阅读说明：本技术 一种来源于丝状真菌黑曲霉的诱导型启动子及其应用 (Inducible promoter derived from filamentous fungi aspergillus niger and application thereof ) 是由 刘晓光 郭文丽 路福平 于 2020-05-14 设计创作，主要内容包括：本发明专利提供了一种来自丝状真菌黑曲霉的新型诱导型启动子及其质粒载体构建,其氨基酸序列如SEQ ID NO：1所示,该启动子能够诱导外源基因在黑曲霉细胞中高效诱导表达,如P450甾体羟化酶基因的高水平表达,考察了赭曲霉C11α-羟化酶基因68J5在黑曲霉ATCC1015细胞中高水平表达以及重组菌对甾体底物16,17α-环氧黄体酮的转化效率,因此可用于构建黑曲霉高效基因工程菌。(The invention provides a novel inducible promoter from filamentous fungi Aspergillus niger and construction of a plasmid vector thereof, and an amino acid sequence of the promoter is shown as SEQ ID NO: 1, the promoter can induce the high-efficiency induction expression of exogenous genes in Aspergillus niger cells, such as the high-level expression of P450 steroid hydroxylase genes, and the high-level expression of Aspergillus ochraceus C11 alpha-hydroxylase gene 68J5 in Aspergillus niger ATCC1015 cells and the conversion efficiency of recombinant bacteria on steroid substrates 16, 17 alpha-epoxyprogesterone are examined, so that the promoter can be used for constructing Aspergillus niger high-efficiency genetic engineering bacteria.)

1. An inducible promoter, wherein the nucleotide sequence of the promoter is as set forth in SEQ ID NO: 1 is shown.

2. A recombinant plasmid, vector or host strain comprising the inducible promoter of claim 1.

3. The recombinant vector or expression vector of claim 2, wherein the recombinant vector is pBG-CYP68J5 and the expression plasmid vector is pG-CYP68J 5.

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 an Aspergillus niger or a Aspergillus niger species.

6. Upstream and downstream primers for amplifying the full length of the inducible promoter DNA of claim 1, wherein the primer pair sequences, C7-F: AAGGCGATGAGGTTGTAATG, C7-R: GGATTACGGCACAGACAC, respectively; 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 hydroxylase gene comprises 11 α -hydroxylase, 11 β -hydroxylase, 15 α -hydroxylase, 16 α -hydroxylase, 7 α -hydroxylase, 14 α -hydroxylase, 9 α -hydroxylase, but not limited to steroid hydroxylase.

8. Use of an inducible promoter as claimed in claim 7 for the inducible expression of an exogenous gene in a filamentous fungus.

9. The use of the inducible promoter of claim 7, wherein the inducible promoter is used for constructing a high efficiency Aspergillus niger steroid hydroxylation genetically engineered bacterium.

The technical field is as follows:

the invention belongs to the technical field of genetic engineering, and particularly relates to an inducible promoter and application thereof.

Background art:

the steroid hormone medicine has wide application in clinic. The medicine can be used for treating inflammation and allergy, cancer, hormone secretion disorder of human body, and fertility control. The physiological and pharmacological activity of steroid drugs depends on the introduction of functional groups at specific sites of the steroid skeleton. The steroid structure is modified by chemical synthesis or microbial conversion, or by a combination of both methods. The total chemical synthesis method has the defects of complex synthesis process, difficult separation of byproducts and the like, so that the key synthesis step which is difficult to complete by the chemical synthesis method industrially utilizes microbial transformation reaction to introduce various functional groups, particularly hydroxyl groups, on the basic skeleton of the steroid.

Important microbial steroid hydroxylation reactions include the introduction of hydroxyl groups at positions C9, C11, C15, C17 of the carrier backbone. Aspergillus ochraceus (Aspergillus ochracea) has good steroid hydroxylation reaction specificity and high hydroxylation activity, and is commonly used for industrial steroid C11 alpha-hydroxylation reaction. Hydroxylating enzymes catalyzing C11 alpha-hydroxylation reaction in aspergillus ochraceus strains belong to a P450 enzyme system, and a gene encoding C11 alpha-hydroxylase is cloned and identified. However, a large amount of brick red pigment is easily generated in the fermentation process of transforming the steroid by using aspergillus ochraceus, which causes difficulty in separation and purification of a transformation product, and moreover, ochratoxin can also be generated by fermenting the aspergillus ochraceus.

Aspergillus niger (Aspergillus niger) is a safe strain (GRAS) certified by the FDA in the United states, is vigorous in growth and metabolism, simple in nutritional requirements, free from mycotoxin production, clear in genetic background and mature in molecular genetic manipulation technology, and is an ideal host bacterium for constructing a filamentous fungus cell factory. Meanwhile, the mode aspergillus niger strain is converted into a steroid substrate, and pigment which interferes product separation and purification is not easy to generate. Aspergillus niger ATCC1015 has steroid C11 alpha-hydroxylase activity, but has low conversion activity and many byproducts, and is difficult to use in industrial production. We have cloned and identified the DNA sequence of the gene Ana100 encoding steroid C11 alpha-hydroxylase and its promoter C7 in the strain A.niger ATCC1015, the expression of which is efficiently induced by steroid substrate at the transcriptional level.

In view of the high inducibility of the gene Ana100 expression of the a.niger steroid C11 a-hydroxylase by steroid substrates, the promoter C7 can be used for high-efficiency expression of different target steroid hydroxylase genes and other target genes in a.niger cell.

According to the invention, an aspergillus ochraceus steroid C11 alpha-hydroxylase gene recombinant expression plasmid is constructed, homologous recombination is utilized to replace an aspergillus ochraceus steroid C11 alpha-hydroxylase gene with a C11 alpha-hydroxylase gene Ana100 of an aspergillus niger strain through an agrobacterium-mediated method, so that an aspergillus niger efficient steroid inducible promoter C7 drives the expression of a target aspergillus ochraceus steroid C11 alpha-hydroxylase gene, and thus the aspergillus niger efficient C11 alpha-hydroxylation engineering bacterium is obtained, and the recombinant expression plasmid has important significance for efficient green production of steroid medicines and intermediates thereof by biological catalysis hydroxylation.

The invention content is as follows:

aiming at the problem that interference pigments are generated in the prior industrial strain Aspergillus ochraceus TCCC41060 steroid C11 alpha hydroxylation process, an inducible promoter C7 of Aspergillus niger is utilized to over-express an Aspergillus ochraceus C11 alpha hydroxylase gene CYP68J5 (68J 5 for short) in Aspergillus niger cells to construct a genetic engineering strain for efficiently catalyzing steroid hydroxylation reaction, and the genetic engineering strain has important significance for improving the biotransformation efficiency of steroids.

The nucleotide sequence of the inducible promoter C7 is shown as SEQ ID NO: 1 is shown.

The inducible promoter C7 of the present invention is derived from Aspergillus niger ATCC 1015.

An expression cassette, an expression vector, a recombinant vector or a host cell comprising the inducible promoter C7 according to the present invention is also within the scope of the present invention.

The primer sequence for gene amplification containing the full-length or partial fragment of the inducible promoter C7 DNA also belongs to the protection scope of the invention.

The inducible promoter C7 can be used for inducing expression of genes including but not limited to 11 alpha-hydroxylase, 11 beta-hydroxylase, 7 alpha-hydroxylase, 14 alpha-hydroxylase and 9 alpha-hydroxylase or expressing steroid 15 alpha-hydroxylase and 16 alpha-hydroxylase in Aspergillus niger host cells to construct corresponding Aspergillus niger high-efficiency steroid hydroxylase genetic engineering bacteria.

Has the advantages that:

the embodiment of the invention examines the high-level expression of the aspergillus ochraceus C11 alpha-hydroxylase gene 68J5 in aspergillus niger ATCC1015 cells and the conversion efficiency of a recombinant bacterium on a steroid substrate 16, 17 alpha-epoxyprogesterone. 68J5 gene recombination aspergillus niger strains are obtained by constructing an inducible expression vector and applying a homologous recombination method. Steroid conversion tests show that the conversion rate of the Aspergillus niger recombinant strain is obviously higher than that of the existing Aspergillus ochraceus production strain, and the research result of the invention has important commercial application value.

The attached drawings of the specification:

FIG. 116, C11 alpha-hydroxylation reaction of 17 alpha-epoxyprogesterone

FIG. 2 agarose gel electrophoresis verification of promoter C7 band

FIG. 3 schematic diagram of recombinant plasmid pBG-68J5 construction

FIG. 4 restriction enzyme validation recombinant plasmid pBG-68J5

(a) M: 10kb DNA ladder; 1: xba I + EcoRI double enzyme digestion verification;

(b) m: 10kb DNA ladder; 2: and (5) carrying out single-enzyme digestion verification on SacI.

FIG. 5 construction scheme of overexpression recombinant plasmid pG-68J5

FIG. 6 restriction enzyme digestion verification electrophoretogram of recombinant plasmid pG-68J5

(a) M: 10kb DNA ladder; 1: carrying out double enzyme digestion verification on Knp I + Xho I;

(b) m: 10kb DNA ladder; 2: and (5) carrying out single enzyme digestion verification on EcoRI.

FIG. 7 agarose gel electrophoresis validation of HYG electrophoretograms

FIG. 8 is a schematic diagram of fixed point integration

FIG. 9 agarose gel electrophoresis verification

FIG. 10 TLC results chart

FIG. 11 results of steroid substrate conversion by the species

The specific implementation mode is as follows:

the process of the invention is described below by means of specific embodiments. Unless otherwise specified, all technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example-promoter sequence acquisition

An upstream sequence of an Aspergillus niger P450 enzyme gene is obtained by using an NCBI database (http:// www.ncbi.nlm.nih.gov /), PCR upstream and downstream primers are designed, and an Aspergillus niger ATCC1015 bp promoter fragment is obtained by PCR amplification by using an Aspergillus niger ATCC1015 genome as a template, as shown in figure 2.

C7-F: AAGGCGATGAGGTTGTAATG (upstream primer)

C7-R: GGATTACGGCACAGACAC (downstream primer)

The promoter fragment is connected to a T vector, and sequencing is carried out, so that the nucleotide sequence of the inducible promoter C7 is shown as SEQ ID NO: 1 is shown.

EXAMPLE two construction of overexpression vectors

The primer sequences required for construction were as follows:

pPZP-HindIII-F: AAGCTTCATTGTTGTCTCCTTC (upstream sequence)

pPZP-XbaI-R: TCTAGAGCCCCGACAG C (downstream sequence)

The plasmid vector L-T was digested with NcotI and HindIII, and purified by gel recovery to obtain a 0.5kb L fragment. The plasmid vector pCSN44-HYG was digested with HindIII and XbaI, and purified by gel recovery to obtain a 2.4kb HYG fragment. The plasmid vector pBlue-HYG was digested with NcotI and XbaI, and the 3.0kb pBlue fragment was obtained by gel recovery and purification. And (3) connecting the 3 fragments in vitro by using recombinase to obtain a recombinant plasmid pBlue-L-HYG. The fragment was digested with SacI enzyme and XbaI enzyme, and recovered from the gel to obtain pBlue-L-HYG fragment. The vector C7-68J5-TT-T was digested with EcoRI and XbaI, and the fragment C7-68J5-TT of 3.24kb was obtained by gel-back purification. The plasmid vector R-T was digested with EcoRI and SacI and purified by gel-back to obtain a 0.51kb R fragment, and 3 fragments were ligated in vitro by recombinase to construct recombinant plasmid pBG-68J5, the construction scheme being shown in FIG. 3. The recombinant plasmid pBG-68J5 was digested with XbaI and EcoRI to obtain 6164bp and 3401bp fragments, and the plasmid was digested with restriction endonuclease SacI to obtain 9565bp band, as shown in FIG. 4.

The recombinant plasmid pBG-68J5 is used as a template, an upstream primer pPZP-HindIII-F and a downstream primer pPZP-XbaI-R with HindIII and XbaI recognition sites are designed to carry out PCR reaction so as to obtain a target fragment, XbaI enzyme and HindIII enzyme are used for double digestion to obtain a target fragment with a sticky end, and then the target fragment is connected with a vector skeleton pPZP fragment with the same sites by a DNA in vitro recombination technology to construct an expression vector pG-68J5, wherein the construction schematic diagram is shown in figure 5. Through Kana resistance screening, single colony is selected to extract plasmid. The recombinant plasmid pG-68J5-TT-R was double-digested with Knp I and Xho I, and then single-digested with EcoRI enzyme, followed by 0.8% agarose gel electrophoresis for verification, the results of which are shown in FIG. 6.

EXAMPLE three recombinant plasmids transferred to Agrobacterium and screening

(1) Preparation of Agrobacterium competence

a. Taking three areas of an agrobacterium glycerol tube, streaking and inoculating the three areas of the agrobacterium glycerol tube on an LB (lysogeny broth) plate, and culturing at 28 ℃ until a single colony grows out;

b. selecting agrobacterium tumefaciens to divide single colony, inoculating the single colony in 5mL LB liquid culture medium, and carrying out shaking culture at 28 ℃ and 200rpm for about 12 hours;

c. inoculating the bacterial liquid into 50mL LB liquid culture medium with the inoculation amount of 2%, and carrying out shaking culture at 28 ℃ and 200rpm for 4-6h until the OD600 value is 0.8;

d. the cultured bacterial liquid is iced for 30min and then transferred into a precooled sterilized 50mL centrifuge tube, centrifuged for 10min at the temperature of 4 ℃ and the rpm of 5000, and the supernatant is discarded;

e. resuspending the cells in 10mL of pre-cooled sterile water, centrifuging at 4 ℃ and 5000rpm for 10min, and discarding the supernatant;

f. resuspending the cells with 20mL of pre-cooled 10% glycerol, centrifuging at 4 ℃ and 5000rpm for 10min, and discarding the supernatant;

g. repeating the previous operation;

h. the cells were resuspended in 1mL of 10% glycerol, and the cells were dispensed into 1.5mL of EP tubes at 90. mu.L/tube and stored at-70 ℃ until use.

(2) Recombinant plasmid pG-68J5 electric shock transformed Agrobacterium tumefaciens

a. Washing the electric rotary cup with anhydrous ethanol for 3 times, especially for corner and sample adding slot, washing with deionized water for 3 times, air drying, and placing on ice for ice bath.

b. The Agrobacterium competent cells were thawed on ice, 3. mu.L of plasmid was added to the competence, mixed well and ice-cooled for 5 min.

c. And transferring the competent cells into a precooled electric rotating cup, wiping water outside the electric rotating cup, and placing the electric rotating cup in an electroporation container to perform pulse with the voltage of 200 omega and 1800V.

d. Immediately taking out the electric rotating cup, adding 1mL of LB into the electric rotating cup, uniformly mixing, transferring the bacterial liquid to a 1.5mL sterile EP tube, and recovering at 180r/min for 3h at 28 ℃.

f. And (3) coating 200 mu L of bacterial liquid on an LB plate containing Kana, and carrying out inverted culture at 28 ℃ for 2d until transformants grow out.

g. And selecting a single colony to perform colony PCR verification on the size of the band, wherein the universal primers are HYG-F and HYG-R.

HYG-F: CTTTTTCGGACTTGAGTGGCG (upstream primer)

HYG-R: TAAGGAAACGGGAGCCTG (downstream primer)

The HYG band was verified by agarose gel electrophoresis, and the results are shown in FIG. 7.

EXAMPLE four Co-culture of Aspergillus niger and Agrobacterium and its validation

The expression cassette of the C11 alpha-hydroxylase gene 68J5 was transferred into Aspergillus niger by Agrobacterium mediated transformation of wild Aspergillus niger ATCC1015, and site-directed integration into Aspergillus niger was achieved by homologous recombination to allow heterologous expression of the 68J5 gene (see FIG. 8 for a schematic).

The experimental steps of the agrobacterium-mediated co-culture method are as follows:

a. single colony of Agrobacterium is picked up and cultured in 5mL LB liquid containing 100ug/mL kana at 28 ℃ and 180r/min for 20-24h with shaking.

b. 1.5mL of overnight cultured Agrobacterium liquid was centrifuged at 5000r/min for 10min in a sterile EP tube, the supernatant was discarded, the cells were rinsed with IM broth containing 100. mu.g/mL kana and 0.2. mu. mol/L AS, resuspended at 4000r/min, centrifuged for 5min, and the supernatant discarded. Then 1mL of IM liquid is used to resuspend the thalli, the thalli is transferred to a sterile test tube, 4mL of IM liquid culture medium is added, the temperature is 28 ℃, 100r/min, and the thalli is cultured for 5 h.

c. At OD₆₀₀Under the condition (1), measuring the OD value of the bacteria to 0.8, wherein the concentration of the agrobacterium is about 4-5 multiplied by 10⁸one/mL.

d. A0.45 μm microfiltration membrane was placed on an IM plate containing 0.2 μmol/L AS and 100 μ g/mL kana with sterile forceps to avoid air bubbles AS much AS possible.

e. 100 μ L of OD was taken₆₀₀About 0.8 of Agrobacterium and a concentration of 1.0X 10 of 100. mu.L⁷The black koji spores are mixed evenly.

f. Uniformly coating the mixed solution of the agrobacterium and the aspergillus niger spores on an IM (instant messenger) plate stuck with a microporous filter membrane by using a coater, air-drying, and culturing for 2-5 days at 25 ℃.

g. After the agrobacterium is induced by an IM (instant messenger) culture medium, the agrobacterium is co-cultured with an Aspergillus niger ATCC1015, the copy number is increased in the process of homologous recombination fixed-point integration, a single colony is selected from a CM plate and cultured on a HYG-PDA (hypg-PDA) slant culture medium for 72 hours, a genome of the single colony is extracted as a verified template, and a PCR (polymerase chain reaction) verification result is carried out by taking L-F, HYG-R as an upstream primer and a downstream primer, wherein the result is shown in figure 9.

L-F: AAAGCAAGTAGCGAGTAG (upstream primer)

HYG-R: TAAGGAAACGGGAGCCTG (downstream primer)

EXAMPLE five detection of conversion efficiency of steroid substrates

Activating the aspergillus niger strain with the inducible promoter, and grinding the steroid substrate into particles with the diameter of 10-15 mu m by a ball mill. Mixing it with surfactant Tween 80 at a volume ratio of 1: 10, adding into Aspergillus niger growth shake flask, shaking uniformly, placing into shaking table (28 deg.C, rotation speed 200r/min), culturing, sampling for 72h, and determining conversion rate, wherein TLC result is shown in FIG. 10. So that the promoter drives the hydroxylase gene 68J5 for functional expression. Meanwhile, when the feeding concentration is 0.1%, compared with the conversion rate of the wild Aspergillus niger ATCC1015 strain, the conversion efficiency of the recombinant Aspergillus niger strain is improved for about 72 hours, and byproducts are obviously reduced.

Example six transformation analysis of recombinant A.niger strains at different times

In order to determine the conversion efficiency of the aspergillus niger recombinant strain to the steroid substrate 16, 17 alpha-epoxyprogesterone, the conversion situation of the starting strain wild aspergillus niger ATCC1015 when the substrate feeding amount is 1g/L is compared, and the result is shown in fig. 11, the conversion efficiency of the recombinant strain is obviously higher than that of the starting strain wild aspergillus niger ATCC1015, the conversion efficiency of the starting strain wild aspergillus niger ATCC1015 is obviously lower than that of the recombinant strain at 48h, and the conversion efficiency of the recombinant strain after 72h reaches 74.6%.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.