A sequential statistical approach was applied to optimizing the fermentation medium of epothilones(Epos) production by means of a mutant which was obtained by treating polyangium cellulosum ATCC 15384 with nitrite a...A sequential statistical approach was applied to optimizing the fermentation medium of epothilones(Epos) production by means of a mutant which was obtained by treating polyangium cellulosum ATCC 15384 with nitrite and ultraviolet. The effects of different carbon sources and nitrogen sources on the fermentation medium were tested, and the suitable ones were selected. Then a uniform design was employed to design the experiments. A linear model was developed for identifying the significant components in fermentation medium, while a third degree polynomial model was used for studying the relationship between the concentration of the components in fermentation medium and the yield of Epos(YEPs). A pattern search method was used for searching the optimum fermentation medium in the test space, which was as follows(g/L): potassium nitrate 8.00, soybean peptone 17.60, potassium hydrogen phos- phate 1.00, beef extraction 6.46, yeast extraction 1.00, calcium chloride 0.25, sodium chloride 1.00 and ferric chloride 0.02. The optimum fermentation medium was expected to result in a yield of Epos(YEPs) of 2.48 mg/L. The validation experiments with the optimum medium were performed in triplicate and the average yield of Epos was 2.45 mg/L which was 7.78 times higher than that of Epos prepared without optimization.展开更多
[Objective]This study aimed to improve the yield of bacteriocins from lactic acid bacteria by optimizing the fermentation process for production of bacteriocins from lactic acid bacteria.[Method]By single-factor analy...[Objective]This study aimed to improve the yield of bacteriocins from lactic acid bacteria by optimizing the fermentation process for production of bacteriocins from lactic acid bacteria.[Method]By single-factor analysis,fermentation temperature,seed age,inoculation volume,fermentation duration and fermentation media p H were optimized to determine the best fermentation process.The inhibitory zone of bacteriocins from lactic acid bacteria was analyzed with oxford cup method,based on which the fermentation process was evaluated.[Result]The optimal fermentation process was optimized:fermentation temperature 37℃,seed age 14 h,inoculation volume 2%,fermentation duration 48 h,fermentation media p H 5.0.[Conclusion]Under the optimized fermentation conditions,the yield of bacteriocins from lactic acid bacteria was improved significantly.展开更多
Phanerochaete chrysosporium was selected as the production strain of laccase,and the effects of stirring speed,ventilation volume,culture temperature,inoculation amount and initial p H of medium on laccase production ...Phanerochaete chrysosporium was selected as the production strain of laccase,and the effects of stirring speed,ventilation volume,culture temperature,inoculation amount and initial p H of medium on laccase production by liquid fermentation in cylinder were studied. On the basis of single factor test,an orthogonal test was carried out to find optimal conditions for laccase production P. chrysosporium through liquid fermentation. These results showed that the stirring speed of fermentation cylinder had the highest effect on laccase production,and the optimal conditions were shown as follows: the temperature at 28 ℃,the rotating speed at 300 r/min,the ventilation volume of 5 L/min( ventilation ratio of 1.0 vvm),the initial p H of medium of 5,and the inoculation amount of 15%,which gave the highest laccase level of 14. 86 U/ml.展开更多
Strain of Pseudomonas Lip35 producing lipase was isolated in a refrigerator. Lipase production and characterization of this strain were investigated under different conditions. The Pseudomonas was cultivated in shakin...Strain of Pseudomonas Lip35 producing lipase was isolated in a refrigerator. Lipase production and characterization of this strain were investigated under different conditions. The Pseudomonas was cultivated in shaking flasks in a fermentation medium in various nutritional and physical environments. Lipase production has been influenced by the presence of yeast-extract, soybean powder, NaCI, and Tween-80. Maximum lipase productivity was obtained when the physical environment of the fermentation medium was optimal for 67 h. The production of lipase reached 58.9 U·mL^-1. The lipase of Pseudomonas Lip35 can be considered to be inducible, but the inducer had little influence on the production of lipase. The lipase was characterized and showed high lipolytic activity from pH 7.5-8.0. The optimum temperature was observed at 20℃ and the thermal inactivation of lipase was obvious at 60℃. The lipase activity was inhibited by K+, stimulated by Ca^2+, and thermostability decreased in the presence of Ca^2+, therefore the lipase was Ca^2+ -dependent cold-adapted enzyme.展开更多
Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this stu...Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.展开更多
Cordycepin has the potential to be an alternative to the disputed herbicide glyphosate.However,current laborious and time-consuming production strategies at low yields based on Cordyceps militaris lead to extremely hi...Cordycepin has the potential to be an alternative to the disputed herbicide glyphosate.However,current laborious and time-consuming production strategies at low yields based on Cordyceps militaris lead to extremely high cost and restrict its application in the field of agriculture.In this study,Komagataella phaffii(syn.Pichia pastoris)was engineered to biosynthesize cordycepin from methanol,which could be converted from CO_(2).Combined with fermentation optimization,cordycepin content in broth reached as high as 2.68±0.04 g/L within 168 h,around 15.95 mg/(L⋅h)in productivity.Additionally,a deaminated product of cordycepin was identified at neutral or weakly alkaline starting pH during fermentation.Transcriptome analysis found the yeast producing cordycepin was experiencing severe inhibition in methanol assimilation and peroxisome biogenesis,responsible for delayed growth and decreased carbon flux to pentose phosphate pathway(PPP)which led to lack of precursor supply.Amino acid interconversion and disruption in RNA metabolism were also due to accumu-lation of cordycepin.The study provided a unique platform for the manufacture of cordycepin based on the emerging non-conventional yeast and gave practical strategies for further optimization of the microbial cell factory.展开更多
FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No.968 up to 19 mg/L in a pilot industry-scale batch fermentation.Here we report a genomics-guided discovery of Burkholderia tha...FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No.968 up to 19 mg/L in a pilot industry-scale batch fermentation.Here we report a genomics-guided discovery of Burkholderia thailandensis MSMB43 as a new and significantly better source of FK228.The genome of B.thailandensis MSMB43 was found to contain a functional biosynthetic gene cluster highly homologous to that of FK228 in C.violaceum No.968,and the bacterium indeed produces authentic FK228.By simple fermentation in shaking flasks in a preferred M8 medium,B.thailandensis MSMB43 produced FK228 up to 67.7 mg/L;by fedbatch fermentation in a 20-L fermentor in M8 medium,B.thailandensis MSMB43 produced FK228 up to 115.9 mg/L,which is 95 fold higher than that of C.violaceum No.968 under the same laboratory fermentation conditions.RT-PCR analysis indicated that the high FK228 yield of B.thailandensis MSMB43 was due to high expression of biosynthetic genes,represented by Bth_depA,during the fermentation process.Further genetic manipulation resulted in a recombinant strain,B.thailandensis MSMB43/pBMTL3-tdpR,which harbors a broad host-range vector expressing the thailandepsin biosynthetic pathway regulatory gene tdpR.This engineered strain produced up to 168.5 mg/L of FK228 in fed-batch fermentation in a 20-L fermentor in M8 medium.Therefore,the wild-type B.thailandensis MSMB43 or its engineered derivative could potentially be a good starting point for an industrial process to improve FK228 production for its expanding use in therapy.展开更多
To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinan...To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.展开更多
As an important industrial enzyme,protease is widely used in feed,food and other fields.At present,the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production.In th...As an important industrial enzyme,protease is widely used in feed,food and other fields.At present,the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production.In this study,Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method.To improve the production of protease,atmospheric room temperature plasma(ARTP)mutagenesis was used in the first round,protease activity reached 315.0 U/mL.Then,to enhance production of protease,^(60)Co-γirradiation was used for combined mutagenesis,leading to protease activity of B.amyloliquefaciens FMME ZK003 up to 355.0 U/mL.Furthermore,to realize the efficient production of protease,after optimization of fermentation conditions,protease activity was increased to 456.9 U/mL.Finally,protease activity of B.amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter.These results indicate that B.amyloliquefaciens can efficiently produce protease,which provides a good foundation for the industrial production of protease.展开更多
基金Supported by the Science Technology Development Project of Jilin Province,China(No.20020503-2)
文摘A sequential statistical approach was applied to optimizing the fermentation medium of epothilones(Epos) production by means of a mutant which was obtained by treating polyangium cellulosum ATCC 15384 with nitrite and ultraviolet. The effects of different carbon sources and nitrogen sources on the fermentation medium were tested, and the suitable ones were selected. Then a uniform design was employed to design the experiments. A linear model was developed for identifying the significant components in fermentation medium, while a third degree polynomial model was used for studying the relationship between the concentration of the components in fermentation medium and the yield of Epos(YEPs). A pattern search method was used for searching the optimum fermentation medium in the test space, which was as follows(g/L): potassium nitrate 8.00, soybean peptone 17.60, potassium hydrogen phos- phate 1.00, beef extraction 6.46, yeast extraction 1.00, calcium chloride 0.25, sodium chloride 1.00 and ferric chloride 0.02. The optimum fermentation medium was expected to result in a yield of Epos(YEPs) of 2.48 mg/L. The validation experiments with the optimum medium were performed in triplicate and the average yield of Epos was 2.45 mg/L which was 7.78 times higher than that of Epos prepared without optimization.
基金Supported by Research Project of Sichuan University of Science&Engineering(No.2011RC12,2014KY02)Scientific Research Foundation of the Education Department of Sichuan Province(No.15ZA0222)Research Project of Liquor-marking Biological Technology and Application of Key Laboratory of Sichuan Province(NJ2013-06)
文摘[Objective]This study aimed to improve the yield of bacteriocins from lactic acid bacteria by optimizing the fermentation process for production of bacteriocins from lactic acid bacteria.[Method]By single-factor analysis,fermentation temperature,seed age,inoculation volume,fermentation duration and fermentation media p H were optimized to determine the best fermentation process.The inhibitory zone of bacteriocins from lactic acid bacteria was analyzed with oxford cup method,based on which the fermentation process was evaluated.[Result]The optimal fermentation process was optimized:fermentation temperature 37℃,seed age 14 h,inoculation volume 2%,fermentation duration 48 h,fermentation media p H 5.0.[Conclusion]Under the optimized fermentation conditions,the yield of bacteriocins from lactic acid bacteria was improved significantly.
基金Supported by Project of Jiangsu Agri-Animal Husbandry Vocational College(NSFPT201601)
文摘Phanerochaete chrysosporium was selected as the production strain of laccase,and the effects of stirring speed,ventilation volume,culture temperature,inoculation amount and initial p H of medium on laccase production by liquid fermentation in cylinder were studied. On the basis of single factor test,an orthogonal test was carried out to find optimal conditions for laccase production P. chrysosporium through liquid fermentation. These results showed that the stirring speed of fermentation cylinder had the highest effect on laccase production,and the optimal conditions were shown as follows: the temperature at 28 ℃,the rotating speed at 300 r/min,the ventilation volume of 5 L/min( ventilation ratio of 1.0 vvm),the initial p H of medium of 5,and the inoculation amount of 15%,which gave the highest laccase level of 14. 86 U/ml.
基金supported by the Major Program of the Hebei Province Commission of Science and Technology during the 11 th Five-Year-Plan period,China(06220106D)
文摘Strain of Pseudomonas Lip35 producing lipase was isolated in a refrigerator. Lipase production and characterization of this strain were investigated under different conditions. The Pseudomonas was cultivated in shaking flasks in a fermentation medium in various nutritional and physical environments. Lipase production has been influenced by the presence of yeast-extract, soybean powder, NaCI, and Tween-80. Maximum lipase productivity was obtained when the physical environment of the fermentation medium was optimal for 67 h. The production of lipase reached 58.9 U·mL^-1. The lipase of Pseudomonas Lip35 can be considered to be inducible, but the inducer had little influence on the production of lipase. The lipase was characterized and showed high lipolytic activity from pH 7.5-8.0. The optimum temperature was observed at 20℃ and the thermal inactivation of lipase was obvious at 60℃. The lipase activity was inhibited by K+, stimulated by Ca^2+, and thermostability decreased in the presence of Ca^2+, therefore the lipase was Ca^2+ -dependent cold-adapted enzyme.
基金supported by the National Key Research and Development Program of China [grant number 2019YFA0904900]Natural Science Foundation of Jiangsu Province [grant number BK20202002].
文摘Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.
基金support from Open Funding Project of State Key Laboratory of Microbial Metabolism(No.MMLKF20-09)Research Project of Applied Basic Research Program of Department of Science&Technology of Liaoning Province(2022JH2/101300137)+1 种基金the Project of Natural Science Foundation of Liaoning Province(2022-KF-15-02)Science Research Foundation of Educational Department of Liaoning Province(No.J2020099).
文摘Cordycepin has the potential to be an alternative to the disputed herbicide glyphosate.However,current laborious and time-consuming production strategies at low yields based on Cordyceps militaris lead to extremely high cost and restrict its application in the field of agriculture.In this study,Komagataella phaffii(syn.Pichia pastoris)was engineered to biosynthesize cordycepin from methanol,which could be converted from CO_(2).Combined with fermentation optimization,cordycepin content in broth reached as high as 2.68±0.04 g/L within 168 h,around 15.95 mg/(L⋅h)in productivity.Additionally,a deaminated product of cordycepin was identified at neutral or weakly alkaline starting pH during fermentation.Transcriptome analysis found the yeast producing cordycepin was experiencing severe inhibition in methanol assimilation and peroxisome biogenesis,responsible for delayed growth and decreased carbon flux to pentose phosphate pathway(PPP)which led to lack of precursor supply.Amino acid interconversion and disruption in RNA metabolism were also due to accumu-lation of cordycepin.The study provided a unique platform for the manufacture of cordycepin based on the emerging non-conventional yeast and gave practical strategies for further optimization of the microbial cell factory.
基金the University of Wisconsin-Milwaukee Research Foundation and a Public Health Service grant(CA152212)the National Cancer Institute to YQC,the National Science Foundation of China(31430002,31770055,31570031)+1 种基金the Fundamental Research Funds for the Central Universities(22221818014)the Major Basic Program of the Natural Science Foundation of Shandong Province(ZR2017ZB0206)。
文摘FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No.968 up to 19 mg/L in a pilot industry-scale batch fermentation.Here we report a genomics-guided discovery of Burkholderia thailandensis MSMB43 as a new and significantly better source of FK228.The genome of B.thailandensis MSMB43 was found to contain a functional biosynthetic gene cluster highly homologous to that of FK228 in C.violaceum No.968,and the bacterium indeed produces authentic FK228.By simple fermentation in shaking flasks in a preferred M8 medium,B.thailandensis MSMB43 produced FK228 up to 67.7 mg/L;by fedbatch fermentation in a 20-L fermentor in M8 medium,B.thailandensis MSMB43 produced FK228 up to 115.9 mg/L,which is 95 fold higher than that of C.violaceum No.968 under the same laboratory fermentation conditions.RT-PCR analysis indicated that the high FK228 yield of B.thailandensis MSMB43 was due to high expression of biosynthetic genes,represented by Bth_depA,during the fermentation process.Further genetic manipulation resulted in a recombinant strain,B.thailandensis MSMB43/pBMTL3-tdpR,which harbors a broad host-range vector expressing the thailandepsin biosynthetic pathway regulatory gene tdpR.This engineered strain produced up to 168.5 mg/L of FK228 in fed-batch fermentation in a 20-L fermentor in M8 medium.Therefore,the wild-type B.thailandensis MSMB43 or its engineered derivative could potentially be a good starting point for an industrial process to improve FK228 production for its expanding use in therapy.
基金supported by the National Key Research and Development Program of China(2019YFE0114600)National Natural Science Foundation of China(21776114).
文摘To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.
基金the Provincal Outstanding Youth Foundation of Jiangsu Province(BK20211529)the National Science Fund for Excellent Young Scholars(22122806).
文摘As an important industrial enzyme,protease is widely used in feed,food and other fields.At present,the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production.In this study,Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method.To improve the production of protease,atmospheric room temperature plasma(ARTP)mutagenesis was used in the first round,protease activity reached 315.0 U/mL.Then,to enhance production of protease,^(60)Co-γirradiation was used for combined mutagenesis,leading to protease activity of B.amyloliquefaciens FMME ZK003 up to 355.0 U/mL.Furthermore,to realize the efficient production of protease,after optimization of fermentation conditions,protease activity was increased to 456.9 U/mL.Finally,protease activity of B.amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter.These results indicate that B.amyloliquefaciens can efficiently produce protease,which provides a good foundation for the industrial production of protease.