Mycobacterium neoaurum-derived steroid C27-monooxygenase and application thereof

Abstract

The present invention discloses a Mycobacterium neoaurum-derived steroid C27-monooxygenase and an application thereof, which belong to the technical fields of genetic engineering and enzyme engineering. By the method of gene knockout and intensive expression, the present invention screens out three isoenzymes of a key enzyme SMO in the process of degrading sterol side chains from Mycobacterium neoaurum. The three isoenzymes are intensively expressed respectively in the Mycobacterium neoaurum for the high yield of androsta-1,4-diene-3,17-dione (ADD), the yield of ADD is increased remarkably, wherein the effect of SMO2 is most remarkable. By overexpressing SMO2, the final ADD yield is increased from 5.2 g.Math.L.sup.1 to 7.3 g.Math.L.sup.1. The present invention provides a helpful guidance for the industrialization of the microbial fermentation method for increasing the ADD yield.

Claims

1. Recombinant Mycobacterium neoaurum wherein the recombinant Mycobacterium neoaurum overexpresses a steroid C27-monooxygenase gene; wherein the steroid C27-monooxygenase gene is a Mycobacterium neoaurum C27-monooxygenase gene selected from the group consisting of: Mycobacterium neoaurum Smo1 gene, Mycobacterium neoaurum Smo2 gene, and Mycobacterium neoaurum Smo3 gene wherein the amino acid sequences of the steroid C27-monooxygenase genes Smo1, Smo2, and Smo3 are SEQ ID NOS: 4, 5, and 6, respectively, and wherein the recombinant Mycobacterium neoaurum produces an increased yield of androsta-1,4-diene-3,17-dione (ADD) when fermented in the presence of cholesterol at 30 C. in fermentation medium that supports growth of Mycobacterium neoaurum compared to the yield of ADD produced by the Mycobacterium neoaurum lacking overexpression of the steroid C27-monooxygenase gene.

2. The recombinant Mycobacterium neoaurum according to claim 1, wherein the recombinant Mycobacterium neoaurum further comprises a recombinant plasmid comprising the steroid C27-monooxygenase gene.

3. A method for producing ADD which comprises; providing the recombinant Mycobacterium neoaurum of claim 1 as a production strain; and incubating the recombinant Mycobacterium neoaurum with phytosterol or cholesterol or both under conditions that support overexpression of the steroid C27-monooxygenase gene in the recombinant Mycobacterium neoaurum.

Description

DETAILED DESCRIPTION

(1) Main reagents: Phytosterol (soyasterol95%) was purchased from Lilly Biotechnology (Huzhou) Co., Ltd, and ADD was purchased from American SIGMA company.

(2) HPLC analysis of ADD: ADD had characteristic absorption peaks under an ultraviolet wavelength of 254 nm, so the HPLC method was adopted to determine product concentration. Chromatographic conditions: chromatographic column: DimosoilC18 (5 l, 250 mm4.6 mm); mobile phase: methanol-water (V/V=70:30); detector: UV Detector; detection wavelength: 254 nm; column temperature: 30 C.; sample amount: 104; and flow velocity: 1.0 ml.Math.min.sup.1.

Example 1: Construction of SMO Knockout Strains and Corresponding Complementation Strains

(3) By querying the whole genome information of Mycobacterium neoaurum, three SMO isoenzymes were screened out. With the Mycobacterium neoaurum with SMO enzyme activity in a lab as an original strain and its chromosome as a template, the PCR measure was utilized to obtain the genes of the three enzymes. By means of the design of gene knockout primers, the PCR measure was utilized to obtain knocked-out genes, which were connected to a Mycobacterium knockout plasmid p2NIL, so that knockout plasmids were constructed, and after the construction was verified successful by PCR, the knockout plasmids were transformed into the Mycobacterium neoaurum. With cholest-4-en-3-one as a substrate, the degradation of the substrate by knockout strains was tested. On the basis of the knockout strains, knocked-out gene complementation strains were constructed, that was, by designing primers, the PCR measure was utilized to amplify complete SMO genes, which were connected to an integration vector pMV306, so that integrative plasmids were constructed, and after the construction was verified successful by PCR, the integrative plasmids were transformed into the corresponding knockout strains, so that the knocked-out gene complementation strains were constructed; and under the same conditions, with cholest-4-en-3-one as a substrate, the degradation of the substrate by the complementation strains was tested. A test result indicated that the ability of the gene knockout strains in degrading cholest-4-en-3-one was impeded, while the knocked-out gene complementation strains decreased such an impeding effect to a certain degree. The present invention proved in the end that the three enzymes SMO were key enzymes in the process of sterol degradation.

(4) A specific construction method for the combinant strains was as follows:

(5) (1) Cloning the Complete Sequence of a Steroid C27-Monooxygenase (SMO) Gene

(6) According to the whole genome sequence (NC-023036) of Mycobacterium neoaurum VKM Ac-1815D published by the website GENBANK, three SMO genes (Smo1, Smo2, Smo3) were found out, and according to specific gene sequences, corresponding gene primers ere designed. With prepared Mycobacterium neoaurum chromosome DNA as a template, corresponding complete gene sequences were amplified out by PCR.

(7) PCR reaction system: 5 L of 10ExTaq Buffer; 4 L of dNTP; 1 L of template DNA; 0.5 L of upstream primer; 0.5 L of downstream primer; 1 L of ExTaq enzyme; and ddH.sub.2O accounting for the rest of a total volume of 50 L. PCR reaction condition: 94 C. 5 min; 94 C. 30 s; 65 C. 45 s; 72 C. 90 s; 35 times of cycling; 72 C. 10 min; and 12 C. 10 min.

(8) Referring to the instructions of a gel extraction kit of Shanghai Generay Biotech Co., Ltd, a PCR product was recycled. The gel extraction product was connected to pMD18-T vector overnight according to a certain proportion, E. coli JM109 competent cells were transformed, an ampicillin resistant plate was utilized to screen recombinant bacteria, recombinant plasmids underwent restriction enzyme digestion to release gene bands, the sizes of which were about 2.7 kb and 1.3 kb, this indicated that recombinant plasmid construction was successful, and the recombinant plasmids were named as pMD18-T-Smo1, pMD18-T-Smo2 and pMD18-T-Smo3.

(9) (2) Constructing Mycobacterium neoaurum Gene Knockout Plasmids

(10) Specific primers were designed, and by way of PCR, 300 bp sequences were amplified out from the upstream of Smo1, Smo2 and Smo3 and 450 bp sequences were amplified out from the downstream. The gene sequences amplified from the upstream and the downstream were mixed as a template, and the upstream primers of the upstream 300 bp sequences and the downstream primers of the 450 bp sequences were utilized to amplify out 750 bp sequences. The amplified sequences and a knockout plasmid p2NIL underwent double-restriction enzyme (KpnI and Hind III) digestion, gel extraction and purification were conducted, T4 DNA ligase was connected to two segments overnight, conjugates underwent heat shock to transform E. coli JM109 competent cells after overnight, and a kanamycin resistant plate was utilized to screen positive transformants. Transformant plasmids were extracted, and the construction of recombinant plasmids was verified successful by restriction enzyme digestion. Single-restriction enzyme (Pac I) digestion was utilized to cut off a selectable marker gene cassette in a plasmid pGOAL19 and inserted it into the restriction enzyme (Pac I) digestion sites of the constructed recombinant plasmids, so that knockout plasmids p2N-Smo1, p2N-Smo2 and p2N-Smo3 were constructed. The construction of the knockout plasmids was verified successful by PCR.

(11) (3) Constructing Mycobacterium neoaurum Knocked-Out Gene Complementation Plasmids

(12) A plasmid pMV306 was used for the complementation of knocked-out genes. Double-restriction enzyme (Xba Ida I) digestion was utilized to cut off the corresponding gene segments in the recombinant plasmids p261-Smo1, p261-Smo2 and p261-Smo3 along with heat shock promoters hsp60 and connected them onto corresponding restriction enzyme (pMV306) digestion sites, so that knocked-out gene complementation plasmids p306-Smo1, p306-Smo2 and p306-Smo3 were constructed.

(13) (4) Transforming the Recombinant Plasmids into the Mycobacterium neoaurum by an Electrotransformation Method

(14) A. The successfully constructed recombinant plasmids were transformed into the Mycobacterium neoaurum by using an electrotransformation method. The improved Gordhan and Parish method was adopted as the transformation method.

(15) B. Positive transformants of the recombinant strains M. neoaurum were screened;

(16) Colonies which grow out on plates with corresponding antibiotic pressures were chosen, flasks were shaken for fermentation, and the plasmids were extracted to be verified by restriction enzyme digestion.

Example 2: Construction of Recombinant Strains Intensively Expressing SMO

(17) A plasmid pMV261 was utilized to overexpress the gene in Mycobacterium neoaurum. Sac I and Hind III were utilized to carry out double-restriction enzyme digestion on pMD18-T-Smo1, BamH I and EcoR I were utilized to respectively carry out double-restriction enzyme digestion on pMD18-T-Smo2 and pMD18-T-Smo3, meanwhile, a corresponding restriction enzyme digestion site was utilized to carry out restriction enzyme digestion on the plasmid pMV261, gel extraction and purification were carried out on the corresponding gene segments and the plasmid pMV261 segment, T.sub.4 DNA ligase connected the two segments overnight, conjugates underwent heat shock to transform E. coli JM109 competent cells after overnight, and a kanamycin resistant plate was utilized to screen positive transformants. Transformant plasmids were extracted, the construction of recombinant plasmids p261-Smo1, p261-Smo2 and p261-Smo3 was verified successful by restriction enzyme digestion, the successfully constructed recombinant plasmids were electrotransformed respectively into Mycobacterium neoaurum JC-12 for the high yield of ADD (Mycobacterium neoaurum JC-12 had been disclosed by Shao M L, Zhang X, Rao Z M, Xu Mi, Yang T W, Li H, Xu Z H (2015) in the document of Enhanced production of androsta-1,4-diene-3,17-dione by Mycobacterium neoaurum JC-12 using three-stage fermentation strategy. PLoS ONE 10(9): e0137658), and thereby recombinant strains JC-12.sub.S1, JC-12.sub.S2 and JC-12.sub.S3 were obtained respectively.

Example 3: Increase of ADD Yield by Recombinant Strains Intensively Expressing SMO

(18) After being activated in a seed medium, the recombinant strains JC-12.sub.S1, JC-12.sub.S2 and JC-12.sub.S3 were inoculated into a fermentation medium according to an inoculation amount of 5%, fermentation was carried out with 20 g.Math.L.sup.1 cholesterol as a substrate under the conditions of 30 C. and 160 rpm for 168 h, and a fermentative transformation experiment was conducted. The seed medium consisted of 10 g.Math.L.sup.1 glucose, 10 g.Math.L.sup.1 peptone, 6 g.Math.L.sup.1 beef extract and 10 g.Math.L.sup.1 NaCl, and pH was 7.5. The fermentation medium consisted of 20 g.Math.L.sup.1 cholesterol, 20 g.Math.L.sup.1 glucose, 10 g.Math.L.sup.1 peptone, 6 g.Math.L.sup.1 beef extract, 3 g.Math.L.sup.1 K.sub.2HPO.sub.4, 0.5 g.Math.L.sup.1 MgSO.sub.4.Math.7H.sub.2O, 5104 g.Math.L.sup.1 MnCl.sub.2.4H.sub.2O and 60 g.Math.L.sup.1 hydroxypropyl--cyclodextrin, and pH was 7.5.

(19) The change of the ADD yield was detected. A result indicated that after 168 h of fermentative transformation, the ADD yield of the recombinant bacteria JC-12.sub.S2 overexpressing SMO2 was increased most remarkably, and increased from 5.2 g.Math.L.sup.1 Mycobacterium neoaurum JC-12 as the original strain to 7.3 g.Math.L.sup.1. In addition, the ADD yields of the recombinant bacteria JC-12.sub.S1 and JC-12.sub.S3 were increased respectively to 6.5 g.Math.L.sup.1 and 6.1 g.Math.L.sup.1.