and then are subjected to oscillation at the temperature of 30 ℃ for 2min at 170 r/min. Adding 200uL ONPG solution into a centrifuge tube, shaking at 30 deg.C for 20min at 170r/min (the solution becomes yellow), adding 300uL 1M Na₂CO₃Terminating the reaction by the solution;

(5) centrifuging the tube at 12000r/min for 2min, and determining OD of supernatant₅₅₀And the absorbance of the OD 420.

The enzyme activity calculation formula is as follows: the enzyme activity is 1000 × [ A ]₄₂₀-(1.75-A₅₅₀)]/t×0.1×A₆₀₀

The results show that the beta-galactosidase enzyme activities of the recombinant strains T5pEZ, T7pEZ, T12pEZ and T18pEZ constructed by the mutations No. 5,7,12 and 18 with relative fluorescence intensity lower than that of the wild-type TEF promoter are also lower than that of the recombinant strain pEZ of the wild-type TEF promoter, namely 25.10%, 39.92%, 65.02% and 78.19% of pEZ, respectively, and the beta-galactosidase enzyme activities of the recombinant strains T22pEZ, T27pEZ, T29pEZ and T30pEZ constructed by the mutations No. 22,27,29 and 30 with relative fluorescence intensity higher than that of the wild-type TEF promoter are respectively improved by 25.10%, 44.86%, 67.49% and 76.13%, and the results are consistent with the trend of the relative fluorescence intensity of the first reporter gene EGFP. The recombinant strain T30pJN44 has the highest beta-galactosidase activity and relative fluorescence intensity, so that the No. 30 mutant promoter is regarded as a strong promoter for subsequent research.

Example 2: the construction of the yarrowia lipolytica recombinant strain for improving the yield of campesterol comprises the following steps: construction of recombinant strain expressing DHCR7 Gene

The target gene DHCR7 for expression is from zebra fish, the obtained sequence is subjected to codon optimization by Nanjing golden fish, as shown in SEQ No.3, a rare codon with low utilization rate is replaced by a preferred codon in a host, the secondary structure of mRNA after gene transcription is simplified, a motif which is not beneficial to high-efficiency expression is removed, the motif which is beneficial to expression is added, the GC content is adjusted, the target gene can be efficiently expressed in the host, the DHCR7 fragment which is subjected to double enzyme digestion by Xma I and SalI is respectively connected with pJN44 and T30pJN44 fragments to obtain pJN44-lacZ and T30pJN44-lacZ recombinant plasmids, the recombinant plasmids are respectively transferred into engineering bacteria pE of yarrowia lipolytica by a method of a yeast transformation kit after PCR identification, recombinant strains pED and T30pED are obtained, a plurality of single colonies are selected for colony PCR identification, and the result is shown in figure 3.

Example 3: fermentation experiments with recombinant strains

1. Recombinant strain shake flask fermentation

After activating recombinant strains pED and T30pED which express DHCR7 gene respectively, picking single colony to insert into 10mL/50mL small bottle liquid YPD seed culture medium, and culturing for 24h under the condition of 30 ℃ and 200 r/min. Then, the cells were inoculated at 2% into 50mL/250mL of liquid YPD medium, fermented at 30 ℃ and 200r/min for 48 hours, and then the collected cells were transferred to 50mL/250mL of liquid YPD medium and further fermented for 96 hours. Collecting cells, and extracting and detecting campesterol.

The campesterol extraction method comprises the following steps:

(1) placing 1mL of fermentation liquid in an anaerobic tube, and adding 100uL of cholestanol standard (diluted by 100 times with 4mg/mL of stock solution);

(2) adding 2.5mL of potassium hydroxide methanol solution of 3mol/L, saponifying at 90 ℃, taking out the anaerobic tube every 20min, shaking for several seconds, and taking out the cooling test tube after 100 min;

(3) after the test tube is cooled, adding 2mL of distilled water and 2mL of normal hexane, vibrating the test tube on a vortex oscillator for 5min, and standing for layering;

(4) after the reaction liquid is layered, taking the n-hexane layer containing the sample, putting the n-hexane layer into a derivatization bottle, adding 100uL of derivatization reagent (99uL BSTFA +1uL TMCS), reacting at 70 ℃ for 60min, cooling, and storing at 4 ℃ until the gas mass analysis.

The campesterol detection method comprises the following steps:

(1) a chromatographic column: rtx-5MS (30 m.times.0.25. mu. m.times.0.25. mu.m)

(2) Chromatographic conditions are as follows: the sample inlet temperature is 290 ℃, the initial temperature of the column box is 100 ℃, the split ratio is 10:1, the sample injection amount is 1uL, the carrier gas is high-purity helium gas, and the flow rate is 1.2 mL/min; temperature programming: the initial temperature was 100 ℃ and the temperature was raised to 280 ℃ at a rate of 30 ℃/min for 15 min.

(3) Mass spectrum conditions: the ion source is an EI source, the ionization energy is 70eV, the temperature of the ion source is 230 ℃, the interface temperature is 250 ℃, data are collected in an SCAN mode when sterol mixed standard substances are analyzed, after characteristic ions are selected, the data collection mode is an SIM mode, wherein the characteristic ions of cholestanol, ergosterol and campesterol are respectively selected to be m/z 75, m/z 337 and m/z 129.

2. Fermenting in a recombinant strain fermentation tank

The recombinant strains pED and T30pED are subjected to scale-up culture in a 5L fermentation tank by adopting a high cell density fermentation technology, and the culture medium is a fermentation culture medium and is added with 2g/L of yeast powder, 1.25 percent of KH2PO4 and 0.25 percent of MgSO4 & 7H 2O. The initial fermentation conditions are set as follows according to literature reports and the results of early-stage shake flask fermentation experiments: the culture temperature is maintained at 28 ℃, the initial rotating speed is set to be 200r/min, when the thalli grow into the logarithmic growth phase, the rotating speed is set to be 400r/min, the rotating speed and the dissolved oxygen amount are connected after the thalli grow into the stationary phase, the rotating speed is set to be 200-800r/min, the dissolved oxygen amount is maintained at 20%, and the pH value is maintained at about 5.5 by using 10mol/L KOH solution. At the initial stage of fermentation, the sugar content in the fermentation tank is maintained at 30g/L, the sugar content in the fermentation tank is continuously monitored during the fermentation process, and the glucose amount in the fermentation tank is maintained by adopting a glucose fed-batch technology. During the culture process, samples are taken every 8h and stored at-80 ℃ to be measured. The cells were monitored for growth by taking samples at 4h intervals to determine OD600 values. After fermenting for 6 days under the condition, collecting thalli, treating bacterial liquid, and measuring the yield of the campesterol in the sampled samples.

The yield of campesterol by GC-MS is shown in Table 1: the shake flask fermentation yield of the strain T30pED containing the strong promoter reaches 664.1.1mg/L (33.21mg/g DCW), the maximum fermentation yield of a 5L fermentation tank reaches 1305.8mg/L (68.73mg/g DCW), the yield is improved by 2.34 times on the basis of a wild promoter strain (pED strain), and the yield of campesterol is greatly improved.

TABLE 1 comparison of campesterol production by recombinant strains pED and T30pED fermentation experiments

In conclusion, the screened strong promoter TEF30 is used for constructing the yarrowia lipolytica recombinant strain, the highest yield of the obtained recombinant yarrowia lipolytica T30pED in shake flask fermentation reaches 664.1mg/L (33.21mg/g DCW), the highest yield of the campesterol in a 5L fermentation tank reaches 1305.2mg/L (68.73mg/g DCW), the yield of the wild-type promoter strain is improved by 2.34 times on the basis of 372.5mg/L (20.6mg/g DCW), the yield is obviously superior to the highest yield recorded in the prior art, and the method has outstanding industrial application significance.

Sequence listing

<110> Seawa biosciences, Inc

<120> recombinant yarrowia lipolytica T30pED for high yield of campesterol, construction method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 206

<212> DNA

<213> yarrowia lipolytica promoter TEF (yarrowia lipolytica)

<400> 1

tagtttcttt gtctggccat ccgggtaacc catgccggac gcaaaataga ctactgaaaa 60

tttttttgct ttgtggttgg gactttagcc aagggtataa aagaccaccg tccccgaatt 120

acctttcctc ttcttttctc tctctccttg tcaactcaca cccgaaatcg ttaagcattt 180

ccttctgagt ataagaatca ttcaaa 206

<210> 2

<211> 206

<212> DNA

<213> Yarrowia lipolytica Strong promoter TEF30(Yarrowia lipolytica)

<400> 2

tagtttcttt gtctggccat ccgggtaacc catgccgtac gaacgataga ctactgaaaa 60

tttttttgct ttgtggttgg gactttagcc aagggtataa aagaccaccg tccccgaatt 120

acctttcctc ttcttttctc tctctccttg tcaactcaca cccgaaatcg ttaagcattt 180

ccttctgagt ataagaatca ttcaaa 206

<210> 3

<211> 1437

<212> DNA

<213> optimized zebrafish DHCR7(Unknown)

<400> 3

atgatggcct ctgaccgagt gcgaaagcga cacaagggat ctgctaacgg agcccagacc 60

gtcgagaagg agccctctaa ggagcccgcc cagtggggcc gagcctggga ggtggactgg 120

ttctctctgt ctggagtgat cctgctgctg tgtttcgccc ccttcctggt ctccttcttc 180

atcatggcct gcgaccagta ccagtgttct atttctcacc ccctgctgga cctgtacaac 240

ggagatgcca ccctgttcac catctggaac cgagccccct ctttcacctg ggccgccgcc 300

aagatctacg ccatctgggt gaccttccag gtggtgctgt acatgtgcgt gcccgacttc 360

ctgcacaaga tcctccccgg ctacgtggga ggagtgcagg acggcgcccg aacccccgcc 420

ggactgatca acaagtacga ggtgaacggc ctccagtgct ggctgatcac ccacgtcctg 480

tgggtgctga acgcccagca cttccactgg ttctctccca ctatcatcat cgacaactgg 540

atccccctcc tgtggtgcac caacatcctg ggctacgccg tgtctacctt cgctttcatt 600

aaggcctacc tgttccccac taaccccgag gactgtaagt tcaccggcaa catgttctac 660

aactacatga tgggcatcga gttcaacccc cgaatcggca agtggttcga cttcaagctg 720

ttcttcaacg gacgacccgg catcgtggcc tggaccctga ttaacctgtc ctacgctgcc 780

aagcagcagg agctgtacgg atacgtgacc aactccatga tcctggtgaa cgtcctgcag 840

gccgtgtacg tggtggactt cttctggaac gaggcctggt acctgaaaac cattgacatt 900

tgtcacgacc acttcggatg gtacctgggc tggggcgact gcgtgtggct gcccttcctg 960

tacaccctcc agggcctgta cctggtgtac aaccccatcc agctgtccac cccccacgcc 1020

gccggcgtgc tgatcctggg cctggtgggc tactacatct tccgagtgac caaccaccag 1080

aaggacctgt tccgacgaac cgagggcaac tgttccatct ggggcaagaa gcccaccttc 1140

attgagtgtt cttaccagtc tgccgacggc gccatccaca agtccaagct gatgacctct 1200

ggcttctggg gcgtggcccg acacatgaac tacaccggag acctgatggg atctctggcc 1260

tactgcctgg cctgcggcgg caaccacctg ctcccctact tctacatcat ctacatgacc 1320

atcctgctcg tgcaccgatg catccgggac gagcaccgat gctctaacaa gtacggcaag 1380

gactgggagc gatacaccgc cgccgtgtct taccgactgc tgcccaacat cttctaa 1437

Claims

1. The strong promoter TEF30 has the nucleic acid sequence shown in SEQ ID No. 2.

2. Use of the strong promoter TEF30 in claim 1 for increasing the expression level of DHCR7 gene in Yarrowia lipolytica (Yarrowia lipolytica).

3. Use of the strong promoter TEF30 according to claim 1 for increasing the production of campesterol from Yarrowia lipolytica.

4. The construction method of the recombinant yarrowia lipolytica T30pED for high yield of campesterol comprises the following steps:

(1) construction of EGFP Gene expression vector pJN44-EGFP

The recombinant plasmid T30pJN44-EGFP in the step (3) is linearized by restriction enzyme Sma I, and is transferred into yarrowia lipolytica engineering bacterium polf-ERG5 by a yeast transformation kit method^-The obtained recombinant yarrowia lipolytica TpEE is correctly identified by PCR;

The recombinant plasmid T30pJN44-lacZ obtained in the step (5) is linearized by restriction enzyme SmaI, and is transferred into yarrowia lipolytica engineering bacterium polf-ERG5 by a yeast transformation kit method^-The obtained recombinant yarrowia lipolytica TpEZ is correctly identified by PCR;

5. Construction process according to claim 3, characterized in that the DHCR7 gene is derived from zebrafish.

6. The method for producing campesterol by recombinant yarrowia lipolytica T30pED obtained by the construction method of claim 4, characterized in that after the recombinant yarrowia lipolytica T30pED strain is activated at 30 ℃ and 200r/min, single colony is selected and inoculated into 10mL/50mL vial liquid YPD seed culture medium, and shake-flask culture is carried out for 24h at 30 ℃ and 200 r/min; then transferring the cells into 50mL/250mL liquid YPD culture medium with the inoculation amount of 2%, fermenting for 48h under the conditions of 30 ℃ and 200r/min, collecting the cells, transferring the cells into 50mL/250mL liquid YPD culture medium, and further fermenting for 96h under the conditions of 30 ℃ and 200 r/min; collecting cells, and extracting campesterol.

7. The method for producing campesterol by recombinant yarrowia lipolytica T30pED obtained by the construction method of claim 4, characterized in that the recombinant yarrowia lipolytica T30pED is fermented in a 5L fermentation tank at high cell density, the sugar content in the fermentation tank is maintained at 30g/L, after 6 days of fermentation, the thalli are collected, the bacterial liquid is treated, and the yield of the campesterol in the samples is determined;

the culture medium in the fermentation tank comprises the following components in percentage by weight: glucose 4%, (NH)₄)₂SO₄ 1.51％，YNB 0.85％，KH₂PO₄ 1.25％，MgSO₄·7H₂0.25% of O, 1% of uracil and 0.2% of yeast powder;