The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression, and tremendous efforts...The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression, and tremendous efforts have been made to increase heterologous protein productivity by P. pastoris in recent years. When new engineered yeast strains are constructed and are ready to use tot industrial protein production, process control and optimization techniques should be applied to improve the fermentation performance in the following aspects： （1） increase recombinant cell concentrations in fermentor to high density during growth phase; （2） effectively induce heterologous proteins by enhancing/stabilizing titers or concentrations of the proteins during induction phase; （3） decrease operation costs by relieving the working loads of heat-exchange and oxygen supply. This article reviews and discusses the key and commonly used techniques in heterologous protein production by P. pastoris, with the focus on optimizations of fermentation media and basic operation conditions, development of optimal glycerol feeding strategies for achieving high density cultivation of P. pastoris and effective heterologous protein induction methods by regulating specific growth rate, methanol concentration, temperatures, mixture ratio of multi-carbon substrates, etc. Metabolic analysis for recombinant protein production by P. pastoris is also introduced to interpret the mechanism of sub-optimal heterologous protein production and to explore further optimal expression methods.展开更多
The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded wi...The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.展开更多
Foaming reduces the working volume and limits the biosynthesis of macrolide immunosuppressant ascomycin(FK520) in the batch fermentation process of Streptomyces hygroscopicus FS-35 in a 7.5 L bioreactor. To find the r...Foaming reduces the working volume and limits the biosynthesis of macrolide immunosuppressant ascomycin(FK520) in the batch fermentation process of Streptomyces hygroscopicus FS-35 in a 7.5 L bioreactor. To find the relation between FK520 production and foaming, effects of 10 fermentation parameters including organic acids and membrane permeability were investigated. The results suggest that acetate accumulation caused by short period oxygen de ficiency and fast consumption of glucose is the reason for increased foaming and declined FK520 production. Therefore, a fed-batch fermentation strategy was developed to reduce the accumulation of acetate. After optimization, the maximum acetate concentration dropped from 320 mg·L-1to 157 mg·L-1, decreased by 50.8%, and the maximum foam height reduced from 5.32 cm to 3.74 cm, decreased by 29.7%, while the maximum FK520 production increased from 375 mg·L-1to 421 mg·L-1, improved by 12%.展开更多
To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the co...To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ФC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions （P〈0.01）. These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombination展开更多
基金Supported by the Key Agricultral Technology Program of Shanghai Science & Technology Committee(073919108)MajorState Basic Research Development Program of China(2007CB714303)
文摘The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression, and tremendous efforts have been made to increase heterologous protein productivity by P. pastoris in recent years. When new engineered yeast strains are constructed and are ready to use tot industrial protein production, process control and optimization techniques should be applied to improve the fermentation performance in the following aspects： （1） increase recombinant cell concentrations in fermentor to high density during growth phase; （2） effectively induce heterologous proteins by enhancing/stabilizing titers or concentrations of the proteins during induction phase; （3） decrease operation costs by relieving the working loads of heat-exchange and oxygen supply. This article reviews and discusses the key and commonly used techniques in heterologous protein production by P. pastoris, with the focus on optimizations of fermentation media and basic operation conditions, development of optimal glycerol feeding strategies for achieving high density cultivation of P. pastoris and effective heterologous protein induction methods by regulating specific growth rate, methanol concentration, temperatures, mixture ratio of multi-carbon substrates, etc. Metabolic analysis for recombinant protein production by P. pastoris is also introduced to interpret the mechanism of sub-optimal heterologous protein production and to explore further optimal expression methods.
基金Supported by the National Science and Technology Key Funds (2003DA901A32) and the National Natural Science Foundationof China (No.20476085).
文摘The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.
基金Supported by the National Basic Research Program of China(2013CB733600)the Key Program of National Natural Science Foundation of China(21236005)+1 种基金the Natural Science Foundation of Tianjin(12JCZDJC21900)the Program of Introducing Talents of Discipline to Universities(B06006)
文摘Foaming reduces the working volume and limits the biosynthesis of macrolide immunosuppressant ascomycin(FK520) in the batch fermentation process of Streptomyces hygroscopicus FS-35 in a 7.5 L bioreactor. To find the relation between FK520 production and foaming, effects of 10 fermentation parameters including organic acids and membrane permeability were investigated. The results suggest that acetate accumulation caused by short period oxygen de ficiency and fast consumption of glucose is the reason for increased foaming and declined FK520 production. Therefore, a fed-batch fermentation strategy was developed to reduce the accumulation of acetate. After optimization, the maximum acetate concentration dropped from 320 mg·L-1to 157 mg·L-1, decreased by 50.8%, and the maximum foam height reduced from 5.32 cm to 3.74 cm, decreased by 29.7%, while the maximum FK520 production increased from 375 mg·L-1to 421 mg·L-1, improved by 12%.
基金supported by the National Basic Research Program of China (Grant Nos. 2012CB722301 and 2011CB111605)the National High Technology Research and Development Project of China (Grant No. 2011AA10A203)the National Natural Science Foundation of China (Grant No. 31070006)
文摘To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ФC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions （P〈0.01）. These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombination
