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Promoter engineering enables precise metabolic regulation towards efficientβ-elemene production in Ogataea polymorpha

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Synthetic and Systems Biotechnology 2024年第2期9卷 234-241页

作者： Min Ye jiaoqi Gao jingjing Li Wei Yu Fan Bai yongjin j.zhou Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences457 Zhongshan RoadDalian116023PR China CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023PR China Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023PR China University of Chinese Academy of Sciences Beijing100049PR China

Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the ***,promoters in nonconventional yeasts such as Ogataea polymorpha are always limited,which results in incompatible gene ***,we expanded the promoter library in *** 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 ***,2 hybrid growth phase-dependent promoters were constructed and characterized,which had 2-fold higher ***,promoter engineering was applied to precisely regulate cellular metabolism for efficient production ofβ-*** 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)*** 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 *** work characterized and engineered a series of promoters,that can be used to fine-tune genes for constructing efficient yeast cell factories.

关键词： Promoter library Growth phase-dependent promoters Promoter engineering Precise metabolic regulation Ogataea polymorpha

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Advances in engineering methylotrophic yeast for biosynthesis of valuable chemicals from methanol

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Chinese Chemical Letters 2018年第5期29卷 681-686页

作者： Xingpeng Duan jiaoqi Gao yongjin j.zhou Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academic Sciences

Methylotrophic yeasts and bacteria, which can use methanol as carbon and energy source, have beenwildly used as microbial cell factories for biomanufacturing. Due to their robustness in industrial harshconditions, methylotrophic yeasts such as Pichia pastoris have been explored as a cell factory forproduction of proteins and high-value chemicals. Methanol utilization pathway （MUT） is highlyregulated for efficient methanol utilization, and the downstream pathways need extensively constructedand optimized toward target metabolite biosynthesis. Here, we present an overview of methanolmetabolism and regulation in methylotrophic yeasts, among which we focus on the regulation of keygenes involved in methanol metabolism. Besides, the recent progresses in construction and optimizationof downstream biosynthetic pathways for production of high value chemicals, such as polyketides, fattyacids and isoprenoids, are further summarized. Finally, we discuss the current challenges and feasiblestrategies toward constructing efficient methylotrophic cell factories may promote wide applications inthe future.

关键词： Microbial cell factory Methylotrophic yeast P. pastoris Methanol metabolism Biosynthetic pathways Metabolic engineering

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Screening neutral sites for metabolic engineering of methylotrophic yeast Ogataea polymorpha

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Synthetic and Systems Biotechnology 2021年第2期6卷 63-68页

作者： Wei Yu jiaoqi Gao Xiaoxin Zhai yongjin j.zhou Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences457 Zhongshan RoadDalian116023PR China CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023PR China Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023PR China University of Chinese Academy of Sciences Beijing100049PR China

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 ***,the lack of gene integration sites limits its systems metabolic engineering,in particular construction of genome-integrated *** 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 *** profiling the growth and fluorescent intensity in various media,17 genome positions were finally identified as potential neutral ***,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 *** can anticipate that the neutral sites screening method described here can be easily adopted to other eukaryotes.

关键词： Neutral sites Ogataea polymorpha Metabolic engineering CRISPR-Cas9 Fatty alcohol biosynthesis

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An improved CRISPRi system in Pichia pastoris

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Synthetic and Systems Biotechnology 2023年第3期8卷 479-485页

作者： Shujing Qiao Fan Bai Peng Cai yongjin j.zhou Lun Yao Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences457 Zhongshan RoadDalian116023PR China CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023PR China Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical PhysicsCAS457 Zhongshan RoadDalian116023PR China University of Chinese Academy of Sciences Beijing100049China

CRISPR interference(CRISPRi)has been developed and widely used for gene repression in various *** we report an improved CRISPRi system in Pichia pastoris by fusing dCas9 with endogenous transcriptional repressor *** CRISPRi system shows strong repression of eGFP,with the highest efficiency of 85%.Repression of native genes is demonstrated by targeting AOX1 ***1 is efficiently repressed and the mutant strains show much slower growth in methanol *** of gRNA expression and processing on CRISPRi efficiency is also *** is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression,and Csy4 cleavage shows higher repression ***,gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of *** expression of two gRNAs targeting promoters of eGFP and AOX1 in an array together with Cys4 recognition sites,both genes can be repressed ***4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes(FAA1 and FAA2).Both genes are efficiently repressed,demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in ***.

关键词： Pichia pastoris CRISPRi HH/HDV Csy4 gRNA array

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Engineering yeast for bio-production of food ingredients

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Systems Microbiology and Biomanufacturing 2023年第1期3卷 2-11页

作者： Chunyang Cao jiaoqi Gao Beiwei Zhu yongjin j.zhou Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences457 Zhongshan RoadDalian 116023People’s Republic of China National Engineering Research Center of Seafood Collaborative Innovation Center of Seafood Deep ProcessingLiaoning Province Collaborative Innovation Center for Marine Food Deep ProcessingSchool of Food Science and TechnologyDalian Polytechnic UniversityDalian 116034People’s Republic of China

The provision of an adequate and high-quality food supply is a challenging issue due to the continued growth of the population and the reduction of arable land *** solve these problems,new and efficient food manufacturing processes need to be developed to meet the needs for healthy and nutritious *** engineering of microorganisms is a feasible approach to alleviate this problem due to its efficient *** thousands of years,yeast has been used as a cell factory for manufacturing bread,beer and *** the development of synthetic biology expands its ability for synthesis of food ingredients,fuels,pharmaceuticals and chemical *** mini review focuses on metabolic engineering of yeast cell factories to synthesize compounds that have been used as food ingredients with highlighting four food flavors.

关键词： Yeast Bio-products Metabolic engineering Food ingredients Cell factory

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Harnessing sub-organelle metabolism for biosynthesis of isoprenoids in yeast

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Synthetic and Systems Biotechnology 2020年第3期5卷 179-186页

作者： Xuan Cao Shan Yang Chunyang Cao yongjin j.zhou Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences457 Zhongshan RoadDalian116023PR China CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023PR China Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023PR China University of Chinese Academy of Sciences Beijing100049PR China School of Food Science and Technology Dalian Polytechnic UniversityDalian116034PR China

Current yeast metabolic engineering in isoprenoids production mainly focuses on rewiring of cytosolic metabolic ***,the precursors,cofactors and the enzymes are distributed in various sub-cellular compartments,which may hamper isoprenoid *** the other side,pathway compartmentalization provides several advantages for improving metabolic flux toward target *** here summarize the recent advances on harnessing sub-organelle for isoprenoids biosynthesis in yeast,and analyze the knowledge about the localization of enzymes,cofactors and metabolites for guiding the rewiring of the sub-organelle *** review may provide some insights for constructing efficient yeast cell factories for production of isoprenoids and even other natural products.

关键词： Isoprenoids Yeast Compartmentalization Sub-organelle metabolism

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Construction of microbial chassis for terpenoid discovery

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Synthetic and Systems Biotechnology 2022年第4期7卷 1181-1182页

作者： Xiaoxin Zhai Lun Yao yongjin j.zhou Dalian Institute of Chemical Physics Chinese Academy of SciencesDalian116023China CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China

Natural products are one of the major sources of small-molecule pharmaceutical drugs[1,2].However,discovery of novel natural products to expand the pool of potential drug candidates is becoming more and more *** of the traditional molecule discovery methods are based on direct separation of natural product from its natural hosts or homology search of biosynthetic ***,there are currently two main obstacles in discovery of natural products from their native hosts:1)the difficulty in cultivation of the native hosts under laboratory conditions,2)no or not enough production of target natural products due to the silence of the biosynthetic pathway genes in the native ***,novel platforms are required for efficient discovery of new natural products.

关键词： becoming enough separation

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Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

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Synthetic and Systems Biotechnology 2022年第1期7卷 498-505页

作者： Chunxiao Yan Wei Yu Xiaoxin Zhai Lun Yao Xiaoyu Guo jiaoqi Gao yongjin j.zhou School of Biological Engineering Dalian Polytechnic UniversityDalian116034PR China Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences457 Zhongshan RoadDalian116023PR China CAS Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023PR China Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical PhysicsCAS457 Zhongshan RoadDalian116023PR China University of Chinese Academy of Sciences Beijing100049China

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic *** polymorpha,one of the methylotrophic yeasts,possesses advantages in broad substrate spec-trum,thermal-tolerance,and capacity to achieve high-density ***,a limited number of available promoters hinders the engineering of *** for ***,we systematically characterized native promoters in *** by both GFP fluorescence and fatty alcohol *** 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 ***,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 ***,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 ***,this study provided a group of constitutive,inducible,and hybrid promoters for metabolic engineering of ***,and also a feasible strategy for rationally regulating the promoter strength.

关键词： Ogataea polymorpha Promoter Hybrid promoter Upstream activation sequence Metabolic engineering Fatty alcohols

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Comparative proteomics analysis of Pichia pastoris cultivating in glucose and methanol

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Synthetic and Systems Biotechnology 2022年第3期7卷 862-868页

作者： Rui Hou Linhui Gao jianhui Liu Zhen Liang yongjin j.zhou Lihua Zhang Yukui Zhang CAS Key Laboratory of Separation Science for Analytical Chemistry National Chromatographic R&A CenterDalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical PhysicsCAS457 Zhongshan RoadDalian116023China Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences457 Zhongshan RoadDalian116023China University of Chinese Academy of Sciences Beijing100049China

The methylotrophic yeast Pichia pastoris(*** phaffii)has been extensively engineered for protein production,and is attracting attention as a chassis cell for methanol biotransformation toward production of small ***,the relatively unclear methanol metabolism hampers the metabolic rewiring to improve the biosynthetic *** here performed a label-free quantitative proteomic analysis of Pichia pastoris when cultivated in minimal media containing methanol and glucose,*** were 243,158 up-regulated proteins and 244,304 down-regulated proteins in log and stationary phase,respectively,when cultivated in methanol medium compared with that of glucose *** enrichment further improved the characterization of more differentially expressed proteins(481 proteins in log phase and 524 proteins in stationary phase).We demonstrated the transaldolase isoenzyme(Tal2,Protein ID:C4R244)was highly up-regulated in methanol medium cultivation,which plays an important role in methanol *** work provides important information for understanding methanol metabolism in methyltrophic yeast and will help to engineer methanol biotransformation in ***.

关键词： Proteomics Peroxisome Methanol metabolism Systems biology

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甲醇酵母代谢工程研究进展

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生物工程学报 2021年第3期37卷 966-979页

作者：高琳惠蔡鹏周雍进中国科学院大连化学物理研究所生物技术研究部辽宁大连116023 大连市能源生物技术重点实验室辽宁大连116023

甲醇酵母由于独特优点被认为是绿色生物制造的潜在宿主。特别是其天然甲醇利用性能有望建立甲醇生物转化路线,拓展生物炼制底物,具有重要经济价值和环保意义。文中综述了代谢工程改造甲醇酵母合成蛋白质和化学品的最新研究进展,并比较... 详细信息

甲醇酵母由于独特优点被认为是绿色生物制造的潜在宿主。特别是其天然甲醇利用性能有望建立甲醇生物转化路线,拓展生物炼制底物,具有重要经济价值和环保意义。文中综述了代谢工程改造甲醇酵母合成蛋白质和化学品的最新研究进展,并比较了其与模式生物酿酒酵母作为细胞工厂的优缺点。随后,分析了甲醇酵母代谢工程改造面临的挑战,并展望了潜在解决方案。随着基因操作工具开发和细胞代谢阐释,甲醇酵母将在未来绿色生物制造发挥越来越重要的作用。

关键词：绿色生物制造代谢工程合成生物学基因编辑甲醇生物转化

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