Despite the long availability of a traditional prophylactic vaccine containing the HBV surface antigen(HBsA g) and aluminum adjuvant, nearly 10% of the population remains unable to generate an effective immune respons...Despite the long availability of a traditional prophylactic vaccine containing the HBV surface antigen(HBsA g) and aluminum adjuvant, nearly 10% of the population remains unable to generate an effective immune response. Previous studies have indicated that hepatitis B virus(HBV) PreS 2-S is abundant in T/B cell epitopes, which induces a stronger immune response than HBsA g, particularly in terms of cytotoxic T lymphocyte(CTL) reaction. In the current study, the HBV PreS 2-S gene encoding an extra26 amino acids(PreS 2 C-terminus) located at the N-terminus of HBsA g was cloned into the pV CH1300 expression vector. Pre S2-S expressed in the methylotrophic yeast, Hansenula polymorpha, was produced at a yield of up to 250 mg/L. Subsequent purification steps involved hydrophobic adsorption to colloidal silica, ion-exchange chromatography and density ultracentrifugation. The final product was obtained with a total yield of ~15% and purity of ~99%. In keeping with previous studies, ~22 nm viruslike particles were detected using electron microscopy. The generated PreS 2-S antigen will be further studied for efficacy and safty in animals.展开更多
Marchantia polymorpha,a model liverwort,provides a valuable system for investigating the evolution of plant sexual reproduction.To explore the cellular landscape of its reproductive structures,we generate a single-nuc...Marchantia polymorpha,a model liverwort,provides a valuable system for investigating the evolution of plant sexual reproduction.To explore the cellular landscape of its reproductive structures,we generate a single-nucleus transcriptomic atlas of the antheridiophore,archegoniophore,and sporophyte.Using singlenucleus RNA sequencing(snRNA-seq),we capture over 30,000 high-quality nuclei and identify distinct cel populations.In the male organ,we characterize stages of spermatogenesis from early antheridium cells to mature sperm,revealing dynamic transcriptional programs including cell cycle regulation,chromatin remodeling,and calcium signaling.In the female organ,we define cell types including archegonial layers and secondary central cells.Sporophyte clusters are annotated as spores,elaters,capsule wall,foot,and seta cells,with transcriptional signatures related to structural support,stress response,and reproductive functions.Cross-species analysis indicates that capsule wall cells in liverworts are similar to tapetum cells.Notably,foot cells exhibit high expression of genes involved in sporopollenin biosynthesis and signaling pathways,serving as a central hub that mediates communication between the maternal gametophyte and the developing sporophyte.This study provides a comprehensive cellular and molecular map of M.polymorpha reproductive organs and sporophyte,establishing a framework for investigating the development and evolution of sexual reproduction in early land plants.展开更多
Hansenula polymorpha DL-1 is a thermotolerant yeast capable of utilizing multiple renewable carbon sources,making it a promising microbial cell factory for sustainable manufacturing.However,advanced metabolic en-ginee...Hansenula polymorpha DL-1 is a thermotolerant yeast capable of utilizing multiple renewable carbon sources,making it a promising microbial cell factory for sustainable manufacturing.However,advanced metabolic en-gineering efforts have been constrained by its strong non-homologous end joining(NHEJ)mechanism and limited choice of suitable genetic tools.This study presents an optimized synthetic biology toolkit to address these limitations.A high-efficiency CRISPR-Cas9-based genome editing system was established,achieving an editing efficiency of 97.2%.To further enhance homologous recombination(HR),the NHEJ pathway was partially suppressed by knocking out KU80 and overexpressing HR-related genes from Saccharomyces cerevisiae.This increased HR rates to 88.9%.In addition,36 neutral sites were identified for stable single-copy gene integration without disrupting native gene expression cassettes.Finally,multi-copy integration tools were developed by targeting rDNA and Ty elements,leading to a~60-fold increase inβ-carotene production compared with single-copy integrants.Furthermore,squalene titers were increased from 0.1 mg/L in the wild-type strain to 187.2 mg/L through iterative multi-copy integration.These advances significantly expand the genetic tractability of H.polymorpha DL-1,underscoring its potential as a versatile platform for efficient and sustainable production of value-added compounds.展开更多
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
Lysozyme is an enzyme that is essential for protection against bacterial infections.In this study,a T4 lysozyme gene was cloned into the yeast expression vector pPIC9K under the control of the Pichia pastoris glyceral...Lysozyme is an enzyme that is essential for protection against bacterial infections.In this study,a T4 lysozyme gene was cloned into the yeast expression vector pPIC9K under the control of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP).A Hansenula polymorpha-derived ribosomal DNA (rDNA)-targeting element was inserted into the expression vector and was critical for stable DNA integration into the H.polymorpha chromosome.Recombinant T4 lysozyme was successfully expressed in the yeast H.polymorpha A16;0.49 g L-1 secreted recombinant T4 lysozyme was obtained 72 h after incubation in culture broth that had an initial pH of 6.0.Recombinant T4 lysozyme showed lytic activity against the cell walls of the gram positive bacteria,Micrococcus lysodeikticus,and the gram negative bacteria Xanthomonas campestris pv.malvacearum and Xanthomonas oryzae pv.oryzae.The zone of inhibition assay was used to evaluate antimicrobial activity.Mass spectrometry showed the N-terminal sequence of recombinant T4 lysozyme was identical to that of the native enzyme.SDS-PAGE indicated that the molecular mass of recombinant T4 lysozyme was 18.7 kD which corresponds to a monomer of the native enzyme.SDS-PAGE without 0.2 mol L-1 dithiothreitol treatment detected two bands (15 and 31 kD) suggesting that some recombinant T4 lysozyme formed interand intra-molecular disulfide bonds which resulted in loss of enzyme activity.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
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
文摘Despite the long availability of a traditional prophylactic vaccine containing the HBV surface antigen(HBsA g) and aluminum adjuvant, nearly 10% of the population remains unable to generate an effective immune response. Previous studies have indicated that hepatitis B virus(HBV) PreS 2-S is abundant in T/B cell epitopes, which induces a stronger immune response than HBsA g, particularly in terms of cytotoxic T lymphocyte(CTL) reaction. In the current study, the HBV PreS 2-S gene encoding an extra26 amino acids(PreS 2 C-terminus) located at the N-terminus of HBsA g was cloned into the pV CH1300 expression vector. Pre S2-S expressed in the methylotrophic yeast, Hansenula polymorpha, was produced at a yield of up to 250 mg/L. Subsequent purification steps involved hydrophobic adsorption to colloidal silica, ion-exchange chromatography and density ultracentrifugation. The final product was obtained with a total yield of ~15% and purity of ~99%. In keeping with previous studies, ~22 nm viruslike particles were detected using electron microscopy. The generated PreS 2-S antigen will be further studied for efficacy and safty in animals.
基金supported by the 10 KP project(https://db.cngb.org/1Okp/)and the Scientific Foundation of the Urban Management Bureau of Shenzhen(202403).
文摘Marchantia polymorpha,a model liverwort,provides a valuable system for investigating the evolution of plant sexual reproduction.To explore the cellular landscape of its reproductive structures,we generate a single-nucleus transcriptomic atlas of the antheridiophore,archegoniophore,and sporophyte.Using singlenucleus RNA sequencing(snRNA-seq),we capture over 30,000 high-quality nuclei and identify distinct cel populations.In the male organ,we characterize stages of spermatogenesis from early antheridium cells to mature sperm,revealing dynamic transcriptional programs including cell cycle regulation,chromatin remodeling,and calcium signaling.In the female organ,we define cell types including archegonial layers and secondary central cells.Sporophyte clusters are annotated as spores,elaters,capsule wall,foot,and seta cells,with transcriptional signatures related to structural support,stress response,and reproductive functions.Cross-species analysis indicates that capsule wall cells in liverworts are similar to tapetum cells.Notably,foot cells exhibit high expression of genes involved in sporopollenin biosynthesis and signaling pathways,serving as a central hub that mediates communication between the maternal gametophyte and the developing sporophyte.This study provides a comprehensive cellular and molecular map of M.polymorpha reproductive organs and sporophyte,establishing a framework for investigating the development and evolution of sexual reproduction in early land plants.
基金supported by the National Key Research and Development Program of China(2023YFF1103700)the Jiangsu Funding Program for Excellent Postdoctoral Talent(2024ZB296)the Post-doctoral Fellowship Program of CPSF(under grant number GZC20240609).
文摘Hansenula polymorpha DL-1 is a thermotolerant yeast capable of utilizing multiple renewable carbon sources,making it a promising microbial cell factory for sustainable manufacturing.However,advanced metabolic en-gineering efforts have been constrained by its strong non-homologous end joining(NHEJ)mechanism and limited choice of suitable genetic tools.This study presents an optimized synthetic biology toolkit to address these limitations.A high-efficiency CRISPR-Cas9-based genome editing system was established,achieving an editing efficiency of 97.2%.To further enhance homologous recombination(HR),the NHEJ pathway was partially suppressed by knocking out KU80 and overexpressing HR-related genes from Saccharomyces cerevisiae.This increased HR rates to 88.9%.In addition,36 neutral sites were identified for stable single-copy gene integration without disrupting native gene expression cassettes.Finally,multi-copy integration tools were developed by targeting rDNA and Ty elements,leading to a~60-fold increase inβ-carotene production compared with single-copy integrants.Furthermore,squalene titers were increased from 0.1 mg/L in the wild-type strain to 187.2 mg/L through iterative multi-copy integration.These advances significantly expand the genetic tractability of H.polymorpha DL-1,underscoring its potential as a versatile platform for efficient and sustainable production of value-added compounds.
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supported by the National High Technology Research & Development Program of China (Grant No. 2007AA02Z111)National Technology for the 10th Five-year Plan of China (Grant No. 2006BAD31B01-04)+1 种基金National Biotechnology Development Plan (Grant Nos. 2008ZX08005-004 and 2009ZX08005-004B)the Researcher Foundation of the Chinese Academy of Agricultural Sciences
文摘Lysozyme is an enzyme that is essential for protection against bacterial infections.In this study,a T4 lysozyme gene was cloned into the yeast expression vector pPIC9K under the control of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP).A Hansenula polymorpha-derived ribosomal DNA (rDNA)-targeting element was inserted into the expression vector and was critical for stable DNA integration into the H.polymorpha chromosome.Recombinant T4 lysozyme was successfully expressed in the yeast H.polymorpha A16;0.49 g L-1 secreted recombinant T4 lysozyme was obtained 72 h after incubation in culture broth that had an initial pH of 6.0.Recombinant T4 lysozyme showed lytic activity against the cell walls of the gram positive bacteria,Micrococcus lysodeikticus,and the gram negative bacteria Xanthomonas campestris pv.malvacearum and Xanthomonas oryzae pv.oryzae.The zone of inhibition assay was used to evaluate antimicrobial activity.Mass spectrometry showed the N-terminal sequence of recombinant T4 lysozyme was identical to that of the native enzyme.SDS-PAGE indicated that the molecular mass of recombinant T4 lysozyme was 18.7 kD which corresponds to a monomer of the native enzyme.SDS-PAGE without 0.2 mol L-1 dithiothreitol treatment detected two bands (15 and 31 kD) suggesting that some recombinant T4 lysozyme formed interand intra-molecular disulfide bonds which resulted in loss of enzyme activity.
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.
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.
