Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment

Steroid medication is used extensively in clinical applications and comprises a large and vital part of the pharmaceutical industry. However, the difficulty of separating 4-androstene-3,17-dione （AD） from 1,4-androstadiene- 3,17-dione （ADD） restricts the application of the microbial transformation of phytosterols in the industry. A novel at- mospheric and room temperature plasma （ARTP） treatment, which employs helium as the working gas, was used to generate Mycobacterium neoaurum mutants producing large amounts of AD. After treatment of cultures with ARTP, four mutants were selected using a novel screening method with a color assay. Among the mutants, M. neoaurum ZADF-4 was considered the best candidate for industrial application. When the fermentation medium contained 15 g/L phytosterols and was cultivated on a rotary shaker at 160 r/min at 30 ~C for 7 d, （6.28＋0.11） g/L of AD and （0.82＋0.05） g/L of ADD were produced by the ZADF-4 mutant, compared with （4.83＋0.13） g/L of AD and （2.34＋0.06） g/L of ADD by the original strain, Iv~. neoaururn ZAD. Compared with ZAD, the molar yield of AD increased from 48.3% to 60.3% in the ZADF-4 mutant. This result indicates that ZADF-4 may have potential for industrial production of AD.

A NEW METHOD FOR THE AROMATIZATION OF 18-METHYL-ESTRA-4(5), 9(10)-DIEN-3,17-DIONE

An aromatization reaction was found when 18--methyl--estra--4(5), 9(10)--dien--3, 17--dione(6) was treated with triethyl orthoforrnate in dioxane in the presence of catalytic amount of p--toluenesulfonic acid (TsOH), and 18--methyl--9(11) --dehydroestrone--3--ethyl ether (8) was obtained in 70% yield. The mechanism was found to be a process of etherification--oxidation reaction.

Efficient production of 14α‑OH‑AD by engineered Mycolicibacterium neoaurum via coupled cofactor and reconstructed electron transport system

14α-hydroxy-androst-4-ene-3,17-dione(14α-OH-AD)is an important precursor for the synthesis of steroid drugs with anticancer and carcinolytic activity.Initially,14α-OH-AD was mostly synthesized by whole-cell fermentation of mold fungi using androstenedione(AD)as a substrate,which had difficulties in product isolation and purification as well as problems of high production cost.In this study,the source of the 14α-hydroxylase gene was expanded.And 14α-hydroxylase genes were heterologously expressed in Mycolicibacterium neoaurum(MNR)M3ΔksdD,which enabled the one-step biotransformation from the cheap substrate phytosterols(PS)to 14α-OH-AD,reducing the difficulty of product purification and production cost.What is more,to alleviate the problem of poor activity of 14α-hydroxylase,the 14α-hydroxylase gene was co-expressed with the electron transport chain element genes and the coenzyme regeneration genes,and a superior engineered strain MNR M3ΔksdD/pMV261-14α-G6PDH was obtained.Finally,the transformation conditions were optimized for the transformation of PS by the engineered strain.The molar yield of 14α-OH-AD reached to 60.4±2.3%(about 0.22 g/L productivity).This study investigated for the first time the effects of the tandem electron transport chain element genes and the tandem coenzyme regeneration genes on the 14α-hydroxylation reaction,providing a theoretical basis for the industrial production of 14α-OH-AD.

A streamlined high throughput screening method for the Mycobacterium neoaurum mutants with expected yield of biotransformation derivatives from sterols

Steroidal drugs have wide indications such as anti-inflammation, anti-tumor, endocrine regulation,fertility management. Phytosterol is the main starting materials for the industrial synthesis of steroid drugs. Microbial transformation of phytosterol is a simple and environmentally friendly process. Efficient microbial strains for industrial phytosterol transformation are critical for the commercial success. To this end, a 96-well plate based method was developed to discriminate the mixture of 4-androstene-3,17-dione, androsta-1,4-diene-3,17-dione, and bisnoraldehyde, with different ratio of the 3 components in the mixture, which mimics the sterol bioconversion products by using the Mycobacterium neoaurum. The M. neoaurum bioconversed broth test using phytosterol as substrate also found that the spectrum methodology can evaluate the relative content of different compounds. The method is practical, high throughput and can replace the conventional HPLC-based assay for rapid selection of desired microbial strains.