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
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
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
AIM: To investigate the safety and efficacy of a Hansenula-derived PEGylated (polyethylene glycol) interferon (IFN)-alpha-2a (Reiferon Retard) plus ribavirin customized regimen in treatment-naïve and previ...AIM: To investigate the safety and efficacy of a Hansenula-derived PEGylated (polyethylene glycol) interferon (IFN)-alpha-2a (Reiferon Retard) plus ribavirin customized regimen in treatment-naïve and previously treated (non-responders and relapsers) Egyptian children with chronic hepatitis C infection.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 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 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Supported by Yassin Abdel-Ghaffar Charity Center for LiverDisease and Research,Cairo,Egypt,in collaboration with the National Liver Institute,Menofiya University,Egypt and Cairo University Pediatric Hospital,Cairo,EgyptAntiviral medications(PEG-IFN-alpha-2a and ribavirin)and HCV genotyping were of-fered as donation from Yassin Abdel-Ghaffar Charity Center for Liver Disease and Research,Cairo,Egypt
文摘AIM: To investigate the safety and efficacy of a Hansenula-derived PEGylated (polyethylene glycol) interferon (IFN)-alpha-2a (Reiferon Retard) plus ribavirin customized regimen in treatment-naïve and previously treated (non-responders and relapsers) Egyptian children with chronic hepatitis C infection.
