CN111484964A

CN111484964A - High-yield 9-OHAD mycobacterium and construction method thereof

Info

Publication number: CN111484964A
Application number: CN202010178401.1A
Authority: CN
Inventors: 宋浩; 孙鸿
Original assignee: Tianjin University Marine Technology Research Institute
Current assignee: Tianjin University Marine Technology Research Institute
Priority date: 2020-03-14
Filing date: 2020-03-14
Publication date: 2020-08-04

Abstract

The invention discloses a high-yield 9-OHAD mycobacterium and a construction method thereof, which are characterized by comprising the following steps of (I) RNA extraction and RT-qPCR analysis, (II) knockout of kstd strain construction, (III) construction of sterol metabolism related gene expression vector, (IV) mycobacterium electrotransformation, and (V) sample extraction, detection and quantification.

Description

High-yield 9-OHAD mycobacterium and construction method thereof

The technical field is as follows:

the invention relates to a mycobacterium with high 9-OHAD yield and a construction method thereof, belonging to the field of biological genetic engineering.

Background art:

steroids are a natural chemical component widely existing in nature, and with the appearance of more and more diseases, steroid hormone drugs have gradually developed into an important class in the field of medicine. Actinomycetes exhibit natural advantages over other species in their utilization and metabolism of xenobiotics, and in some cases sterols can also serve as carbon and energy sources for microorganisms. The mycobacterium may use natural sterols or some other steroid as the sole carbon and energy source. Disruption of the mycobacterial catabolic pathway results in the excessive accumulation of some important intermediates, such as AD and 9-OHAD, which can be used as precursors for the production of steroid hormone drugs.

The study shows that the complete degradation of a steroid molecule into a small molecule energy substance comprises four steps, namely the uptake of the steroid molecule, the degradation of the steroid nucleus, the oxidation of the side chain group, the biological contact of the steroid substance in the uptake environment of actinomycetes, the efficiency of the hydrophobic sterol molecule into the phospholipid layer by producing and secreting specific surfactants, the cholesterol oxidase or dehydrogenase present outside the cell is one of the positive factors for the transport of cholesterol, the branched degradation of the sterol is similar to that of fatty acid β -oxidation, the cholesterol retention is based on 1 unit β -cholesterol, the cholesterol oxidase or dehydrogenase is induced by the degradation of the steroid coenzyme, the cholesterol dehydrogenase is induced by the cholesterol oxidase or dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase, the cholesterol oxidase, the cholesterol dehydrogenase.

Disclosure of Invention

In order to obtain high yield of 9-OHAD, the present invention focuses on the molecular modification of mycobacteria mainly on the ring-opening of the steroid nucleus, such as overexpression of 3-steroid-9 a-hydroxylase (KSH) and knock-out of 3-steroid-D1-dehydrogenase (KstD), with little uptake and side chain degradation of phytosterols, both of which are equally important for high yield of 9-OHAD, and both of which are required for rapid uptake of phytosterols and high yield of 9-OHAD.Studies show that the complete degradation of a steroid molecule into small molecular energy substances takes place in four steps, uptake of sterol molecules, ring-opening of the steroid nucleus, degradation of the ring-opened steroid nucleus, oxidation of side chain groups, determination of key genes affecting 9-OHAD production by quantitative fluorescence PCR, knock-out of genes accumulating 9-OHAD, and simultaneous overexpression of some of genes 3 β -KDs, 5 α -std, side chain degradation and ring-opened nuclear degradation of the three modules, and results show that the original yield of mycobacteria is increased by 9-OHAD 366.6.366.

The invention provides a method for constructing a mycobacterium with high 9-OHAD yield, which comprises the following steps: RNA extraction and RT-qPCR analysis

RNA was isolated from mycobacterial sterol transformation media using RNAprep pure cell/bacteria kit (TIANGEN.) total RNA from different samples was concentrated in 30 μ L RNase free water according to kit instructions.

Reverse transcription of 1. mu.g total RNA using the FastQuant cDNA kit (TIANGEN). according to the kit instructions, total RNA samples were pretreated with Dnase I2. mu. L5 × g DNA buffer, 1. mu.g total RNA, adjusted to 10. mu. L with ddH2O, incubated for 3min at 42 ℃ and then immediately cooled ice.A reverse transcription reaction mixture was then prepared 2. mu. L10 × Fast RT buffer, 1. mu. L RTEnzymaMix, 2. mu. L FQ-RT primer MIX, 1. mu.g total RNA, adjusted to 20. mu. L with ddH2O, incubated for 15min at 42 ℃ and then incubated for 3min at 95 ℃ and then immediately cooled on ice-20 ℃ and the mixture was diluted 10-fold prior to NanoDrop quantitation.

Gene-specific qRT-PCR primers were designed and qRT-PCR was performed in cfx96(BioRad) using the SuperReal PreMix Plus kit (SYBRGreen) (Tiangen). 2. mu. L of the above cDNA samples were mixed with 0.6. mu. L + 0.6. mu. L specific oligonucleotide, 10. mu. L2 × SuperReal PreMix Plus and 6.8. mu. L RNase-free ddH₂O was thoroughly mixed in 20. mu. L reaction mixture RT-qPCR reactions were performed for 15min of pre-denaturation at 95 ℃ for 10 sec for 40 cycles, 20 sec at 60 ℃, 20 sec at 72 ℃, 20 sec, followed by a melting curve phase at 55 ℃ to 95 ℃Groups are strains that grow on a phytosterol carbon source. Each sample was analyzed in triplicate. Relative expression levels were calculated using the 2- Δ Δ CT method with the 16SrRNA gene amplicon as an internal control.

Construction of (two) knock-out kstd Strain

To delete kstd1 or kstd3, two fragments of upstream sequences 1112bp or 1361bp were first cloned by primers kstd1-up-f/kstd3-up-r or kstd 1-down-respectively, and downstream sequences 1057bp or 1293 bp. tdks 3-down/-r. the fragments were then ligated to 2175bp or 2656 by XbaI enzyme, then 2175bp or 2656bp fragments were digested with HindIII/NotI and then cloned into plasmid p2NI L. finally, a selection marker cassette digested with PacI from pGoal19 was inserted into the PacI site of p2NI L to generate homologous recombinant pdel-tdks 1 or pdel-kstd after alkali treatment, as described by gouhan and parkh, after successful transfer of the gene from tdtd strain 54 bp to tdtd 3978 bp or tdks 73 bp, or after deletion of the original recombinant pdel-tdks 3626 gene by electroporation with primers kstd 3978-9 or 99 bp, finally verified by PCR with primers 16820-20. the deletion of the original recombinant plasmid.

(III) construction of sterol metabolism-related Gene expression vector

The gene 3 β -Hsd was cloned from the genome of MS136 using Hsd-pf & pr, BamHI-HindIII and ligated into plasmid pMV261 at the BamHI-HindIII plasmid pMV261-Hsd was introduced into MS136 to obtain MS136 and MS136-H respectively, 5 α -reductase-pf & pr, cyp125-pf & pr, fade 19-pf & pr, fadA5-pf & pr, kshA-pf & pr and kshB-pf & pr. were used to clone the gene 5 α -4std, cyp125, fade 19, fadA5, hA and kshB from MS136 the genome using 5 α -redase-pf & pr, cys 6319, plasmid XbXgV 261-dli + plasmid XgHa + XgSk-hrsg & gt, plasmid XgHa + XgHa-hA + XgHa-hA & gH & pr..

(IV) Mycobacterium electrotransformation

And (2) treating the electric rotating cup (2mm), namely spraying 75% alcohol on the electric rotating cup, drying the electric rotating cup under ultraviolet rays on a clean bench, putting the electric rotating cup into an ice box for ice bath for 10min, taking a branched bacillus sensitive state tube, putting the branched bacillus sensitive state tube on the ice box ice, standing the electric rotating cup on the ice for 10min, adsorbing the competence added with the plasmid into the electric rotating cup, setting the parameter voltage of an electric rotating instrument to be 1800V, putting the electric rotating cup into an electric rotating instrument tank, performing electric rotation for 2 times, performing electric rotation, then performing ice bath for 3min, adding a seed culture medium of 800u L into the clean bench, centrifuging for 30min, recovering the electric rotating cup in a shaking table for 2-3 h.4000rpm for 5min, discarding supernatant, reserving about 100u L, resuspending the cells, sucking the cells by a liquid transfer gun onto a solid culture medium plate containing corresponding antibiotics, coating the solid culture medium plate uniformly until the cells are dry, observing the growth after about 5-7 days, and selecting bacteria for verification.

(V) sample extraction, detection and quantification

And (3) treating a product, namely putting 500u L of fermentation liquor into a centrifuge tube, centrifuging for 15min at 4000rpm, sucking supernatant liquid by using an injector, filtering the supernatant liquid into a clean centrifuge tube through a 0.22um water system membrane for later use, sucking 10u L of filtrate, adding the filtrate into 990u L of diluent, fully mixing the filtrate uniformly for subsequent analysis and detection, and storing the rest filtrate at-20 ℃.

And (3) detecting a product 9-OHAD, namely the 9-OHAD has a characteristic absorption peak under the ultraviolet wavelength of 240nm, and a substrate phytosterol has no characteristic absorption, so that the product is quantitatively analyzed by adopting an HP L C method standard curve, wherein the chromatographic conditions comprise that a chromatographic column is C18(250mm × 4.6.6 mm), the detection wavelength is 240nm, the column temperature is 50 ℃, the flow rate is 1.0m L/min, and mobile phases comprise ethanol, water, glacial acetic acid (520:480:0.1), a diluent comprises acetonitrile, water (50:50) and the sample injection amount is 20u L.

The invention has the beneficial effects that:

the invention firstly provides a method for modularly transforming mycobacteria to construct a high-yield 9-OHAD strain, and identifies the relationship between a plurality of genes and 9-OHAD accumulation, and the method improves the yield of 9-OHAD by over-expressing 3 β -HSD, 5 α -redutase and KSH genes in mycobacteria and simultaneously knocking off the KSTD gene, thereby providing a theoretical basis for future industrial production.

Description of the drawings:

FIG. 1 is a schematic illustration of the metabolism of phytosterols by mycobacteria.

FIG. 2 is a gene transcription analysis and engineering of Module A (a) transcriptional expression levels of genes 3 β -HSD and 5 α -reductase in the sterol uptake module (Module-A) in response to phytosterols (b) ligation of target genes (3 β -HSD or 5 α -reductase) into the pMV261 plasmid to overexpress a single gene to form two recombinant plasmids, (c) 9-OHAD was produced after overexpression of each gene using 13 g/L phytosterols as substrate and 9-OHAD production was detected 84 hours after inoculation.the control group transferred MS136 with blank pMV261 plasmid.

FIG. 3 is the transcriptional analysis and engineering of the Module B gene. (a) Transcriptional expression levels of the genes cyp125, fadD19, fadA5 and hsd4A in the side chain degradation module (module-B) in response to phytosterols. (b) Each target gene (cyp125, fadD19, or fadA5) was ligated into the pMV261 plasmid to form three recombinant plasmids. Only individual genes are overexpressed. (c) Production of 9-OHAD overexpressing each gene. Control MS136 was transformed with the blank pMV261 plasmid. The values in each curve are expressed as mean ± standard deviation (error bars) from three independent determinations.

FIG. 4 is a graph of gene transcription analysis and engineering of module-C, (a) the level of transcription expression of gene KSH in sterol nuclear open cycle module (module-C) in response to phytosterol, (b) the level of transcription expression of gene KStD in sterol nuclear open cycle module (module-C) in response to phytosterol, (C) ligation of target gene (KSH) into pMV261 plasmid to form a recombinant plasmid, (d) production of 9-OHAD overexpressing KSH gene control MS136 transferred into blank pMV261 plasmid, the value in each curve is expressed as the mean. + -. standard deviation (error bars) from three independent determinations, (e) phenotypic analysis of Mycobacterium 136 recombinant cells MS136 in medium with 10 g/L glycerol, the growth curve in each curve is observed in medium with 10 g/L glycerol, the value in each curve is spectroscopically determined by OD600 values of TDOD nm (f) the mean value in real-time production of 9-OHAD in 13 g/L phytosterol aerobic fermentation process, the value of Mycobacterium is expressed as the mean. + -. standard deviation of. + -. MS 136. 20 g/delks19 M.54. Del MS 136. 19. the error bars 363. 36136-Del MS 136.

FIG. 5 is a graph of construction of recombinant strain Mycobacterium sp.MS136-delkstd1+ H +4s + AB for enhanced 9-OHAD production (a) construction of recombinant plasmids.A selected gene 3 β -HSD (in module-A), 5 α -reductase (in module-A) and KSH (in module-C) was ligated into pMV261 plasmid: (b) time curves for 9-OHAD production by each recombinant strain in aerobic fermentation.the values in each curve are expressed as mean. + -. standard deviation from three independent determinations (error bars). MS136-pMV 261: MS136 strain with pMV261 plasmid; MS136-delkstd1-pMV 261: recombinant MS136 strain which knockouts KstD1 in chromosome and together with pMV 261. MS136-delkstd1+ H +4s + MS136 strain 136, and KSD 6746 strain which knockouts in chromosome 3-HSD, KSAB 261, KS5-dHA, and KSV 6778 in chromosome 6747, and KSV 6747-plasmid knockouts.

The specific implementation mode is as follows:

example 1

1 Material

1.1 strains, plasmids and media

All plasmids and strain information referred to in this example are detailed in Table 1 plasmids pMV261, p2NI L and pGOA L19 were purchased from Biosci.

L B liquid culture medium containing peptone 10 g/L, yeast extract 5 g/L10 g/L, pH 7.5, sterilized at 121 deg.C for 15min, L B solid culture medium containing peptone 10 g/L, yeast extract 5 g/L10 g/L, agar powder 15 g/L, pH 7.5, sterilized at 121 deg.C for 15 min.

The proportion of the trace elements is 10 g/L of magnesium sulfate, 0.5 g/L of ferrous sulfate, 0.2 g/L0 of zinc sulfate and pH 3-3.5, the seed culture medium comprises 1.5 g/L1 of ammonium dihydrogen phosphate, 10 g/L2 of glycerol, 10 g/L3 of yeast powder, 0.5 g/L4 of sodium dihydrogen phosphate, 0.5 g/L5 of disodium hydrogen phosphate, 802.5 g/L of tween, 10m L of trace elements and pH 7.0-7.3, the slant culture medium comprises 1.5 g/L of ammonium dihydrogen phosphate, 10 g/L of glycerol, 10 g/L of yeast powder, 0.5 g/L of sodium dihydrogen phosphate, 0.5 g/L of disodium hydrogen phosphate, 10m L of trace elements and pH 7.0-7.3.

The transformation medium comprises 0.8 g/L g of sodium dihydrogen phosphate, 2.52 g/L g of disodium hydrogen phosphate, 1.8 g/L g of ammonium sulfate, 800.2 g/L g of tween, 0.3 g/L g of urea, 13.2 g/L g of phytosterol-70 g/L g of cyclodextrin, 10m of trace elements L, 5 g/L g of glycerol and 7.0-7.3 of pH.

TABLE 1 strains and plasmids involved in the experiments

1.2 reagent and apparatus restriction enzymes BamHI, HindIII, EcoRI, NotI and PacI from Fermentas; a bacterial RNA extraction kit, a reverse transcription cDNA synthesis kit, a Superreal Premix Plus kit, a plasmid miniextraction kit, a common DNA product purification kit and an agarose gel DNA recovery kit are purchased from Tiangen corporation; 9-OHAD was purchased from Shanghai Boton Biochemical industries, Inc. The apparatus used was: high performance liquid chromatography (Waters), electrotransformer (Ependorf), Real-time PCR (BioRad), NanoDrop (NanoDrop 2000, Thermo).

1.3 primers used for PCR are shown in Table 2.

TABLE 2 primer sequences

2 method

2.1 Gene sequence analysis

Gene sequencing was done with the help of Novogene Bioinformatics technology.A comparative analysis of nucleotide and amino acid sequences was performed by the B L AST program of NCBI (National Center for Biotechnology Information Server, http:// blast.ncbi.nlm.nih.gov.) multiple sequence alignment was performed by DANMAN 6.0 conserved domains in proteins were also searched on the NCBI website (http:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.) A phylogenetic tree was constructed using the MEGA 5.1 software package with ClustalW and the adjacency algorithm.

The construction method of the high-yield 9-OHAD mycobacterium provided by the embodiment specifically comprises the following steps:

2.2 RNA extraction and RT-qPCR analysis

Total RNA samples were pretreated with Dnase I, 2 μ L5 × g DNA buffer, 1 μ g total RNA, adjusted to 10 μ L with ddH2O, incubated at 42 ℃ for 3min, then immediately cooled ice according to kit instructions, then reverse transcription reaction mixtures were prepared 2 μ L10 × Fast RT buffer, 1 μ L RTEnzymaMix, 2 μ L FQ-RT primer MIX, 1 μ g total RNA, adjusted to 20 μ L with ddH2O, incubated at 42 ℃ for 15min, then incubated at 95 ℃ for 3min, then immediately cooled on ice-20 ℃ for storage for the next experiment-the mixtures were diluted 10 fold before NanoDrop quantitation.

Gene-specific qRT-PCR primers (Table 2) were designed and qRT-PCR was performed in cfx96(BioRad) using the SuperReal PreMix Plus kit (SYBRGreen) (Tiangen). 2. mu. L of the above cDNA samples were mixed with 0.6. mu. L + 0.6. mu. L specific oligonucleotides, 10. mu. L2 × SuperReal PreMix Plus and 6.8. mu. L RNase-free ddH₂O was thoroughly mixed in 20. mu. L reaction mixture RT-qPCR was performed for 15min of pre-denaturation at 95 ℃ for 10 sec for 40 cycles, at 60 ℃ for 20 sec, at 72 ℃ for 20 sec, followed by a melting curve phase at 55 ℃ to 95 ℃The strains grown in the source, the test group were strains grown in a phytosterol carbon source. Each sample was analyzed in triplicate. Relative expression levels were calculated using the 2- Δ Δ CT method with the 16SrRNA gene amplicon as an internal control.

2.3 construction of knock-out kstd Strain

To delete kstd1 or kstd3, first two fragments of upstream sequences 1112bp or 1361bp, respectively, were cloned by primers kstd1-up-f/kstd3-up-r or kstd 1-down-then fragments were ligated to 2175bp or 2656 by XbaI enzyme, then fragments of upstream sequences 1057bp or 1293 bp. kstd 3-down/-r, respectively, subsequently the fragments of upstream sequences 1055 bp or 2656bp were digested with HindIII/NotI, then cloned into plasmid p2NI L. finally, the PacI site of p2NI L was inserted using the PacI single digested selection marker cassette from pGoal19 to generate homologous recombinant plasmids pdel-td 1 or pdel-tdks 3. alkali treated, after having been treated with parkin, the pckscd 19, after having been successfully deleted from the pckstd 3978 to 99 bp, or after having been confirmed by electroporation using primers kstd 3978-99 bp, or deletion of the original gene by PCR with primers kstd 99 bp, finally plasmid 3-99 bp, after having been deleted by electroporation with the pckstd 99, the plasmid 3-20 bp, the plasmid 3-tdksd strain, or after having been successfully deleted by PCR.

2.4 construction of sterol metabolism-related Gene expression vector

2.5 Mycobacterium electrotransformation

2.6 extraction, detection and quantification of samples

Example 2 transcriptional analysis and engineering of Module genes

1. Transcriptional analysis and engineering of Module A genes

In bacteria, the oxidation of the 3' -hydroxyl group catalyzes the isodehydrogenase of cholesterol 5-en-3-one to cholesterol 4-en-3-one from hydroxysteroids (Hsd). A putative 3-oxo-5 α -steroid-4-dehydrogenase protein coding sequence, designated 5 α -reductase, was found to encode a putative protein of 263 amino acids.

In order to explore the potential functions of 3 β -hsd and 5 α -reductases in sterol bioconversion processes, transformation media such as sterol were transferred as carbon sources after activation of the strains with glycerol as a carbon source phytosterols were used as a key period for carbon source growth after 0-120 hours of inoculation of the transformation media.the expression response of both genes was systematically analyzed by quantitative PCR in FIG. 2a, the exploration of 3 β -hsd and 5 α -reductases detected in this study generally showed induction profiles.

Two genes, 3 β -hsd and 5 α -reductase, were selected to construct expression vectors based on the gene expression reaction of bacterial strains phytosterol as a carbon source to enhance mycobacteria MS136 sterol uptake capacity when the genes for 3 β -hsd and 5 α -reductase were overexpressed, 9-OHAD production increased, control was the strain of MS136 strain with the pMV261 plasmid (fig. 2c), MS136-H and MS 136-S9-OHAD production increased 8.5% and 9.1%, respectively.

2-Modular B Gene transcriptional analysis and engineering

The deletion of cytochrome P450 cyp125 gene will lead to the accumulation of 4-cholest-4-en-3-one, which cannot continue to convert cholesterol.fadD 19 encodes an acyl-CoA synthetase.fadA is an enzyme that catalyzes the thiolation of the linear fatty acid CoA (or acetoacetyl-CoA). 17 β -hydroxysteroid dehydrogenase also plays a role in the oxidation of the sterol side chain β.Hydroxysteroid dehydrogenase (17 β -hydroxysteroid dehydrogenase) protein is found to be named hsd 4A.

Side chain oxidation of sterols involves a variety of enzymatic catalysis. We selected four genes, cyp125, fadD19, fadA5 and hsd4A, for sterol expression response testing. Phytosterols were used as the key carbon source for growth after 0-120 hours of inoculation of the transformation medium. The expression responses of these four genes were systematically analyzed by quantitative PCR. In FIG. 3a, four genes, cyp125, fadD19, fadA5 and hsd4A, show expression responses to sterols. Cyp125, fadA5 and hsd4A showed expression responses occurring at 72 hours. FadD19 showed two expression responses at 72 and 120 hours.

Based on 1 unit β -sitosterol, when it degrades to intermediate AD or 9-OHAD, it undergoes a dozen steps of enzymatic reactions that can produce 3 units of propionyl-coa and 1 unit of acetyl-coa, to enhance the ability of the strain to degrade sterol side chains, we chose three genes (cyp125, fadD19 and fadA5) overexpression treatment to up-regulate sterol metabolism, unfortunately, we did not get the pressure to successfully express fadA 5.

Transcriptional analysis and engineering of 3-module C genes

Module C mainly comprises 3-ketosteroid-1-dehydrogenase (KstD) and 3-ketosteroid-9-hydroxylase (KSH), forming the structure of 9-hydroxy-1, 4-dien steroid (9-OHADD).

In screening industrial steroid microorganisms, KstD and KSH are key enzymes for the production of important precursors of sterols, such as AD and 9-OHAD. In order to search potential functions in the KSH and KstD sterol biotransformation process, glycerol is used as a carbon source to activate the strain, sterol is used as a carbon source of a culture medium, and the key period of phytosterol in the culture medium can be transformed within 0-120h after inoculation. Growth of carbon source, and systematic analysis of gene expression of two reactions by quantitative PCR method. In fig. 4c, KstD1 and KstD3 showed expression responses at 96 hours, while KstD2 showed no expression responses. In FIG. 4b, the expression response of KSHA1 and KSHB appeared at 72 hours, the expression peak appeared at 120 hours, and the expression level of KSHA2 was almost unchanged.

In 9 α -hydroxylation of steroids, KSH activity consists of two parts, terminal oxygenase (KSHA of KSH) and iron oxidase (KSHB of KSH). multiple KSHA activities were observed in some strains using strong sterols, indicating an important role for KSHA in 9 α -hydroxylation.

Because of the transcriptional silencing of KstD2, knock-down of KstD homologs was constructed based on KstD1 and KstD3 in this fraction for accumulation of 9-OHAD. In FIG. 4e, it can be seen that the MS136-delkstd deletion strain showed similar growth rate on glycerol as its parent strain for the first 48 hours. However, from 60 hours on, the growth rate of MS136-delkstd was significantly faster than MS 136. The strains MS136-delkstd1 and MS136-delkstd1+3 were not viable in phytosterols, whereas MS136 and MS136-delkstd2 were able to grow normally. In FIG. 4f, the 9-OHAD yield started to increase significantly after 72 hours, the 9-OHAD yields of MS136 and MS136-delkstd3 started to decrease rapidly, and the yields of MS136-delkstd1 and MS136-delkstd1+3 continued to increase. The yield reached the highest at 120 h. Knock-out of KstD1 had a significant effect on 9-OHAD accumulation. KstD3 had no significant effect on 9-OHAD accumulation.

Modular construction of 49-OHAD high-yield recombinant strains

Based on the molecular modification results of each module, we selected 3 genes (3 β -HSD, 5 α -reductase and KSH) with significantly increased 9-OHAD accumulation, constructed a common overexpression vector of the three genes and knocked-out kstD1 to construct a high-yield 9-OHAD strain, namely MS136-delkstd1+ H + S + AB fermentation results (FIG. 5b), and showed that the accumulation of MS136-delkstd1+ H + S + AB9-OHAD is slightly smaller than MS136-delkstd1 before 60 hours, but showed similar growth rate, and after 60 hours, the yield of MS136-delkstd1+ H + S + AB was significantly increased, 108 hours was highest, 6.6 g/L, and the yield of MS136-delkstd-pMV261 was also highest at 108 hours, 5.8 g/L.

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<400>27

gctctagagg tggaaccgcc gaagtgaga 29

<210>28

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

gctctagagt ggacgggctc tacgcggtcg 30

<210>29

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>29

ataagcggcc gccgtcccgc tcgatgaact cgg 33

<210>30

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>30

atgacctcag acatcaaggt gttcg 25

<210>31

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>31

cgaacacctt gatgtctgag gtcat 25

<210>32

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>32

gctcgtgtcg tgagatgtt 19

<210>33

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>33

tgtagcccag gtcataagg 19

<210>34

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>34

accaaagtcg tggcggagaa at 22

<210>35

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>35

gatgttcttg ttgccgacca gc 22

<210>36

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>36

tcattgcccg gttgtctgtt gg 22

<210>37

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>37

cgatctgacg agcgagttct tcca 24

<210>38

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>38

ggattcccca agggtgtcat 20

<210>39

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>39

catccaggtg gccgactgcg 20

<210>40

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>40

gccgttctgc tcaacatgga cg 22

<210>41

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>41

gttcttggcg atctgctgtg cg 22

<210>42

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>42

aggaagcggt caacaagtac aac 23

<210>43

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>43

gtcttctcca gcgcatattg agtg 24

<210>44

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

acctatgaca cggagatcat c 21

<210>45

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

tcaacaacat tcgcgcatcc 20

<210>46

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

ttctacatcc acttcggtct gc 22

<210>47

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>47

cttgtagccg ctgtagttgt tg 22

<210>48

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>48

gcctggacgc cgactttctg t 21

<210>49

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>49

tagcacaaga ccacccgacc g 21

<210>50

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>50

ccgattacta ccccgagacc cc 22

<210>51

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>51

ggagcggacg ccgaccttga tg 22

<210>52

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>52

gcctgcccac catcatcaag cg 22

<210>53

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>53

cagttccgtc agtgcggtgt cg 22

<210>54

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>54

tgctggatga ggcgtggtgg tt 22

<210>55

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>55

ttgatgaatc gcctgccctc gc 22

Claims

1. A method for producing 9-OHAD-highly productive mycobacteria comprising the steps of:

RNA extraction and RT-qPCR analysis

RNA was isolated from mycobacterial sterol transformation media using RNAprep pure cell/bacteria kit (TIANGEN), total RNA from different samples was concentrated in 30. mu. L RNase-free water according to kit instructions, RNA quality was verified using NanoDrop (NanoDrop 2000, Thermo) and 1% (w/v) agarose gel electrophoresis;

reverse transcribing 1. mu.g total RNA using the FastQuant cDNA kit (TIANGEN), pretreating the total RNA sample with DnaseI according to the kit instructions, 2. mu. L5 × g DNA buffer, 1. mu.g total RNA, adjusted to 10. mu. L with ddH2O, incubating at 42 ℃ for 3min, then immediately cooling the ice, then preparing a reverse transcription reaction mixture, 2. mu. L10 × Fast RT buffer, 1. mu. L RTEnzyma Mix, 2. mu. L FQ-RT primer MIX, 1. mu.g total RNA, adjusted to 20. mu. L with ddH2O, incubating at 42 ℃ for 15min, then incubating at 95 ℃ for 3min, then immediately cooling on ice, -20 ℃ preservation, -diluting the mixture 10-fold before quantitation in NanoDrop;

gene-specific qRT-PCR primers were designed and qRT-PCR was performed in cfx96(BioRad) using the SuperReal PreMix Plus kit (SYBR Green) (Tiangen), 2. mu. L of the above cDNA samples were mixed with 0.6. mu. L + 0.6. mu. L specific oligonucleotide, 10. mu. L2 × SuperReal PreMix Plus and 6.8. mu. L RNase-free ddH₂O is mixed completely in a 20 μ L reaction mixture, RT-qPCR reaction is carried out for 15 minutes of pre-denaturation at 95 ℃ for 10 seconds for 40 cycles, at 60 ℃ for 20 seconds, at 72 ℃ for 20 seconds, followed by a melting curve phase at 55 ℃ to 95 ℃, control is a strain grown in a glycerol carbon source, test is a strain grown in a phytosterol carbon source, each sample is analyzed in triplicate, relative expression levels are calculated using the 2- Δ Δ CT method with the amplicon of the 16SrRNA gene as an internal control;

construction of (two) knock-out kstd Strain

Constructing a targeted gene deletion mutant of M.mycobacterium MS136, cloning first two fragments of upstream sequences 1112bp or 1361bp, downstream sequences 1057bp or 1293bp, tdks 3-down/-r, in order to delete kstd1 or kstd3, respectively, by primers kstd1-up-f/kstd3-up-r or kstd1-down-, then ligating the fragments to p 5bp or 2656 by XbaI enzyme, subsequently digesting the 2175bp or 2656bp fragment with HindIII/NotI, then cloning into plasmid p2NI L, finally inserting a PacI site of p2 using a PacI single digested selection marker cassette from pGoI 19 to generate homologous recombinant plasmids 1 or tdksel-tdpdpdpdp 3, after treatment with alkali, inserting the PacI site of the PacI gene into the PacI site of p2, as described above, after successful gene deletion of the original plasmid transfected cells from Gordks 3, or after deletion of the original plasmid 1bp, deletion of the mutant by PCR, finally deleting the mutant from the mutant with primers kstd 1bp 3-9-99, the sequence of the mutant, which was confirmed by electroporation, deletion of the mutant by PCR, using primers kstd1, or the primer kstd 369-19 bp, or the primer, the sequence of the deletion of the mutant after deletion of the mutant, which was determined by PCR, which was found to find the deletion of the original plasmid 3bp, or after deletion of the deletion mutant was found by PCR;

(III) construction of sterol metabolism-related Gene expression vector

Cloning of the gene 3 β -Hsd from the genome of MS136 using Hsd-pf & pr, BamHI-HindIII and ligating it to the plasmid pMV261 at the BamHI-HindIII site, introducing the plasmids pMV261 and pMV261-Hsd into MS136 to obtain MS136 and MS136-H, respectively, using 5-reductase-pf & pr, cyp125-pf & pr, fade 19-pf & pr, fadA5-pf & pr, kshA-pf & pr and kshB-pf & 829, cloning of the gene 5 α -4std, cyp125, fade 19, fadA5, hA and kshB from the genome of MS136, constructing the plasmid containing the genes HindIII + XbXksH + XksH, XksH & BamHI + XshIXksH & gt, XshIK & gt, XshIfSfS 136-AB 136-F, MS136-AB 136-S and MS136 gf-AB, respectively, using Hsd-pfIXshIJVdV 261-9, XshIJVdXshIJV, XshIJVdXshIJK & dSfI + XshIfI & dXhA & dSfI, plasmid, BamHI & dXshIfXshIfXshIfXshIfXshIfI & dXshIfI & dXpr829 & dXshIfI & dXshIfS, BamHI & d;

(IV) Mycobacterium electrotransformation

Treating an electric rotating cup (2mm), namely spraying 75% alcohol on the electric rotating cup, drying the electric rotating cup under ultraviolet light on a superclean bench, putting the electric rotating cup into an ice box for ice bath for 10min, putting a mycobacteria sensitive state tube on the ice box ice, standing the plasmid on the ice for 10min, adsorbing the competence added with the plasmid into the electric rotating cup, setting an electric rotating instrument parameter voltage 1800V, putting the electric rotating cup into an electric rotating instrument groove, performing electric rotation for 2 times, performing electric rotation, then performing ice bath for 3min, adding a seed culture medium of 800u L into the superclean bench, recovering for 2-3h in a 170r shaking table, centrifuging for 5min at 4000rpm, discarding supernatant, leaving about 100u L, resuspending the cells, sucking the cells onto a solid culture medium plate containing corresponding antibiotics by a liquid transfer gun, uniformly coating the solid culture medium plate until the cells are dry, allowing the colonies to grow, and selecting the bacteria for verification after 5-7 days;

(V) sample extraction, detection and quantification

The product is treated by placing 500u L fermentation liquor in a centrifuge tube, centrifuging for 15min at 4000rpm, sucking supernatant with an injector, filtering with 0.22um water system membrane into a clean centrifuge tube for standby, sucking 10u L filtrate, adding into 990u L diluent, mixing well for subsequent analysis and detection, and storing the rest filtrate at-20 deg.C;

the detection of the product 9-OHAD is that the 9-OHAD has a characteristic absorption peak under 240nm ultraviolet wavelength, but the substrate phytosterol has no characteristic absorption, so the product is quantitatively analyzed by adopting a HP L C method determination standard curve, the chromatographic conditions comprise that a chromatographic column is C18(250mm × 4.6.6 mm), the detection wavelength is 240nm, the column temperature is 50 ℃, the flow rate is 1.0m L/min, the mobile phase comprises ethanol, water, glacial acetic acid (520:480:0.1), the diluent comprises acetonitrile, water (50:50), and the sample injection amount is 20u L;

the primer sequences are as follows: