CN111733122A

CN111733122A - Mutant strain HGMS2 for preparing 4-androstene-3, 17-dione (4-AD)

Info

Publication number: CN111733122A
Application number: CN202010694128.8A
Authority: CN
Inventors: 苏正定; 成细瑶; 周曦
Original assignee: Wuhan Emojiahua Biotechnology Co ltd
Current assignee: Wuhan Emojiahua Biotechnology Co ltd
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-10-02

Abstract

The present application provides mutants of strain HGMS2 for the production of 4-androstene-3, 17-dione (4-AD). The KstD and Ksh genes of the mutant strain HGMS2 were knocked out. Compared with the wild type HGMS2 strain, the HGMS2 strain with the KstD and Ksh genes knocked out has the advantage that the generation of ADD and 9-OH-AD is remarkably reduced by the knocked-out strain. In addition, the mutant strain with the KsthA and KstD genes knocked out in the fermentation process improves the yield of 4-AD and reduces impurities.

Description

Mutant strain HGMS2 for preparing 4-androstene-3, 17-dione (4-AD)

Technical Field

The application relates to the field of biology, in particular to mutant HGMS2 strain for preparing 4-androstene-3, 17-dione (4-AD).

Background

Steroids have antibacterial, anti-inflammatory, antiviral and anticancer therapeutic effects, and the core structure of steroids is usually composed of three six-membered cyclohexane rings and one five-membered cyclopentane ring. The synthesis of higher steroid drugs relies on the most important starting materials 4-androstene-3, 17-dione (4-AD) and androsta-1, 4-diene-3, 17-dione (ADD). The production of 4-AD is more important, since the latter can be synthesized from 4-AD.

Currently, 4-AD is an environmentally friendly material produced by bioconversion of phytosterols by bacteria containing incomplete steroid degradation pathways (4-AD is produced in an environmentally friendly manner by bioconversion of phytosterols using bacteria having incomplete steroid degradation pathways). Although many actinomycetes contain steroid degradation pathways, including Pseudomonas NCIB10590 and Nocardia monomorph VKM Ac-2033D, only strains of Mycobacterium are suitable for industrial use. Mycobacterium isolated from nature NRRL B-3805 is the first bacterium to be used for industrial scale conversion of phytosterols to 4-AD. Other strains of Mycobacteria, such as Mycobacterium nwIB-01 and Mycobacterium HGMS2, have subsequently been isolated for industrial use. All these strains belong to the new Mycobacterium aurum group so far. However, these strains produce multiple 4-AD analogues during phytosterol conversion, making increased 4-AD production and its purification in industry difficult.

In the past decade, the disclosure of the molecular mechanisms of the bacterial steroid degradation pathway has benefited from the biochemical properties of key enzymes, the identification of gene clusters, and the comprehensive information annotated to the genomes of different bacteria that break down steroids. In general, the formation of 9, 10-secosteroids is a central pathway and further extends to three pathways, the cholesterol degradation pathway, the cholic acid degradation pathway and the testosterone degradation pathway. The genes involved in regulating bacterial degradation of phytosterol are up to 260. Industrial microorganisms used for bioconversion of phytosterols mainly utilize the cholesterol degradation pathway to oxidize 3-hydroxyl group and degrade the side chain of phytosterol, and then accumulate steroid intermediates. NRRL B-3805 in the case of incomplete degradation of 4-AD, phytosterols were degraded using 11 enzymes in 14 consecutive steps. M. smegmatis has an additional gene cluster, distinct from the cholesterol degradation pathway, for the metabolism of the C and D steroid rings. The cholesterol degradation pathway of Rhodococcus RHA1 strain shows stronger activity on the degradation of ADD and 3, 4-DHSA. The cholesterol degradation pathway in new s.aureus ATCC 25795 uses hydroxysteroid dehydrogenase (Hsd4A) as a molecular switch to distinguish between the two competing branched pathways, the AD and HBC sub-pathways of sterol side chain degradation.

In the bacterial cholesterol degradation pathway, the two most important key enzymes, 3-sterone-1, 2 dehydrogenase (KstD) and 3-sterone-9 α -hydroxylase (Ksh), cause B-ring cleavage at position C9, 10. KstD is a Flavin Adenine Dinucleotide (FAD) -dependent enzyme that introduces a double bond at a position between the C1 and C2 atoms of the A ring of 3-ketosteroid substrates. A search in the NCBI protein database showed that more than 500 different microorganisms contained the putative KstD enzyme gene, mainly in the actinomycetales genus. Importantly, a single species may contain multiple substrate-specific KstD. For example, Rhodococcus strains have three different KstD genes. The M.aureus DSM 1381 also contains three different KstD enzyme genes, while the Gordonia neofelifecisNRRL B-59395 contains up to five KstD enzyme genes. Each KstD enzyme of each species exhibits uniqueness in both amino acid sequence and three-dimensional conformation.

Although traditional UV or chemically mediated mutagenesis strategies and modern metabolic engineering techniques have been employed to increase the production of 4-AD and other sterol compounds, the associated progress has not been significant due to the complexity of the phytosterol degradation pathway.

Disclosure of Invention

In order to solve the above problems, the present application provides a mutant HGMS2 strain for preparing 4-androstene-3, 17-dione (4-AD), wherein KstD and Ksh genes of the mutant HGMS2 strain are knocked out.

Among the above HGMS2 strain mutants, wherein the HGMS2 strain mutant is incapable of producing the KstD enzyme KstD211 and is incapable of producing at least one of the KshA226, KshA395 and KshB122 enzymes.

Compared with the wild type HGMS2 strain, the HGMS2 strain with the KstD and Ksh genes knocked out has the advantage that the knock-out strain obviously reduces the production of ADD and 9-OH-AD. In addition, the mutant strain with the KsthA and KstD genes knocked out in the fermentation process improves the yield of 4-AD and reduces impurities.

Drawings

Fig. 1 shows that the KstD and Ksh enzymes are responsible for the accumulation of 4-AD and its degradation in phytosterol catabolic processes in m.sp.hgms2. Wherein the core structure of the steroid comprises four rings, ring A, B, C and D, which are labeled on phytosterols. The subsequent step involves 9 a-OH-4-anhydroSTAdiene-3, 17-dione (9-OH-ADD), which is a transition intermediate and is converted to HSA. 4-AD: 4-androstenone-3, 17-dione; ADD: 1, 4-androstenone-3, 17-dione; HSA: 3-hydroxy-9, 10-docosyl-1, 3,5(10) -triene-9, 17-dione; 9 OH-AD: 9 alpha-hydroxyandrostan-4-ene-3, 17-dione.

Figure 2 shows the HPLC profile of a large scale m.sp.hgms2 extract. Fermentation using beta-sitosterol as sole carbon source was at 144h compared to 4-AD and ADD. 1.4-AD; 2, ADD; 3.9 OH-AD; BA; *: unknown metabolites when the strain is cultured in the absence of phytosterols. BA: 21-hydroxy-20-methylpregn-4-en-3-one.

FIG. 3 shows the construction of a gene knockout vector for knocking out the KstD and Ksh genes in HGMS 2. a) A homologous recombination vector was constructed based on the p2NIL plasmid. U: an upstream sequence; d: a downstream sequence. b) A list of recombinant vectors constructed for knocking out four targeted genes from HGMS2 strain. c) PCR validation of p2NILSac-KstD vector. M: DNA marking; WT: m.sp.hgms2 genomic PCR products, including the KstD gene and its upstream and downstream sequences from amplification; KstD-ko: PCR products amplified from p2NILSac-KstD vector. d) PCR validation of p2NILSac-KshA226 and p2NILSac-KshA395 vectors. M: DNA marking; KshA226-1 and KshA 226-2: two PCR products amplified from putative colonies harboring the p2NILSac-KshA226 vector; KshA395-1 and KshA 395-2: two PCR products amplified from putative colonies harboring the p2NILSac-KshA395 vector. WT: m.sp.hgms2 genomic PCR products, including the KshA226 gene and its upstream and downstream sequences from amplification. e) PCR validation of knock-out p2NILSac-KshB122 vector. M: DNA marking; KshB 122: PCR products amplified from putative colonies harboring the p2NILSac-KshB122 vector.

FIG. 4 shows that knocking out the KstD211 gene from HGMS2 abolished ADD production during phytosterol transformation. a) PCR products amplified from putative KstD knockout colonies were analyzed by agarose gel electrophoresis and compared to PCR products amplified from the WT strain. M: DNA marking; WT: PCR products amplified from m.sp.hgms2 genome; KstD1 and KstD 2: two putative KstD knockout mutants. b) TLC assay of catabolites extracted from KstD1 mutant phytosterol conversion, compared to WT strain and standards for 4-AD and ADD. Bands for 9OH-AD are marked with arrows. c) HPLC profile of phytosterol fermented extract of HGMS2 hKstD1 mutant at 144h using β -sitosterol as sole carbon source compared to WT strain. 1.4-AD; 2, ADD; 3.9 OH-AD; BA; *: unknown metabolites when the HGMS2 strain was cultured in the absence of phytosterols.

FIG. 5 shows that the deletion of a single Ksh gene from the mutant HGMS2KstD1 eliminates ADD and 9-OH-AD during phytosterol transformation. PCR products amplified from putative Ksh knock-out colonies were analyzed using agarose gel electrophoresis and compared to products amplified from three Ksh knock-out vectors and WT strains. M: DNA marking; KshA 226: the putative HGMS2KstD1+ KshA226 mutant. KOA 226-Pl: PCR products amplified from p2NILSac-KshA 226; WT: PCR products amplified from m.sp.hgms2 genome; KshA 395: the putative mutant HGMS2KstD1+ KshA 395. KOA 395-Pl: PCR products amplified from p2NILSac-KshA 395; shKshB122a and KshB122 b: two putative HGMS2KstD1+ KshB122 mutants; KOB 122-P1: PCR products amplified from p2NILSac-KshB 122; d) TLC assay of extracted catabolites in wild type strains, single mutants of the KstD gene and double mutants plus one of the three Ksh genes knocked out. KstD1, KshB122, KshA226 and KshA395 refer to samples from HGMS2KstD1, HGMS2KstD1+ KshB122, HGMS2KstD1+ KshA226 and HGMS2KstD1+ KshA395 mutants, respectively. The 9OH-AD weaknesses of the HGMS2KstD1 and HGMS2KstD1+ KshA226 mutant samples are marked with arrows.

FIG. 6 shows HPLC profiles of β -sitosterol fermentation extracts at 144h for the three double mutants compared to the WT strain and the HGMS2KstD1 mutant. 1.4-AD; 2, ADD; 3.9-OH-AD and 4. BA. *: an unknown metabolite produced when the m.sp.hgms2 strain is cultured in the absence of phytosterols.

Detailed Description

The following examples are presented to enable those skilled in the art to more fully understand the present application and are not intended to limit the present application in any way. Unless otherwise indicated, percentages herein refer to mass percentages.

Ksh is an iron-sulfur monooxygenase consisting of two components, the oxidase KshA and the reductase KshB. Ksh homologs are distributed across multiple developmental populations. Mycobacterium VKM Ac-1817D efficiently converts phytosterols to 9-OH-AD using five KshAs and two KshBs. Rhodococcus DSM43269 contains five KshA homologues, and each KshA exhibits a different specificity for a different substrate. Among the current strains producing 4-AD and ADD, a number of predicted Ksh genes have been identified. Thus, removal or reduction of this Ksh activity can hinder steroid ring opening, allowing 4-AD and ADD to accumulate.

The new mycobacterium aureum HGMS2(mycobacterium. sp. hgms2) is currently one of the most important strains used in the pharmaceutical industry for the transformation of phytosterols. M.sp.hgms2 can efficiently convert phytosterols to androstenedione (4-androstene-3, 17-dione, 4-AD), which is one of the important steroids for the synthesis of higher steroid drugs. However, m.sp.hgms2 produces many 4-AD analogs during phytosterol conversion, not only reducing the yield of 4-AD, but also hindering its purification process. Recently, the inventors have identified a 3-ketosteroid 1, 2-dehydrogenase (KstD) and three 3-ketosteroid-9 α -hydroxyzymes (Khss) that remain active in Mycobacterium HGMS 2. In this work, the KstD and Ksh genes were knocked out from m.sp.hgms2 using an internally designed homologous recombination approach. The resulting mutants can significantly reduce or eliminate 4-AD analogs or other by-products, such as 1, 4-androstene 3,17-dione (ADD) and 9 α -hydroxyandrostene 4-ene-3, 17-dione (9 OH-AD). One double mutant (M.sp.HGMS2 KstD1+ KshB122) significantly enhanced the yield of phytosterols converted to 4-AD. Compared with wild strains, under pilot scale fermentation, the conversion rate of phytosterol to 4-AD and 4-AD yield is respectively improved by 1.4 times and 1.1 times. More importantly, the double mutant retained the same phytosterol catabolic efficiency as the wild type strain, while not producing ADD and 9 OH-AD. Thus, a highly efficient m.sp.hgms2 strain for the conversion of phytosterols to 4-AD is provided.

One active KstD enzyme (KstD211) and three active Ksh enzymes (KshA226, KshA395 and KshB122) were identified in a strain m.sp.hgms2 used in the pharmaceutical industry for the production of 4-AD by whole genome functional annotation and enzymatic analysis. These active enzymes are associated with the production of 4-AD analogs and 4-AD degradation during phytosterol fermentation (FIG. 1). In this work, the effect of these genes on 4-AD accumulation during phytosterol transformation was investigated using an internally designed homologous recombination approach. Compared with the wild type HGMS2 strain, the HGMS2 strain with the KstD and Ksh genes knocked out has the advantage that the knock-out strain obviously reduces the production of ADD and 9-OH-AD. In addition, the mutant strain with the KsthA and KstD genes knocked out in the fermentation process improves the yield of 4-AD and reduces impurities. Thus, highly efficient bacterial strains for the conversion of phytosterols to 4-AD are provided.

Some m.sp.hgms2 strain mutants were generated by knocking out the KstD and Ksh genes. The mutant shows improved performance in phytosterol conversion to 4-AD compared to the wild type strain. After only one KstD enzyme, namely KstD211, is knocked out from the HGMS2 strain, as the solid phase mutant HGMS2KstD1 strain loses the capability of converting 4-AD into ADD, the main impurity, namely ADD, in the phytosterol fermentation liquor disappears. In addition, three Ksh genes were knocked out from the HGMS2KstD1 mutant, the HGMS2KstD1+ KshA395 and HGMS2KstD1+ KshB122 mutants completely blocked the occurrence of 9OH-AD, while the HGMS2KstD1+ KshA226 mutant still converted 4-AD to 9 OH-AD. These observations are consistent with previous studies, although KshA226 is inactive against 4-AD, as compared to KshA395 and KshB122, despite the higher activity of these three Ksh enzymes against ADD. Thus, knockout of the KstD gene followed by knockout of the Ksh gene is beneficial for phytosterol conversion to 4-AD. Indeed, the HGMS2MSKstD1+ KshB122 mutant increased the conversion by an average of 1.4-fold, reaching 89.7%, as tested in pilot scale fermentations. The yield of 4-AD of the double mutant increased to 7.2g/L, i.e., 1.1-fold. More importantly, the KstD and Ksh gene knockout m.sp.hgms2 strains were found to have no significant effect on cell growth and primary metabolism compared to the wild-type strain.

In summary, the present application provides a highly efficient mutant strain of Mycobacterium HGMS2 for use in the pharmaceutical industry for the production of 4-AD. In addition, the only pair of KshA395/KshB122 enzymes in the Mycobacterium HGMS2 strain exhibited activity against 4-AD. This gene knockout study helped understand the phytosterol catabolism mechanism in the m.sp.hmgs2 strain.

The following description is given in conjunction with specific examples to better understand the present application.

Example 1 fermentation Process for converting wild bacteria and mutant bacteria into beta-sitosterol by HGMS2

This experiment was performed as a pilot experiment in a 15L fermentor. Yeast extract 15g/L, glucose 6g/L, (NH)₄)₂HPO₄0.6g/L,NaNO₃5.4g/L, β -sitosterol 8%, dispersant 8g/L, soybean oil 180g/L, and lecithin 3g/L of fermentation medium before fermentation, β -sitosterol and dispersant are mixed and emulsified, all the raw materials are added into a 15L fermentation tank, water is added until the total fermentation volume is 10L, the fermentation medium is sterilized at 121 ℃ for 30 minutes, after sterilization, the temperature of the fermentation liquid is controlled at 30 ℃, the rotation speed is 500rpm/min, the prepared seed solution is inoculated into the fermentation tank by a flame inoculation method, the temperature and the speed of the container are maintained, and sampling detection is carried out periodically until the fermentation is finished.

EXAMPLE 2 HPLC analysis of 4-AD analogues in fermentation extracts

M.sp.hgms2 has good conversion and 4-AD yield. Fermentation was carried out for 7-10 days in soybean oil-based medium using β -sitosterol as substrate, accumulation of 4-AD major catabolite from m.sp.hgsm2. After fermentation, the fermentation broth after 7 days of beta-sitosterol fermentation was analyzed by HPLC assay. As shown in FIG. 2, the main component was 4-AD. Byproducts accumulated during fermentation include ADD, 9OH-AD and BA. The average contents of the four components are 80.21%, 10.73%, 1.83% and 1.94%, respectively, and the remaining components are unknown metabolites not related to phytosterol catabolism.

EXAMPLE 3 construction of Gene knockout homologous recombination vector

In a phytosterol fermentation process performed by m.sp.hgms2 strain. Active KstD and Ksh enzymes reduce the accumulation of 4-AD by forming 9, 10-secosteroids. Therefore, the present application uses a method of homologous recombination to knock out these genes from the genome of m.sp HGMS2 strain. Prior to construction of the homologous recombination vector, the m.sp.hgms2 strain was tested for antibiotic resistance and appropriate antibiotic markers were selected. Although many mycobacteria are resistant to a variety of antibiotics, the present application demonstrates that the m.sp.hgms2 strain is not resistant to kanamycin (Km), tetracycline (Tc), streptomycin (Sm) and spectinomycin (Sm). Thus, the present application selects Km as an antibiotic resistance marker and selects a sucrose lethal gene (SacB) as a second selection marker to construct a knock-out vector.

SacB was integrated into this plasmid p2NILSac using plasmid p2NIL containing the Km gene as template (FIG. 3). To knock out the KstD and Ksh genes, DNA fragments containing 1000bp flanking each target gene were amplified using PCR and inserted into the BamHI and HindIII sites of the p2 nildac vector. As a result, four recombinant vectors were constructed for knocking out KstD211, KshA226, KshA395 and KshB122 genes from the m.sp.hgms2 genome. The four vectors were designated p2 NILSac-. DELTA.KstD, p2 NILSac-. DELTA.KshA 226, p2 NILSac-. DELTA.KshA 395, and p2 NILSacB-. DELTA.KshB 122, respectively. The constructed vector was confirmed by PCR assay.

Table 1 shows the homologous recombination sequences used to knock out the targeted gene in the HGMS2 mutant.

TABLE 1

The primers used for the construction of the knock-out vectors are shown in table 2.

TABLE 2

Example 4 identification of knockout mutants by PCR method and Gene sequencing

The bacteria grown on sucrose solid medium are all mycobacteria that have undergone secondary recombination. Single colonies were picked for PCR validation. The content of GC in the mycobacteria genome is high, the annealing temperature is not lower than 65 ℃, and the interference of nonspecific amplification is prevented. And taking an upstream homology arm front primer and a downstream homology arm rear primer of the knocked-out gene as a front and rear pair of primers, and taking a bacterial colony as a template for verification. Because the secondary recombination has two types of knockout and wild type, the size of the band is different after PCR amplification, and the wild type fragment is about 1600bp larger than the knockout, so the knockout strain is screened out through PCR identification. Transferring the successfully verified knockout strain into 5mL LB liquid medium containing 0.05% Tween-80, culturing at 30 ℃ and 200rpm for 2 days, extracting genome, performing PCR identification by using the genome as a template and the same primer, and sequencing the knockout genome. Genome-verified knockout strains were stored at-80 ℃.

Example 5 characterization of Mycobacterium KstD knockout mutants

Since ADD is a major byproduct, presumably caused by active KstD211, the present application first identified knockout of the KstD211 gene from the m.sp.hgms2 genome. Knock-out of the KstD211 gene was accomplished by electroporation of the p2 nildac- Δ KstD plasmid into fresh m.sp.hgms2 competent cells. Screening was then performed in two steps using a kanamycin and sucrose based method. Positive recombinant colonies were identified by colony PCR. Once the KstD211 gene was knocked out from the m.sp.hgms2 genome, the PCR product size became shorter (1600bp) compared to the wild type strain (3600 bp). As shown in fig. 4, two positive colonies, HGMS2 Δ KstD1 and HGMS2 Δ KstD2 mutants, were identified. The results of DNA sequencing confirmed that the KstD211 gene was knocked out from both HGMS2 Δ KstD1 and HGMS2 Δ KstD2 mutants. Thus, the present application randomly selected the Δ KstD1 mutant to further study small-scale fermentation of β -sitosterol.

After 7 days of fermentation of β -sitosterol, the catabolites were extracted with solvent and evaluated by Thin Layer Chromatography (TLC) analysis. As shown in FIG. 4, ADD was almost completely disappeared as compared with the WT strain. However, 9OH-AD was detectable on TLC plates. In addition, based on TLC density, the extract was diluted to the same concentration as the WT sample and subjected to HPLC assay. As shown in fig. 4, the knock-out of the KstD211 gene resulted in HGMS2 Δ KstD1 strain with almost no ADD (peak 2 in fig. 4). In addition, an increase in 9OH-AD production was also observed (peak 3 of FIG. 4), indicating that 9OH-AD accumulation is associated with KstD211 activity. When 9, 10-ring-opened steroid formation involving KstD211 is hindered, accumulation of 9-OH-AD occurs.

Example 6 characterization of Mycobacterium KstD and Ksh Gene knock-out mutants

As previously described, m.sp HGMS2 contains two pairs of KshA/KshB modules that share one KshB. To explore which Ksh enzymes are responsible for the formation of 9-OH-AD, the present application knocked out each Ksh gene based on the HGMS2 Δ KstD1 mutant using the same knockout strategy as for making the HGMS2 Δ KstD1 mutant. As shown in fig. 5, positive recombinant colonies, i.e., HGMS2KstD1+ Δ KshA226, HGMS2KstD1+ Δ KshA395, and HGMS2KstD1+ Δ KshB122 mutants, were identified by colony PCR, in which the KstD211 gene was also knocked out. DNA sequencing also confirmed that these Ksh genes had been knocked out of the three mutants.

The three Ksh mutants were further tested for phytosterol conversion using β -sitosterol as substrate. After 7 days of fermentation of the beta-sitosterol, the catabolites in the fermentation broth were extracted with solvent and shown by TLC analysis. As shown in FIG. 5, the three Ksh mutants showed different effects on 9-OH-AD accumulation. One spot was found for the sample from the HGMS2KstD1+ Δ KshA226 mutant, while no detectable 9-OH-AD spot was visible for the other two mutants. This observation is consistent with previous results, i.e., KshA226 is inactive against 4-AD, as compared to KshA395 and KshB 122. Furthermore, HPLC analysis of these extracts clearly showed that the 9-OH-AD generation was completely blocked during phytosterol conversion using the HGMS2KstD1+ Δ KshA395 and HGMS2KstD1+ Δ KshB122 mutants, as shown in FIG. 6.

Those skilled in the art will appreciate that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the application.

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Claims

1. An HGMS2 strain mutant for preparing 4-androstene-3, 17-dione (4-AD), wherein the KstD and Ksh genes of the HGMS2 strain mutant are knocked out.

2. The mutant HGMS2 strain of claim 1, wherein the mutant HGMS2 strain is incapable of producing the KstD enzyme KstD211 and at least one of the KshA226, KshA395, and KshB122 enzymes.