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
Using plants to produce heterologous proteins makes it very attractive due to the potentially low costs. Using this procedure it is possible to produce medicinal protein for clinical applications with the plants biore...Using plants to produce heterologous proteins makes it very attractive due to the potentially low costs. Using this procedure it is possible to produce medicinal protein for clinical applications with the plants bioreactors increasing gradually. The paper proposes the five major systems of the plant bioreactor as well as their advantage and disadvantage and the development of each system. Focuses on the five major systems of the plant bioreactor to produce vaccines, antibodies and medical protein and the research achievement at the present stage and the research on my laboratory. The key technology research of plant bioreactor such as new genes, new biological components, new technologies and new research methods related with plant bioreactor offer a work foundation for a long-term development in future.展开更多
Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins.Improving the yield in K.marxianus remains a challenge and incorporating large-scale functional modules poses a tec...Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins.Improving the yield in K.marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering.To address these issues,linear and circular yeast artificial chromosomes of K.marxianus(KmYACs)were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K.marxianus.These modules contained up to seven genes with a maximum size of 15 kb.KmYACs carried telomeres either from K.marxianus or Tetrahymena.KmYACs were transferred successfully into K.marxianus and stably propagated without affecting the normal growth of the host,regardless of the type of telomeres and configurations of KmYACs.KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins.In high-density fermentation,the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l,the highest reported level to date in K.marxianus.Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis,enhanced flux entering the tricarboxylic acid cycle,and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins.Consistently,supplementing lysine or arginine further improved the yield.Therefore,KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research.Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins,and this strategy may be applied to optimize other microbial cell factories.展开更多
Xylanase,an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat,has been found to improve the organizational structure of dough and thus increase its volume.In our past work,o...Xylanase,an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat,has been found to improve the organizational structure of dough and thus increase its volume.In our past work,one promising xylanase FXYL derived from Fusarium oxysporum Fo47 and first expressed 779.64 U/mL activity in P.pastoris.It has shown significant potential in improving the quality of whole wheat bread,making it become a candidate for development as a new flour improver.After optimization of expression elements and gene dose,the xylanase activity of FXYL strain carrying three-copies reached 4240.92 U/mL in P.pastoris.In addition,12 factors associated with the three stages of protein expression pathway were co-expressed individually in order in three-copies strain,and the translation factor Pab1 co-expression increased FXYL activity to 8893.53 U/mL.Nevertheless,combining the most effective or synergistic factors from three stages did not exhibit better results than co-expressing them alone.To further evaluate the industrial potential,the xylanase activity and protein concentration reached 81184.51 U/mL and 11.8 g/L in a 5 L fed-batch fermenter.These engineering strategies improved the expression of xylanase FXYL by more than 104-fold,providing valuable insights for the cost-effective industrial application of FXYL in the baking field.展开更多
Bacillus subtilis,as a model microorganism with a clear background,has the advantages of strong secretion ability and a generally recognized as safe status.Although the production of heterologous proteins is increasin...Bacillus subtilis,as a model microorganism with a clear background,has the advantages of strong secretion ability and a generally recognized as safe status.Although the production of heterologous proteins is increasing with the development of biotechnology,the expression level of many heterologous proteins could not meet the requirements for industrial applica-tion.Here,to further enhance the production ofα-amylase from Bacillus stearothermophilus(AmyS)which is industrially important due to its wide application,12 potential expression-related genes were selected due to upregulation in high pro-duction strain and,respectively,overexpressed to evaluate their function in the expression of AmyS in B.subtilis 1A976.The highest enzyme activity was obtained by overexpression of ponA(1.58-fold),which was the major penicillin-binding protein in cell wall synthesis.In addition,the sources and expression level of ponA were investigated.Subsequently,to exert the superb secretion ability of B.subtilis WS9,a host strain with excellent expression capability,these identified enhancers were,respectively,investigated in this strain.Due to the extremely low transformation efficiency of B.subtilis WS9,many attempts were taken to improve transformation efficiency of B.subtilis WS9.As a decisive regulator of the competent cell formation,comK was integrated into the genome of B.subtilis WS9,named as WS9C.The easy-transformable WS9C highly facilitated the subsequent genetic manipulation.Integration of ponA also increased the production of AmyS(1.37-fold)in WS9C.On this basis,combinatorial overexpression of ponA with other five screened genes liaH,oppA,secA,prsA,and ltaS was performed,respectively,and the most suitable combination was overexpression of ponA combined with ltaS,which facilitated the AmyS activity(1.53-fold).Finally,the highest enzyme activity of recombinant strain reached 2901.6 U/mL.This study provided many manipulated targets for improving the production of recombinant proteins and laid foundations for functional annotation of genes in B.subtilis.展开更多
Objective: To observe the expression and cellular localization of interleukin 8 (IL 8) mRNA and protein in the area of xenogenic bone implant. Methods: The bovine cancellous bone granules were implanted into the ...Objective: To observe the expression and cellular localization of interleukin 8 (IL 8) mRNA and protein in the area of xenogenic bone implant. Methods: The bovine cancellous bone granules were implanted into the thigh muscles of mice. The samples were taken 4, 7, 14 and 21 days after implantation. IL 8mRNA and protein in the site of implant were assayed by in situ hybridization and immunocytochemical techniques. Results: The expression of IL 8mRNA and protein were observed in all specimens 4, 7, 14 and 21 days after implantation. IL 8mRNA was expressed mainly by the neutrophils, monocytes, macrophages and fibroblasts at 7th day post implantation. Some mesenchymal cells, multinucleated giant cells, vascular endothelial cells and smooth muscle cells also expressed IL 8mRNA in the area of xenogenic bone implant at 14th and 21st days. Immunocytochemical studies demonstrated the same results as that of in situ hybridization. Conclusions: Many different kinds of cells express IL 8mRNA and secret IL 8 in the area of xenogenic bone implant, suggesting that IL 8 may play an important role in local immunity of xenogenic bone graft.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National Ministry of Science and Technology "836" Project grant number:2007AA100503
文摘Using plants to produce heterologous proteins makes it very attractive due to the potentially low costs. Using this procedure it is possible to produce medicinal protein for clinical applications with the plants bioreactors increasing gradually. The paper proposes the five major systems of the plant bioreactor as well as their advantage and disadvantage and the development of each system. Focuses on the five major systems of the plant bioreactor to produce vaccines, antibodies and medical protein and the research achievement at the present stage and the research on my laboratory. The key technology research of plant bioreactor such as new genes, new biological components, new technologies and new research methods related with plant bioreactor offer a work foundation for a long-term development in future.
基金supported by the National Key Research and Development Program of China(Nos.2021YFA0910601 and 2021YFC2100203)Shanghai Municipal Education Commission(2021-03-52)Science and Technology Research Program of Shanghai(19DZ2282100).
文摘Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins.Improving the yield in K.marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering.To address these issues,linear and circular yeast artificial chromosomes of K.marxianus(KmYACs)were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K.marxianus.These modules contained up to seven genes with a maximum size of 15 kb.KmYACs carried telomeres either from K.marxianus or Tetrahymena.KmYACs were transferred successfully into K.marxianus and stably propagated without affecting the normal growth of the host,regardless of the type of telomeres and configurations of KmYACs.KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins.In high-density fermentation,the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l,the highest reported level to date in K.marxianus.Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis,enhanced flux entering the tricarboxylic acid cycle,and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins.Consistently,supplementing lysine or arginine further improved the yield.Therefore,KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research.Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins,and this strategy may be applied to optimize other microbial cell factories.
基金supported by the Key-Area Research and Development Program of Guangdong Province(2020B020226007)National Key Research and Development Program of China(2021YFC2100405,2022YFC2105501).
文摘Xylanase,an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat,has been found to improve the organizational structure of dough and thus increase its volume.In our past work,one promising xylanase FXYL derived from Fusarium oxysporum Fo47 and first expressed 779.64 U/mL activity in P.pastoris.It has shown significant potential in improving the quality of whole wheat bread,making it become a candidate for development as a new flour improver.After optimization of expression elements and gene dose,the xylanase activity of FXYL strain carrying three-copies reached 4240.92 U/mL in P.pastoris.In addition,12 factors associated with the three stages of protein expression pathway were co-expressed individually in order in three-copies strain,and the translation factor Pab1 co-expression increased FXYL activity to 8893.53 U/mL.Nevertheless,combining the most effective or synergistic factors from three stages did not exhibit better results than co-expressing them alone.To further evaluate the industrial potential,the xylanase activity and protein concentration reached 81184.51 U/mL and 11.8 g/L in a 5 L fed-batch fermenter.These engineering strategies improved the expression of xylanase FXYL by more than 104-fold,providing valuable insights for the cost-effective industrial application of FXYL in the baking field.
基金supported by grants from the National Natural Science Foundation of China(32272264,31730067,and 31901633)the Jiangsu Provincial Key Research and Development Program(BE2023686)+1 种基金the Fundamental Research Funds for the Central Universities(JUSRP122012)Shenzhen Institute of Synthetic Biology Scientific Research Program(DWKF20210004).
文摘Bacillus subtilis,as a model microorganism with a clear background,has the advantages of strong secretion ability and a generally recognized as safe status.Although the production of heterologous proteins is increasing with the development of biotechnology,the expression level of many heterologous proteins could not meet the requirements for industrial applica-tion.Here,to further enhance the production ofα-amylase from Bacillus stearothermophilus(AmyS)which is industrially important due to its wide application,12 potential expression-related genes were selected due to upregulation in high pro-duction strain and,respectively,overexpressed to evaluate their function in the expression of AmyS in B.subtilis 1A976.The highest enzyme activity was obtained by overexpression of ponA(1.58-fold),which was the major penicillin-binding protein in cell wall synthesis.In addition,the sources and expression level of ponA were investigated.Subsequently,to exert the superb secretion ability of B.subtilis WS9,a host strain with excellent expression capability,these identified enhancers were,respectively,investigated in this strain.Due to the extremely low transformation efficiency of B.subtilis WS9,many attempts were taken to improve transformation efficiency of B.subtilis WS9.As a decisive regulator of the competent cell formation,comK was integrated into the genome of B.subtilis WS9,named as WS9C.The easy-transformable WS9C highly facilitated the subsequent genetic manipulation.Integration of ponA also increased the production of AmyS(1.37-fold)in WS9C.On this basis,combinatorial overexpression of ponA with other five screened genes liaH,oppA,secA,prsA,and ltaS was performed,respectively,and the most suitable combination was overexpression of ponA combined with ltaS,which facilitated the AmyS activity(1.53-fold).Finally,the highest enzyme activity of recombinant strain reached 2901.6 U/mL.This study provided many manipulated targets for improving the production of recombinant proteins and laid foundations for functional annotation of genes in B.subtilis.
文摘Objective: To observe the expression and cellular localization of interleukin 8 (IL 8) mRNA and protein in the area of xenogenic bone implant. Methods: The bovine cancellous bone granules were implanted into the thigh muscles of mice. The samples were taken 4, 7, 14 and 21 days after implantation. IL 8mRNA and protein in the site of implant were assayed by in situ hybridization and immunocytochemical techniques. Results: The expression of IL 8mRNA and protein were observed in all specimens 4, 7, 14 and 21 days after implantation. IL 8mRNA was expressed mainly by the neutrophils, monocytes, macrophages and fibroblasts at 7th day post implantation. Some mesenchymal cells, multinucleated giant cells, vascular endothelial cells and smooth muscle cells also expressed IL 8mRNA in the area of xenogenic bone implant at 14th and 21st days. Immunocytochemical studies demonstrated the same results as that of in situ hybridization. Conclusions: Many different kinds of cells express IL 8mRNA and secret IL 8 in the area of xenogenic bone implant, suggesting that IL 8 may play an important role in local immunity of xenogenic bone graft.
