Cholesterol and phytosterols can be served as the substrates for Actinobacteria to produce the precursors used in the synthesis of steroidal medicines.Cytochrome P450s from CYP125 and CYP142 families initiates the met...Cholesterol and phytosterols can be served as the substrates for Actinobacteria to produce the precursors used in the synthesis of steroidal medicines.Cytochrome P450s from CYP125 and CYP142 families initiates the metabolism of cholesterol and/or phytosterols.In this study,we demonstrate the functional redundancy and substrate preference of five cytochrome CYP125s and CYP142s by the unmarked cyp genes deletion strains of Mycobacterium neoaurum NRRL B-3805.CYP125-3 was found to be responsible for the catalytic hydroxylation of both cholesterol and the C24-branched side-chains of phytosterols,while CYP142-2 can only oxidize cholesterol and/or cholest-4-en-3-one.The strain harboring the CYP125-3 produced 6.47 g l^(-1)and 5.13 g l^(-1)of 4-androstene-3,17-dione(AD)when 15 g l^(-1)cholesterol or 17.5 g l^(-1)phytosterols used,respectively.While,the strain harboring the CYP142-2 produced 7.10 g l^(-1)AD when 15 g l^(-1)cholesterol used.The other three cytochrome P450s have a smaller contribution for the degradation of cholesterol and phytosterols.CYP125-3 has the hydroxylation activity on more substrates than that of CYP142-2.This study provided possible guidance to improve the efficiency of AD and other metabolites production from different kinds of sterols by the genetic manipulation.展开更多
Biotransformation of phytosterol(PS) by a newly isolated mutant Mycobacterium neoaurum ZJUVN-08 to produce androstenedione has been investigated in this paper.The parameters of the biotransformation process were optim...Biotransformation of phytosterol(PS) by a newly isolated mutant Mycobacterium neoaurum ZJUVN-08 to produce androstenedione has been investigated in this paper.The parameters of the biotransformation process were optimized using fractional factorial design and response surface methodology.Androstenedione was the sole product in the fermentation broth catalyzed by the mutant M.neoaurum ZJUVN-08 strain.Results showed that molar ratio of hydroxypropyl-β-cyclodextrin(HP-β-CD) to PS and substrate concentrations were the two most significant factors affecting androstenedione production.By analyzing the statistical model of three-dimensional surface plot,the optimal process conditions were observed at 0.1 g/L inducer,pH 7.0,molar ratio of HP-β-CD to PS 1.92:1,8.98 g/L PS,and at 120 h of incubation time.Under these conditions,the maximum androstenedione yield was 5.96 g/L and nearly the same with the non-optimized(5.99 g/L),while the maximum PS conversion rate was 94.69% which increased by 10.66% compared with the non-optimized(84.03%).The predicted optimum conditions from the mathematical model were in agreement with the verification experimental results.It is considered that response surface methodology was a powerful and efficient method to optimize the parameters of PS biotransformation process.展开更多
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-andros...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.展开更多
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. Microbi...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.展开更多
基金supported by the National Key R and D Program of China(NO.2019YFA0905300)National Natural Science Foundation of China(No.32171477)Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(No.TSBICIP-KJGG-001 and TSBICIP-PTJJ-007).
文摘Cholesterol and phytosterols can be served as the substrates for Actinobacteria to produce the precursors used in the synthesis of steroidal medicines.Cytochrome P450s from CYP125 and CYP142 families initiates the metabolism of cholesterol and/or phytosterols.In this study,we demonstrate the functional redundancy and substrate preference of five cytochrome CYP125s and CYP142s by the unmarked cyp genes deletion strains of Mycobacterium neoaurum NRRL B-3805.CYP125-3 was found to be responsible for the catalytic hydroxylation of both cholesterol and the C24-branched side-chains of phytosterols,while CYP142-2 can only oxidize cholesterol and/or cholest-4-en-3-one.The strain harboring the CYP125-3 produced 6.47 g l^(-1)and 5.13 g l^(-1)of 4-androstene-3,17-dione(AD)when 15 g l^(-1)cholesterol or 17.5 g l^(-1)phytosterols used,respectively.While,the strain harboring the CYP142-2 produced 7.10 g l^(-1)AD when 15 g l^(-1)cholesterol used.The other three cytochrome P450s have a smaller contribution for the degradation of cholesterol and phytosterols.CYP125-3 has the hydroxylation activity on more substrates than that of CYP142-2.This study provided possible guidance to improve the efficiency of AD and other metabolites production from different kinds of sterols by the genetic manipulation.
基金Project (No. 31130042) support by the National Natural ScienceFoundation of China
文摘Biotransformation of phytosterol(PS) by a newly isolated mutant Mycobacterium neoaurum ZJUVN-08 to produce androstenedione has been investigated in this paper.The parameters of the biotransformation process were optimized using fractional factorial design and response surface methodology.Androstenedione was the sole product in the fermentation broth catalyzed by the mutant M.neoaurum ZJUVN-08 strain.Results showed that molar ratio of hydroxypropyl-β-cyclodextrin(HP-β-CD) to PS and substrate concentrations were the two most significant factors affecting androstenedione production.By analyzing the statistical model of three-dimensional surface plot,the optimal process conditions were observed at 0.1 g/L inducer,pH 7.0,molar ratio of HP-β-CD to PS 1.92:1,8.98 g/L PS,and at 120 h of incubation time.Under these conditions,the maximum androstenedione yield was 5.96 g/L and nearly the same with the non-optimized(5.99 g/L),while the maximum PS conversion rate was 94.69% which increased by 10.66% compared with the non-optimized(84.03%).The predicted optimum conditions from the mathematical model were in agreement with the verification experimental results.It is considered that response surface methodology was a powerful and efficient method to optimize the parameters of PS biotransformation process.
基金supported by the National Basic Research Program(973)of China(No.2012CB725202)the National High-Tech R&D Program(863)of China(No.2011AA02A211)+4 种基金the National Natural Science Foundation of China(No.21276110)the Fundamental Research Funds for the Central Universities(Nos.JUSRP51306A and JUSRP11545)the National 111 Project of China’s Higher Education(No.111-2-06)the Program of the Key Laboratory of Industrial Biotechnology,Ministry of Education,China(No.KLIB-KF201406)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD),China
文摘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.
文摘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.
