(-)-α-Bisabolol is a plant-derived sesquiterpene derived from Eremanthus erythropappus,which can be used as a raw material in cosmetics and has anti-inflammatory function.In this study,we designed six mutation sites ...(-)-α-Bisabolol is a plant-derived sesquiterpene derived from Eremanthus erythropappus,which can be used as a raw material in cosmetics and has anti-inflammatory function.In this study,we designed six mutation sites of the(-)-α-bisabolol synthase BOS using the plmDCA algorithm.Among these,the F324Y mutation demonstrated exceptional performance,increasing the product yield by 73%.We constructed a de novo(-)-α-bisabolol biosynthesis pathways through systematic synthetic biology strategies,including the enzyme design of BOS,selection of different linkers in fusion expression,and optimization of the mevalonate pathway,weakening the branching metabolic flow and multi-copy strategies,the yield of(-)-α-bisabolol was significantly increased,which was nearly 35-fold higher than that of the original strain(2.03 mg/L).The engineered strain was capable of producing 69.7 mg/L in shake flasks.To the best of our knowledge,this is the first report on the biosynthesis of(-)-α-bisabolol in Komagataella phaffii,implying this is a robust cell factory for sustainable production of other terpenoids.展开更多
Komagataella phaffii is a methylotrophic yeast harboring a tightly regulated alcohol oxidase promoter(PAOX1),which is now widely used for recombinant protein production.During PAOX1 expression phase by methanol induct...Komagataella phaffii is a methylotrophic yeast harboring a tightly regulated alcohol oxidase promoter(PAOX1),which is now widely used for recombinant protein production.During PAOX1 expression phase by methanol induction,a methanol metabolism organelle peroxisome enlarged and occupied 80%of K.phaffii cell through peroxins functions of matrix protein import and organelle division.Using a K.phaffii expressing xylanase in this study,each of all 23 PEX genes of K.phaffii,encoding peroxin,was knockout to influence the peroxisome size,leading to changes of K.phaffii physiological status and recombinant xylanase expression.It was observed that PEX3 knockout reduced peroxisome size by 54.3%,increased xylanase expression by 29%,decreased apoptosis ratio by 70.6%.Transcriptome analysis revealed that PEX3 gene knockout decreased 18 other PEX genes of all three steps of peroxisome propagation,biogenesis,matrix protein import,and peroxisome fission.PEX3 gene knockout influenced expression of ribosomal subunit-related and protein transportation significantly based on gene function annotation and enrichment analysis.Additionally,Therefore,PEX3 gene knockout promoted xylanase folding correctly via Sec63 complex,and PDI1 significantly.In a summary,PEX3 gene knockout pro-vided a novel strategy to enhance recombinant xylanase by K.phaffii.展开更多
Patatin,a prominent food protein derived from potatoes,is renowned for its exceptional nutritional value.Patatin has been characterized for its diverse physiological attributes,including esterase activity,antioxidativ...Patatin,a prominent food protein derived from potatoes,is renowned for its exceptional nutritional value.Patatin has been characterized for its diverse physiological attributes,including esterase activity,antioxidative properties,cholesterol-lowering effects,and high lysine content,alongside notable physicochemical traits such as foaming,emulsification,and gelation capabilities.Conventional methods for patatin extraction are fraught with inefficiencies,elevated costs,and detrimental impacts on protein structural and functional integrity.Herein,we leveraged an optimized strategy integrating an expression cassette toolbox and regulation of protein secretion to harness Komagataella phaffii as the expression host and achieved an expression level of 3.2 g per litre(g/L)in a 5-Litre bioreactor,which is the highest yield of patatin production using engineered bacteria and funguses that has been reported thus far.In this study,we innovatively refined the endogenous promoter PCAT1,and its efficacy in driving heterologous protein expression under methanol induction surpassed that of the conventional AOX1 promoter.Furthermore,crucial nodes for patatin heterologous expression in yeast were identified,substantially curtailing the production costs associated with patatin synthesis.展开更多
The development of green biomanufacturing technologies centered around advanced microbial cells has emerged as a hot research field in synthetic biology.As known,Komagataella phaffii is an unconventional yeast with un...The development of green biomanufacturing technologies centered around advanced microbial cells has emerged as a hot research field in synthetic biology.As known,Komagataella phaffii is an unconventional yeast with unique biological characteristics,which is generally regarded as safe(GRAs).Owing to its wide substrate spectrum,K.phaffi has been widely genetically modified for valuable chemicals such as organic acids,fatty acids and some nature products.Actually,K.phaffii is very well known for the protein expression,and few reviews have systemically addressed the value-added chemicals by K.phaffii.Accordingly,this re-view will introduce the most advanced development of genetic editing tools of K.phaffi.Recent progresses and bottlenecks in the production of high-value chemicals using K.phaffii will also be summarized.Finally,future perspectives for the utilization of K.phaffii as a chassis cell used for high-value products synthesis will be discussed.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
Myoglobin produced by fermentation using engineered Komagataella phaffii is an important color additive in meat analogue products,but its allergenicity is poorly understood.Here,we initially searched the Allergen Onli...Myoglobin produced by fermentation using engineered Komagataella phaffii is an important color additive in meat analogue products,but its allergenicity is poorly understood.Here,we initially searched the Allergen Online database and did not find any allergic or cross-reactive proteins in porcine myoglobin(PM).In vitro simulated digestion demonstrated that PM did not exhibit notable acid-base resistance or anti-digestion capabilities.However,sensitization was observed in BALB/c mice,including a significant increase in specific antibodies and biomarkers for allergic reactions,as well as alterations in gut microbiome and serum metabolome.Interestingly,the intensity of sensitization exhibited a negative correlation with the purity of PM.60%and 88%purities showed weaker sensitization compared to the ovalbumin control group.These allergic reactions were likely due to the non-myoglobin protein portion,highlighting the importance of purification processes and the urgent need to assess the allergenicity of this portion.展开更多
Efficient methanol assimilation was crucial for methanol-based biomanufacturing of high-valued products.Given the low methanol utility in native Komagataella phaffii cells,we disrupted the genes encoding formaldehyde ...Efficient methanol assimilation was crucial for methanol-based biomanufacturing of high-valued products.Given the low methanol utility in native Komagataella phaffii cells,we disrupted the genes encoding formaldehyde dehydrogenase and formate dehydrogenase and incorporated heterologous RuMP into K.phaffii to drive more flux into central metabolic pathways.We also performed transcriptome analysis to evaluate the metabolic impact of this genetic modification.The results showed that the biomass ofΔfldh mutant strain was 8.3%higher than that of the wild type control strain.The ratio of biomass accumulation from methanol was respectively 5.754 and 6.209 in WT andΔfldh.Recombinant RuMP-Δfdh and hps-Δfdh rescued the unlivingΔfdh and were able to growth in medium with methanol as sole carbon source.In addition,the transcription revealed the impact of disrupted fldh or fdh on TCA,PPP and the respiratory chain.Our results suggested that inactivation of fldh and expressing RuMP were beneficial for methanol utility.It was fdh rather than fldh that was indispensable for K.phaffii cell growth.This study provided new insights into how to reprogram K.phaffii to enhance its methanol assimilation rate,also the theoretical bases for the mechanism underlying the better methanol digestion.展开更多
The collection and utilization of CO_(2) are recognized as the key strategies for mitigating global climate change.Recently,numerous microorganisms capable of utilizing CO_(2) as a carbon source for growth have been e...The collection and utilization of CO_(2) are recognized as the key strategies for mitigating global climate change.Recently,numerous microorganisms capable of utilizing CO_(2) as a carbon source for growth have been explored and engineered for biomanufacturing.However,these processes are typically initiated by exposing microorganisms to high concentrations of CO_(2),which significantly limits the application of microbial carbon fixation in synthetic biology.Here,we demonstrate that Komagataella phaffii(K.phaffii)can fix CO_(2) via the Reductive Glycine Pathway(RGlyP)at a low concentration(0.5%CO_(2)).We propose that the endogenous RGlyP in glycine auxotroph K.phaffii A01 can effectively operate under growth pressure and utilize CO_(2) to synthesize glycine.The molecular mechanisms involved are elucidated at the transcriptional level.This is the most efficient RGlyP reported so far,demonstrating the great potential of the endogenous RGlyP in K.phaffii for CO_(2) fixation and utilization research and will further promote the development of synthetic biology,contributing to the mitiga-tion of global climate and food crises.展开更多
基金supported by the key scientific research and development project of Zhejiang Province(2024SSYS0103)the Start-up funds of Xianghu Laboratory(2023C4S02002)the National Natural Science Foundation of China(32301255)。
文摘(-)-α-Bisabolol is a plant-derived sesquiterpene derived from Eremanthus erythropappus,which can be used as a raw material in cosmetics and has anti-inflammatory function.In this study,we designed six mutation sites of the(-)-α-bisabolol synthase BOS using the plmDCA algorithm.Among these,the F324Y mutation demonstrated exceptional performance,increasing the product yield by 73%.We constructed a de novo(-)-α-bisabolol biosynthesis pathways through systematic synthetic biology strategies,including the enzyme design of BOS,selection of different linkers in fusion expression,and optimization of the mevalonate pathway,weakening the branching metabolic flow and multi-copy strategies,the yield of(-)-α-bisabolol was significantly increased,which was nearly 35-fold higher than that of the original strain(2.03 mg/L).The engineered strain was capable of producing 69.7 mg/L in shake flasks.To the best of our knowledge,this is the first report on the biosynthesis of(-)-α-bisabolol in Komagataella phaffii,implying this is a robust cell factory for sustainable production of other terpenoids.
基金supported by the Science and Technology Commission of Shanghai Municipality(No.22dz1205800)National Natural Science Foundation of China(No.31870045)Open Funding Project of the State Key Laboratory of Bioreactor Engineering.
文摘Komagataella phaffii is a methylotrophic yeast harboring a tightly regulated alcohol oxidase promoter(PAOX1),which is now widely used for recombinant protein production.During PAOX1 expression phase by methanol induction,a methanol metabolism organelle peroxisome enlarged and occupied 80%of K.phaffii cell through peroxins functions of matrix protein import and organelle division.Using a K.phaffii expressing xylanase in this study,each of all 23 PEX genes of K.phaffii,encoding peroxin,was knockout to influence the peroxisome size,leading to changes of K.phaffii physiological status and recombinant xylanase expression.It was observed that PEX3 knockout reduced peroxisome size by 54.3%,increased xylanase expression by 29%,decreased apoptosis ratio by 70.6%.Transcriptome analysis revealed that PEX3 gene knockout decreased 18 other PEX genes of all three steps of peroxisome propagation,biogenesis,matrix protein import,and peroxisome fission.PEX3 gene knockout influenced expression of ribosomal subunit-related and protein transportation significantly based on gene function annotation and enrichment analysis.Additionally,Therefore,PEX3 gene knockout promoted xylanase folding correctly via Sec63 complex,and PDI1 significantly.In a summary,PEX3 gene knockout pro-vided a novel strategy to enhance recombinant xylanase by K.phaffii.
基金supported by the National Key Research and Devel-opment Program(2021YFC2104000)the National Natural Science Foundation of China(32272276)the Fundamental Research Funds for the Central Universities.
文摘Patatin,a prominent food protein derived from potatoes,is renowned for its exceptional nutritional value.Patatin has been characterized for its diverse physiological attributes,including esterase activity,antioxidative properties,cholesterol-lowering effects,and high lysine content,alongside notable physicochemical traits such as foaming,emulsification,and gelation capabilities.Conventional methods for patatin extraction are fraught with inefficiencies,elevated costs,and detrimental impacts on protein structural and functional integrity.Herein,we leveraged an optimized strategy integrating an expression cassette toolbox and regulation of protein secretion to harness Komagataella phaffii as the expression host and achieved an expression level of 3.2 g per litre(g/L)in a 5-Litre bioreactor,which is the highest yield of patatin production using engineered bacteria and funguses that has been reported thus far.In this study,we innovatively refined the endogenous promoter PCAT1,and its efficacy in driving heterologous protein expression under methanol induction surpassed that of the conventional AOX1 promoter.Furthermore,crucial nodes for patatin heterologous expression in yeast were identified,substantially curtailing the production costs associated with patatin synthesis.
基金supported by the National Key R&D Program of China(Grant No.2022YFC210590O)National Natural Science Foundation of China(Grant Nos.22178169,22008113)+2 种基金Shandong Taishan Industrial Experts Program(Grant No.202306155)China Postdoctoral Science Foundation(Grant No.2023M740370)the State Key Laboratory of Materials-Oriented Chemical Engineering(Grant No.KL-MCE-23A10).
文摘The development of green biomanufacturing technologies centered around advanced microbial cells has emerged as a hot research field in synthetic biology.As known,Komagataella phaffii is an unconventional yeast with unique biological characteristics,which is generally regarded as safe(GRAs).Owing to its wide substrate spectrum,K.phaffi has been widely genetically modified for valuable chemicals such as organic acids,fatty acids and some nature products.Actually,K.phaffii is very well known for the protein expression,and few reviews have systemically addressed the value-added chemicals by K.phaffii.Accordingly,this re-view will introduce the most advanced development of genetic editing tools of K.phaffi.Recent progresses and bottlenecks in the production of high-value chemicals using K.phaffii will also be summarized.Finally,future perspectives for the utilization of K.phaffii as a chassis cell used for high-value products synthesis will be discussed.
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.
文摘硫酸软骨素A(chondroitin sulfate A,CSA)是关节软骨修复中不可或缺的重要成分。CSA由软骨素4-O-磺基转移酶-1(chondroitin 4-O-sulfotransferase-1,C4ST-1)催化软骨素中N-乙酰氨基半乳糖胺(N-acetylgalactosamine,GalNAc)4号位羟基磺酸化合成。然而,由于C4ST-1酶活性较低,其催化能力受限,进而阻碍了CSA的工业化生产进程。为此,该研究旨在通过结合重组菌构建和培养基优化提升C4ST-1酶活性。首先在筛选确定了以毕赤酵母GS115为底盘细胞的基础上,进一步优化了以OST1-α分泌信号肽和SUMO Pro 3促溶标签的组合方式进行C4ST-1分泌表达,经摇瓶发酵C4ST-1最高酶活性为1889.2 U/L。鉴于前期研究发现无机盐对C4ST-1酶活性的抑制以及现有培养基成本较高问题,该研究通过优化培养基组分,发现不添加昂贵成分酵母无氨基氮源(yeast nitrogen base without amino acids,YNB)时,C4ST-1酶活性较原培养基提高68.4%。此外,结合碳源、其他氮源以及生物素的筛选与优化使C4ST-1酶活性进一步提高。最终,在5L发酵罐补料分批发酵72 h时,获得最高酶活性为5040.7 U/L。该研究不仅为CSA规模化生产奠定了基础,也将为其他糖胺聚糖(如肝素、硫酸皮肤素)合成所需的磺基转移酶发酵生产提供借鉴。
基金supported by the National Natural Science Foundation of China(32125031)the National Key Research and Development Program of China(2021YFC2101400)+1 种基金the Fundamental Research Funds for the Central Universities(JUSRP222001)Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province,Jiangnan University.
文摘Myoglobin produced by fermentation using engineered Komagataella phaffii is an important color additive in meat analogue products,but its allergenicity is poorly understood.Here,we initially searched the Allergen Online database and did not find any allergic or cross-reactive proteins in porcine myoglobin(PM).In vitro simulated digestion demonstrated that PM did not exhibit notable acid-base resistance or anti-digestion capabilities.However,sensitization was observed in BALB/c mice,including a significant increase in specific antibodies and biomarkers for allergic reactions,as well as alterations in gut microbiome and serum metabolome.Interestingly,the intensity of sensitization exhibited a negative correlation with the purity of PM.60%and 88%purities showed weaker sensitization compared to the ovalbumin control group.These allergic reactions were likely due to the non-myoglobin protein portion,highlighting the importance of purification processes and the urgent need to assess the allergenicity of this portion.
基金supported by the National Natural Science Foundation of China(32370054)the national first-class discipline program of Light Industry Technology and Engineering(LITE2018-24).
文摘Efficient methanol assimilation was crucial for methanol-based biomanufacturing of high-valued products.Given the low methanol utility in native Komagataella phaffii cells,we disrupted the genes encoding formaldehyde dehydrogenase and formate dehydrogenase and incorporated heterologous RuMP into K.phaffii to drive more flux into central metabolic pathways.We also performed transcriptome analysis to evaluate the metabolic impact of this genetic modification.The results showed that the biomass ofΔfldh mutant strain was 8.3%higher than that of the wild type control strain.The ratio of biomass accumulation from methanol was respectively 5.754 and 6.209 in WT andΔfldh.Recombinant RuMP-Δfdh and hps-Δfdh rescued the unlivingΔfdh and were able to growth in medium with methanol as sole carbon source.In addition,the transcription revealed the impact of disrupted fldh or fdh on TCA,PPP and the respiratory chain.Our results suggested that inactivation of fldh and expressing RuMP were beneficial for methanol utility.It was fdh rather than fldh that was indispensable for K.phaffii cell growth.This study provided new insights into how to reprogram K.phaffii to enhance its methanol assimilation rate,also the theoretical bases for the mechanism underlying the better methanol digestion.
基金supported by the National Natural Science Foundation of China(32370054)the national first-class discipline program of Light Industry Technology and Engineering(LITE2018-24).
文摘The collection and utilization of CO_(2) are recognized as the key strategies for mitigating global climate change.Recently,numerous microorganisms capable of utilizing CO_(2) as a carbon source for growth have been explored and engineered for biomanufacturing.However,these processes are typically initiated by exposing microorganisms to high concentrations of CO_(2),which significantly limits the application of microbial carbon fixation in synthetic biology.Here,we demonstrate that Komagataella phaffii(K.phaffii)can fix CO_(2) via the Reductive Glycine Pathway(RGlyP)at a low concentration(0.5%CO_(2)).We propose that the endogenous RGlyP in glycine auxotroph K.phaffii A01 can effectively operate under growth pressure and utilize CO_(2) to synthesize glycine.The molecular mechanisms involved are elucidated at the transcriptional level.This is the most efficient RGlyP reported so far,demonstrating the great potential of the endogenous RGlyP in K.phaffii for CO_(2) fixation and utilization research and will further promote the development of synthetic biology,contributing to the mitiga-tion of global climate and food crises.
