Methylotrophic yeast Ogataea polymorpha is capable to utilize multiple carbon feedstocks especially methanol as sole carbon source and energy,making it an ideal host for bio-manufacturing.However,the lack of gene inte...Methylotrophic yeast Ogataea polymorpha is capable to utilize multiple carbon feedstocks especially methanol as sole carbon source and energy,making it an ideal host for bio-manufacturing.However,the lack of gene integration sites limits its systems metabolic engineering,in particular construction of genome-integrated pathway.We here screened the genomic neutral sites for gene integration without affecting cellular fitness,by genomic integration of an enhanced green fluorescent protein(eGFP)gene via CRISPR-Cas9 technique.After profiling the growth and fluorescent intensity in various media,17 genome positions were finally identified as potential neutral sites.Finally,integration of fatty alcohol synthetic pathway genes into neutral sites NS2 and NS3,enabled the production of 4.5 mg/L fatty alcohols,indicating that these neutral sites can be used for streamline metabolic engineering in O.polymorpha.We can anticipate that the neutral sites screening method described here can be easily adopted to other eukaryotes.展开更多
Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production.However,promoters in nonconventional yeasts such as Ogataea polymorpha are always limited,which res...Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production.However,promoters in nonconventional yeasts such as Ogataea polymorpha are always limited,which results in incompatible gene modulation.Here,we expanded the promoter library in O.polymorpha based on transcriptional data,among which 13 constitutive promoters had the strengths ranging from 0–55%of PGAP,the commonly used strong constitutive promoter,and 2 were growth phase-dependent promoters.Subsequently,2 hybrid growth phase-dependent promoters were constructed and characterized,which had 2-fold higher activities.Finally,promoter engineering was applied to precisely regulate cellular metabolism for efficient production ofβ-elemene.The glyceraldehyde-3-phosphate dehydrogenase gene GAP was downregulated to drive more flux into pentose phosphate pathway(PPP)and then to enhance the supply of acetyl-CoA by using phosphoketolase-phosphotransacetylase(PK-PTA)pathway.Coupled with the phase-dependent expression of synthase module(ERG20∼LsLTC2 fusion),the highest titer of 5.24 g/L with a yield of 0.037 g/(g glucose)was achieved in strain YY150U under fed-batch fermentation in shake flasks.This work characterized and engineered a series of promoters,that can be used to fine-tune genes for constructing efficient yeast cell factories.展开更多
Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engi-neering.Ogataea polymorpha,one of the methylotrophic yeasts,possesses advantages in broad substrate spec-trum,...Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engi-neering.Ogataea polymorpha,one of the methylotrophic yeasts,possesses advantages in broad substrate spec-trum,thermal-tolerance,and capacity to achieve high-density fermentation.However,a limited number of available promoters hinders the engineering of O.polymorpha for bio-productions.Here,we systematically characterized native promoters in O.polymorpha by both GFP fluorescence and fatty alcohol biosynthesis.Ten constitutive promoters(P_(PDH),P_(PYK),P_(FBA),P_(PGM),P_(GLK),P_(TRI),P(GPI),P_(ADH1),P_(TEF1) and P_(GCW14))were obtained with the activity range of 13%–130% of the common promoter P_(GAP)(the promoter of glyceraldehyde-3-phosphate de-hydrogenase),among which P_(PDH) and P_(GCW14) were further verified by biosynthesis of fatty alcohol.Furthermore,the inducible promoters,including ethanol-induced P_(ICL1),rhamnose-induced P_(LRA3) and P_( LRA4),and a bidirectional promoter(P_(Mal)-P_(Per))that is strongly induced by sucrose,further expanded the promoter toolbox in O.polymorpha.Finally,a series of hybrid promoters were constructed via engineering upstream activation sequence(UAS),which increased the activity of native promoter P LRA3 by 4.7–10.4 times without obvious leakage expression.Therefore,this study provided a group of constitutive,inducible,and hybrid promoters for metabolic engineering of O.polymorpha,and also a feasible strategy for rationally regulating the promoter strength.展开更多
基金supported by National Natural Science Foundation of China(21922812 and 21808216)Dalian Science and Technology Innovation Funding(2019J12GX030)+1 种基金DMTO research grant(grant no.DICP DMTO201701)BioChE research grant(grant no.DICP BioChE-X201801)from Dalian Institute of Chemicals Physics,CAS.
文摘Methylotrophic yeast Ogataea polymorpha is capable to utilize multiple carbon feedstocks especially methanol as sole carbon source and energy,making it an ideal host for bio-manufacturing.However,the lack of gene integration sites limits its systems metabolic engineering,in particular construction of genome-integrated pathway.We here screened the genomic neutral sites for gene integration without affecting cellular fitness,by genomic integration of an enhanced green fluorescent protein(eGFP)gene via CRISPR-Cas9 technique.After profiling the growth and fluorescent intensity in various media,17 genome positions were finally identified as potential neutral sites.Finally,integration of fatty alcohol synthetic pathway genes into neutral sites NS2 and NS3,enabled the production of 4.5 mg/L fatty alcohols,indicating that these neutral sites can be used for streamline metabolic engineering in O.polymorpha.We can anticipate that the neutral sites screening method described here can be easily adopted to other eukaryotes.
基金This research was supported by the National Key Research and Development Project(2023YFC3503900)Liaoning Distinguished Scholar Program(2023JH6/100500001)。
文摘Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production.However,promoters in nonconventional yeasts such as Ogataea polymorpha are always limited,which results in incompatible gene modulation.Here,we expanded the promoter library in O.polymorpha based on transcriptional data,among which 13 constitutive promoters had the strengths ranging from 0–55%of PGAP,the commonly used strong constitutive promoter,and 2 were growth phase-dependent promoters.Subsequently,2 hybrid growth phase-dependent promoters were constructed and characterized,which had 2-fold higher activities.Finally,promoter engineering was applied to precisely regulate cellular metabolism for efficient production ofβ-elemene.The glyceraldehyde-3-phosphate dehydrogenase gene GAP was downregulated to drive more flux into pentose phosphate pathway(PPP)and then to enhance the supply of acetyl-CoA by using phosphoketolase-phosphotransacetylase(PK-PTA)pathway.Coupled with the phase-dependent expression of synthase module(ERG20∼LsLTC2 fusion),the highest titer of 5.24 g/L with a yield of 0.037 g/(g glucose)was achieved in strain YY150U under fed-batch fermentation in shake flasks.This work characterized and engineered a series of promoters,that can be used to fine-tune genes for constructing efficient yeast cell factories.
基金National Natural Science Foundation of China(21808216,22161142008 and M-0246)Key project at central government level:The ability establishment of sustainable use for valuable Chinese medicine resources(2060302)DICP innovation grant(DICP I202021 and I201920)from Dalian Institute of Chemicals Physics,CAS.
文摘Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engi-neering.Ogataea polymorpha,one of the methylotrophic yeasts,possesses advantages in broad substrate spec-trum,thermal-tolerance,and capacity to achieve high-density fermentation.However,a limited number of available promoters hinders the engineering of O.polymorpha for bio-productions.Here,we systematically characterized native promoters in O.polymorpha by both GFP fluorescence and fatty alcohol biosynthesis.Ten constitutive promoters(P_(PDH),P_(PYK),P_(FBA),P_(PGM),P_(GLK),P_(TRI),P(GPI),P_(ADH1),P_(TEF1) and P_(GCW14))were obtained with the activity range of 13%–130% of the common promoter P_(GAP)(the promoter of glyceraldehyde-3-phosphate de-hydrogenase),among which P_(PDH) and P_(GCW14) were further verified by biosynthesis of fatty alcohol.Furthermore,the inducible promoters,including ethanol-induced P_(ICL1),rhamnose-induced P_(LRA3) and P_( LRA4),and a bidirectional promoter(P_(Mal)-P_(Per))that is strongly induced by sucrose,further expanded the promoter toolbox in O.polymorpha.Finally,a series of hybrid promoters were constructed via engineering upstream activation sequence(UAS),which increased the activity of native promoter P LRA3 by 4.7–10.4 times without obvious leakage expression.Therefore,this study provided a group of constitutive,inducible,and hybrid promoters for metabolic engineering of O.polymorpha,and also a feasible strategy for rationally regulating the promoter strength.
