阅读说明：本技术 一种来自丝状真菌粗糙脉胞菌的新型诱导型启动子及其应用 (Novel inducible promoter from filamentous fungus neurospora crassa and application thereof ) 是由 刘晓光 张会霞 路福平 于 2020-05-19 设计创作，主要内容包括：本发明针对目前工业上甾体生物转化羟化反应投料量偏低的问题,提供一种诱导型启动子,其核苷酸序列如SEQ ID NO：1所示。该启动子能够用于构建粗糙脉孢工程菌,为构建高效催化甾体化合物羟化反应的基因工程菌株奠定坚实的基础,对于提高甾体化合物的生物转化效率具有重要的意义。(Aiming at the problem of low feeding amount of steroid biotransformation hydroxylation reaction in the current industry, the invention provides an inducible promoter, the nucleotide sequence of which is shown as SEQ ID NO: 1 is shown. The promoter can be used for constructing rough vein spore engineering bacteria, lays a solid foundation for constructing a genetic engineering strain for efficiently catalyzing hydroxylation reaction of steroid compounds, and has important significance for improving biotransformation efficiency of steroid compounds.)

1. A strictly inducible promoter, wherein the nucleotide sequence of said promoter is as set forth in SEQ ID NO: 1, or a sequence similar to SEQ ID NO: 1 and has a promoter function.

2. A gene expression cassette, recombinant vector, expression vector and host cell comprising the inducible promoter of claim 1.

3. The recombinant vector or expression vector of claim 2, wherein the recombinant vector is pYES2-CYP-7 and the expression vector is pMF 272-EGFP.

4. The host cell of claim 2, wherein the host cell is a filamentous fungus.

5. The host cell of claim 4, wherein the filamentous fungus is Neurospora crassa.

6. A primer set for amplifying the full length of the inducible promoter DNA of claim 1, wherein the primer set has the following sequence, 5'-GCCCCCCTCATGTCTAGC-3', 5'-CGGCGCGAAGTAGCGCTG-3'; or a sequence in which a number of bases are successively reduced at the 3' -end of at least one of the primers.

7. Use of an inducible promoter as claimed in claim 1 wherein the strictly inducible promoter is used for inducible expression of a steroid hydroxylase gene.

8. Use of a strictly inducible promoter according to claim 7 for the inducible expression of a steroid hydroxylase gene in a filamentous fungus.

9. Use of a strictly inducible promoter as claimed in claim 7 wherein the hydroxylase gene comprises 11 β -hydroxylase, 15 α -hydroxylase, 11 α -hydroxylase, 16 α -hydroxylase, 7 α -hydroxylase, 14 α -hydroxylase or 9 α -hydroxylase gene.

10. The use of the strictly inducible promoter according to claim 7, wherein the strictly inducible promoter is used for constructing a genetically engineered recombinant bacterium for inducing and expressing hydroxylase.

Background

Steroid hormone drugs are the second main class of drugs which are second only to antibiotics at present, have extremely important medical value in clinic, and have good effects on life maintenance, physiological function regulation, immunologic function, body growth, dermatosis treatment, fertility control and the like. Chemical synthesis still plays a significant role in research and production processes of steroid compounds, and in the production of steroid hormone drugs, microbial transformation technology has become an indispensable key technology in the synthetic routes of a plurality of steroid compounds or intermediates thereof. A number of facts indicate that the steroid industry is an example of the industrial production of a combination of chemical synthesis and biotechnology.

The transformation reaction of the microorganism to the steroid compound is various, and the typical transformation reaction of the microorganism mainly comprises hydroxylation, dehydrogenation, side chain degradation and the like. Among them, the biocatalytic hydroxylation of steroids is particularly important. At present, the biocatalytic hydroxylation of steroids has been widely used in the production of anti-inflammatory drugs, immunosuppressants, progestational agents, diuretics, anabolic agents and contraceptives because of their high efficiency.

Bacteria and fungi can catalyze the hydroxylation reaction of steroids, but most of the filamentous fungi such as aspergillus, penicillium and the like which are successfully applied to industrial production at present. In the process of biological catalysis of the steroid compounds, the catalytic activity of the thalli directly influences the conversion efficiency of the products. With the development of molecular biology, the construction of genetically engineered strains with high transformation capacity to improve steroid transformation efficiency has become a hot research topic in recent years. Hydroxylating enzymes for catalyzing hydroxylation reaction in filamentous fungi belong to a P450 enzyme system, but relatively few researches are carried out on the hydroxylase participating in the reaction at present, related hydroxylase promoters at home and abroad are not reported, and the expression regulation mechanism is not clear.

Hydrocortisol was synthesized by hydroxylation reaction at C11 beta-position of deoxycorticosterol acetate, a steroid compound, using Neurospora crassa as a catalyst (see FIG. 1). Compared with a chemical synthesis method, the method has mild reaction conditions, high regioselectivity, stereoselectivity and chemoselectivity, easy purification, high conversion rate, low production cost and environmental friendliness. Studies have shown that Neurospora crassa (FGSC9717) is a candidate strain excellent in hydroxylation effect. The existing research proves that the steroid C11 beta-hydroxylase of the filamentous fungi is induced by steroid substrates at the transcriptional level, but the induction mechanism of the hydroxylase and related genes thereof are not reported in the literature.

Therefore, for the research from the P450 hydroxylase gene promoter, a good foundation is laid for explaining the molecular mechanism of the biological catalysis reaction of the hydroxylase, and meanwhile, a key gene resource is provided for constructing the next generation of high-efficiency engineering bacteria, so that the method has an important significance for the high-efficiency production of the biological catalysis hydroxylation of the steroid drugs.

The invention content is as follows:

the invention aims to solve the problem of low transformation efficiency of steroid biotransformation hydroxylation reaction in the current industry, provides an inducible promoter Pshn for constructing a rough vein spore engineering bacterium, the promoter can induce the expression of a P450 hydroxylase gene CYP5311B2 (abbreviated as CYP-7), lays a solid foundation for constructing a genetic engineering strain for efficiently catalyzing steroid hydroxylation reaction, and has important significance for improving the biotransformation efficiency of the steroid.

The neurospora crassa promoter Pshn is derived from neurospora crassa FGSC9717, and the nucleotide sequence of the neurospora crassa promoter Pshn is shown as SEQ ID NO: 1 is shown.

The neurospora crassa promoter Pshn of the present invention may also be a promoter that can react with SEQ ID NO: 1 and has a promoter function.

An expression cassette, an expression vector, a recombinant vector or a host cell comprising the neurospora crassa promoter Pshn according to the present invention also belongs to the scope of the present invention.

The primer sequence for gene amplification containing the whole length or partial fragment of the neurospora crassa promoter Pshn DNA also belongs to the protection scope of the invention.

The neurospora crassa promoter Pshn can be used for inducible expression of steroid compound hydroxylase genes.

The neurospora crassa promoter Pshn can be used for inducible expression of steroid compound hydroxylase genes in filamentous fungi neurospora crassa.

The Neurospora crassa promoter Pshn of the present invention may be used for inducible expression of genes including, but not limited to, steroid 11 β -hydroxylase, 15 α -hydroxylase, 11 α -hydroxylase, 16 α -hydroxylase, 7 α -hydroxylase, 14 α -hydroxylase, or 9 α -hydroxylase. The hydroxylase expressed by the gene can catalyze hydroxylation reactions including but not limited to steroid compounds C11 beta, C15 alpha, C11 alpha, C16 alpha, C7 alpha, C14 alpha or C9 alpha.

The inducible promoter Pshn can be used for constructing genetic engineering recombinant bacteria for inducing and expressing hydroxylase. The genetically engineered recombinant bacteria may be filamentous fungi.

Description of the drawings:

FIG. 1: the 11 beta hydroxylation of the deoxycorticosterone acetate catalyzes the biological reaction.

FIG. 2: verifying an electrophoretogram of a promoter Pshn product; m: marker; pshn: the promoter Pshn.

FIG. 3A: a schematic diagram of construction of the Pshn promoter expression vector pMF 272-Pshn-CYP-7.

FIG. 3B: enzyme digestion verification of the recombinant expression vector pMF 272-Pshn-CYP-7; m: 10kb DNA ladder; 1: and (4) enzyme cutting products.

FIG. 4: the expression vector pMF272-Pshn-CYP-7 of the Pshn promoter is linearized by treatment with restriction enzymes; m: 10kb DNA ladder; 1: the product was linearized with the plasmid.

FIG. 5: site-specific integration of 11 beta-hydroxylase CYP-7 gene expression cassettes and gene recombinant bacteria verification; m: 10kb DNA ladder; 1. 2, 3: transformants of the Pshn-CYP-7 heteronuclear recombinant strain. M: 10kb DNA ladder, w: neurospora crassa wild type

FIG. 6: after the recombinant strain is hybridized with 19018, 11 beta-hydroxylase CYP-7 gene expression cassette is integrated at a fixed point and the gene recombinant strain is verified; m: 10kb DNA ladder, w: neurospora crassa wild type, 1, 2: Pshn-CYP-7 pure nuclear recombinant expression strain.

FIG. 7: and comparing the steroid substrate feeding amount of the neurospora crassa recombinant strain and the Absidia coerulea strain.

FIG. 8: conversion rate of the neurospora crassa recombinant bacteria to steroid substrates in the fermentation process.

The specific implementation mode is as follows:

the following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Relates to the strains: neurospora crassa (Neurospora crassa) FGSC9717 fungus genetic collection center, Neurospora crassa (Neurospora crassa)19018, Absidia coerulea blue (Absidia coerulea) As3.65, Microbacterium species collection center of Tianjin science and technology university.

Enzymes and reagents used:

restriction enzymes EcoRI, NotI, XbaI, BamHI, Solution I ligation kit and Pyrobest DNA polymerase were purchased from Takara. Methanol, acetonitrile, ethyl acetate, petroleum ether and dichloromethane were purchased from Tianjin chemical reagent six factories. Other conventional reagents are imported split charging or domestic analytical purification. The synthesis of primers and the determination of the sequence were carried out by Beijing Huada.

The culture medium used:

10 XVogel's: 26g of sodium citrate dihydrate, 25.2g of potassium nitrate, 28.8g of ammonium dihydrogen phosphate, 16g of potassium dihydrogen phosphate, 2g of magnesium sulfate heptahydrate and 1g of calcium chloride dihydrate are weighed, dissolved in 800mL of double-distilled water, 1mL of trace element salt solution, 500uL of biotin and a small amount of chloroform are added, water is added to 1L, a small amount of chloroform is added dropwise, and the mixture is stored at 4 ℃.

10 × FIG's mother liquor: weighing 20g of L-sorbose, 0.5g of D-fructose and 0.5g of D-glucose, adding double distilled water, diluting to 100mL, sterilizing at 115 ℃ for 20min, and storing in a refrigerator at 4 ℃.

20 × BDES solution: weighing 20g of L-sorbose, 1g of sucrose and 1g of D-fructose, dissolving in 80mL of double distilled water, adding water to a constant volume of 100mL, sterilizing at 115 ℃ for 20min, and storing in a refrigerator at 4 ℃ for later use.

YPD medium (g/L): peptone 20, yeast powder 10, glucose 20, sterilizing at 115 deg.C for 20 min.

MM minimal medium (histidine auxotroph selection medium): 10mL of 10 XVogel's buffer, 2g of sucrose, 100mL of double distilled water, and sterilizing at 115 ℃ for 20 min.

MM slant medium: 10mL of 10 XVogel's buffer solution, 2g of sucrose, 1.5g of agar powder, adding double distilled water to a constant volume of 100mL, and sterilizing at 115 ℃ for 20 min.

TopAgar solid medium: 10 XVogel's 10mL, agar powder 1.0g, double distilled water to volume of 90mL, 121 ℃ sterilization for 20 min. Heating and dissolving the mixture in a microwave oven, adding 10mL of 10 XFig's mother liquor before pouring the mixture, and uniformly mixing.

Bottomigar culture medium: 10mL Vogel's 10mL, agar powder 1.5g, double distilled water to 80mL, 121 ℃ sterilization for 20min, before pouring, adding 10mL10 XFig's mother liquor.

BDES solid medium: 10mL Vogel's 10mL, agar powder 1.8g, double distilled water to 80mL, 115 ℃ autoclaving for 20min, adding 5mL20 × BDES mother liquor before pouring.

Synthetic hybridization medium (Synthesis cross, SC): weighing 0.5g of potassium nitrate, 0.7g of dipotassium phosphate, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.1g of anhydrous calcium chloride, 0.1g of sodium chloride, 100uL of trace elements, 100uL of biotin and 2g of sucrose, adding double distilled water to dissolve, continuously adding water to 1L, 15g of agar powder, and sterilizing at 115 ℃ for 20 min.

Enzyme production culture medium: 10 XVogel's 10mL, glucose 0.5g, double distilled water to 100mL, 115 ℃ sterilization for 20 min.

LB medium (g/L): 10g of peptone, 10g of NaCl and 5g of yeast powder, adding water to dissolve, fixing the volume to 1L, and sterilizing at 121 ℃ for 20 min.

EXAMPLE 1 obtaining of promoter sequence

A hydroxylase gene cDNA was identified by transcriptome sequencing and heterologous expression in Saccharomyces cerevisiae. The gene sequence of the hydroxylase gene was obtained by PCR amplification and DNA sequencing (GenBank: X15033.1). Further, the nuclear genome of Neurospora crassa was used as a template, and a primer was designed based on the gene sequence of the obtained hydroxylase gene, wherein the upstream: 5'-GCCCCCCTCATGTCTAGC-3', downstream: 5'-CGGCGCGAAGTAGCGCTG-3', obtaining a product fragment (shown in figure 2) with the length of about 950bp by reverse PCR (Inverse-PCR) amplification, connecting the PCR product with a T vector, designing a universal primer for DNA sequencing, and obtaining the nucleotide sequence of the inducible promoter Pshn, such as SEQ ID NO: 1 is shown.

Example 2 construction of promoter Pshn-inducible expression vector

In this example, the recombinant vector used was pYES2-CYP-7, and the expression vector was pMF 272-GFP.

The primers involved in the construction process were as follows:

Pshn-NotI-F：GCGGCCGCCGTAGCACAACGTCGAGC

Pshn-BamHI-R：GGATCCGATGGCTGTTGTCGCTGA

pshn is obtained by PCR amplification with primers (Pshn-NotI-F/Pshn-BamHI-R), and a recombinant expression vector pMF272-Pshn-CYP-7 is constructed by enzyme digestion/enzyme coupling.

The constructed expression vector was subjected to enzyme digestion analysis as shown in FIG. 3A. The vector pMF272 was 8.4kb in size, with Pccg1 and EGFP fragments 1.6kb in size and Pshn + CYP-7 fragments 2.5 kb. After a single digestion with XbaI, agarose gel electrophoresis results (FIG. 3B) showed fragments of about 7.7kb and 1.5kb, confirming successful construction of the expression vector, which was named pMF 272-Pshn-CYP-7.

The expression plasmid was digested with pfoI and linearized with the neurospora crassa steroid 11 β -hydroxylase expression vector pMF272-Pshn-CYP-7, as shown in fig. 4.

EXAMPLE 3 electroporation and culture of Neurospora crassa

Integrating the target gene of the prepared expression vector (pMF272-Pshn-CYP-7) linearized fragment to a Neurospora crassa genome his3 locus by an electric shock transformation method, primarily screening histidine auxotrophs to obtain a transformant, inoculating the transformant to an MM basic culture medium, culturing at 28 ℃ for 7-8 days, and collecting spores for later use.

On the basis of histidine-deficient screening, whether the target fragment is successfully transformed into the neurospora crassa genome needs to be verified by a colony PCR method. The PCR primer is CYP-7-yan-F/R. The PCR results are shown in FIG. 5.

CYP-7-yan-F：GGCCACAGCTGCAGTCCT

CYP-7-yan-R：AGGACAGGCATGTTTACCC

Example 4 Neurospora crassa transformant was hybridized with Neurospora crassa 19018

The obtained Neurospora crassa positive transformant is hybridized with Neurospora crassa 19018 to obtain a Neurospora crassa homozygote containing 11 beta-hydroxylase gene, and an inducible Pshn recombinant strain is obtained. The recombinant strain target gene was verified and the Neurospora crassa-derived strain (wild type) was used as a control as shown in FIG. 6.

The specific experimental process is as follows: respectively inoculating the neurospora crassa transformant and the neurospora crassa 19018 into MM basic solid slant culture medium, culturing in an incubator at 28 ℃ for 7-8 times until spores are mature, and collecting the spores by using 1mol/mL sorbitol. The spore suspension of the Neurospora crassa transformant is respectively inoculated on one side of rough filter paper of a synthetic hybridization medium and cultured for 5 days at 25 ℃. Then the neurospora crassa 19018 spores are inoculated on the other end of the filter paper, and cultured for 2-3 weeks at 25 ℃ until black ascospores grow out.

Example 5 screening and verification of genetically engineered bacteria and detection of steroid dosage

The rough neurospora obtained by the growth of the ascospores is randomly selected, genome DNA is extracted, and the genome DNA is amplified by using a primer CYP-7-yan-F/R. And the genome of the Neurospora crassa (wild type) was used as a control.

To investigate the 11 β -hydroxylating activity of the recombinant Neurospora crassa on deoxycortisone acetate. The substrate dosage of the Absidia coerulea is known to be 2g/L, and the substrate dosage of the inducible promoter Pshn gene recombination Neurospora crassa is compared with that of Absidia coerulea.

After the neurospora crassa is cultured, firstly, the steroid substrate deoxycrtisone acetate is ground by a ball mill, and then the particle diameter of the deoxycrtisone acetate is 10-15 mu m. Adding a proper amount of methanol into a steroid substrate, deoxidizing cortisone acetate, properly heating to dissolve the substrate, adding the substrate into the recombinant Neurospora crassa fermentation liquid, uniformly shaking, putting the mixture into a shaking table (28 ℃, the rotating speed is 180r/min), continuously culturing, timely sampling, extracting, measuring the conversion condition, and timely supplementing the steroid substrate.

The results are shown in FIG. 7. Under the condition of substrate conversion in the whole sampling detection, when the substrates are completely converted, the dosage of the Absidia coerulea substrate reaches 2g/L, and the dosage of the recombined Neurospora crassa substrate reaches 5 g/L.

Example 6 detection of conversion efficiency of genetically engineered bacteria to steroids

After the neurospora crassa is cultured, firstly, the steroid substrate deoxycrtisone acetate is ground by a ball mill, and then the particle diameter of the deoxycrtisone acetate is 10-15 mu m. Adding a proper amount of methanol into a steroid substrate, namely the deoxycortisone acetate, properly heating to dissolve the substrate, adding the substrate into the recombinant Neurospora crassa fermentation liquid, uniformly shaking, putting the mixture into a shaking table (28 ℃, the rotating speed of 180r/min), continuously culturing, and timely sampling to determine the conversion rate.

The results are shown in FIG. 8. The conversion rate exhibited by the recombinant strain throughout the fermentation period. The conversion rate of the inducible promoter Pshn gene recombinant bacteria reaches 80 percent in about 48 hours. The results show that the conversion rate of the inducible promoter Pshn gene recombinant strain for catalyzing the hydroxylation reaction of the steroid compounds is improved by about 15%, and the conversion time is shortened by 12 hours.

Although the preferred embodiments of the present invention have been disclosed, it should be understood that they are not intended to limit the invention, but various changes and modifications may be made therein by those skilled in the art without departing from the spirit and scope of the invention, and it is intended that the scope of the invention be defined by the appended claims.