The recombinant expression of phycocyanobilin(PCB)was carried out in Escherichia coli,and the best fermentation conditions of recombinant E.coli biosynthesized PCB are optimized in the response surface methodology to ...The recombinant expression of phycocyanobilin(PCB)was carried out in Escherichia coli,and the best fermentation conditions of recombinant E.coli biosynthesized PCB are optimized in the response surface methodology to improve PCB production.The recombinant PCB is extracted,isolated,and purified by methanol and chloroform extraction.Recombinant PCB is validated in UV-vis spectroscopy,high-pressure liquid chromatography,and mass spectrometry.In addition,the anti-oxidant activities of the recombinant PCB are determined.The best induction conditions that optimized by Design Expert 8.0 software include:lactose concentration 4 mmol/L,induction temperature 24.69℃,induction time 4.6 h,and induction duration 13.57 h,under which the PCB expression level reached approximately 13 mg PCB/L,which is more than four times of previously reported 3 mg PCB/L.The maximum absorption peak of the recombinant PCB is located at 680 nm with a high fluorescence intensity of 470 nm.The recombinant PCB has a good ability to scavenge 2,2-diphenyl-1-picrylhydrazyl(DPPH)free radicals.展开更多
Fusidane-type antibiotics,represented by helvolic acid,fusidic acid and cephalosporin P1,are fungi-derived antimicrobials with little cross-resistance to commonly used antibiotics.Generation of new fusidane-type deriv...Fusidane-type antibiotics,represented by helvolic acid,fusidic acid and cephalosporin P1,are fungi-derived antimicrobials with little cross-resistance to commonly used antibiotics.Generation of new fusidane-type derivatives is therefore of great value,but this is hindered by available approaches.Here,we developed a stochastic combinational strategy by random assembly of all the post-tailoring genes derived from helvolic acid,fusidic acid,and cephalosporin P1 biosynthetic pathways in a strain that produces their common intermediate.Among a total of 27 gene combinations,24 combinations produce expected products and afford 58 fusidane-type analogues,of which 54 are new compounds.Moreover,random gene combination can induce unexpected activity of some post-tailoring enzymes,leading to a further increase in chemical diversity.These newly generated derivatives provide new insights into the structure-activity relationship of fusidane-type antibiotics.The stochastic combinational strategy established in this study proves to be a powerful approach for expanding structural diversity of natural products.展开更多
基金Supported by the National Natural Science Foundation of China(No.41906109)the Key Research and Development Program of Yantai(No.Y933021011)
文摘The recombinant expression of phycocyanobilin(PCB)was carried out in Escherichia coli,and the best fermentation conditions of recombinant E.coli biosynthesized PCB are optimized in the response surface methodology to improve PCB production.The recombinant PCB is extracted,isolated,and purified by methanol and chloroform extraction.Recombinant PCB is validated in UV-vis spectroscopy,high-pressure liquid chromatography,and mass spectrometry.In addition,the anti-oxidant activities of the recombinant PCB are determined.The best induction conditions that optimized by Design Expert 8.0 software include:lactose concentration 4 mmol/L,induction temperature 24.69℃,induction time 4.6 h,and induction duration 13.57 h,under which the PCB expression level reached approximately 13 mg PCB/L,which is more than four times of previously reported 3 mg PCB/L.The maximum absorption peak of the recombinant PCB is located at 680 nm with a high fluorescence intensity of 470 nm.The recombinant PCB has a good ability to scavenge 2,2-diphenyl-1-picrylhydrazyl(DPPH)free radicals.
基金financially supported by grants from National Key Research and Development Program of China (2018YFA0903200 and 2018YFA0903201)the National Natural Science Foundation of China (31870032, 81925037, 31761143016, 31670036 and 31800021)+8 种基金the 111 Project of Ministry of Education of the People’s Republic of China (B13038)Chang Jiang Scholars Program (Young Scholar) from the Ministry of Education of China (Hao Gao, 2017)National High-level Personnel of Special Support Program (2017RA2259, China)the Guangdong Natural Science Funds for Distinguished Young Scholar (2019B151502014, China)Guangdong Science and Technology Planning Project (2020A0505100041, China)Guangdong Special Support Program (2016TX03R280, China)Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y036, China)K. C. Wong Education Foundation (Hao Gao, 2016, China)Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant Number JP16H06443, JP20KK0173, and JP20H00490)。
文摘Fusidane-type antibiotics,represented by helvolic acid,fusidic acid and cephalosporin P1,are fungi-derived antimicrobials with little cross-resistance to commonly used antibiotics.Generation of new fusidane-type derivatives is therefore of great value,but this is hindered by available approaches.Here,we developed a stochastic combinational strategy by random assembly of all the post-tailoring genes derived from helvolic acid,fusidic acid,and cephalosporin P1 biosynthetic pathways in a strain that produces their common intermediate.Among a total of 27 gene combinations,24 combinations produce expected products and afford 58 fusidane-type analogues,of which 54 are new compounds.Moreover,random gene combination can induce unexpected activity of some post-tailoring enzymes,leading to a further increase in chemical diversity.These newly generated derivatives provide new insights into the structure-activity relationship of fusidane-type antibiotics.The stochastic combinational strategy established in this study proves to be a powerful approach for expanding structural diversity of natural products.
