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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

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

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

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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葡萄糖和木糖高效共利用代谢工程研究进展

生物工程学报 2024年第8期40卷 2710-2730页

作者：王倩高教琪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

利用可再生生物质高效生产化学品是可持续生物制造的重要途径。木质纤维素来源广泛且可再生,被认为是极具潜力的第二代生物炼制原料,木质纤维素水解物中混合糖的高效共发酵被认为是降低产品成本的关键途径之一。然而,大多数微生物由于... 详细信息

利用可再生生物质高效生产化学品是可持续生物制造的重要途径。木质纤维素来源广泛且可再生,被认为是极具潜力的第二代生物炼制原料,木质纤维素水解物中混合糖的高效共发酵被认为是降低产品成本的关键途径之一。然而,大多数微生物由于碳源阻遏效应会优先消耗葡萄糖而非木糖,严重制约了木质纤维素转化效率。因此,开发能够同时利用葡萄糖和木糖的微生物平台对工业规模生产至关重要。本文综述了基于代谢工程策略促进微生物高效共利用葡萄糖和木糖的关键方法和研究实例,包括葡萄糖抑制缓解、木糖跨膜转运途径改造、木糖代谢途径构建和定向进化等策略。

关键词：微生物木质纤维素生物炼制木糖代谢工程

多形汉逊酵母细胞工厂实现甲醇生物转化合成3-羟基丙酸

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Chinese Journal of Catalysis 2023年第3期46卷 84-90页

作者：禹伟高教琪姚伦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 中国科学院大连化学物理研究所中国科学院分离分析化学重点实验室辽宁大连116023 中国科学院大连化学物理研究所能源生物技术大连市重点实验室辽宁大连116023 中国科学院大学北京100049

3-羟基丙酸(3-HP)是公认的高附加值化学品之一,可用于合成多种化学品,例如丙烯酰胺、丙烯酸、1,3-丙二醇以及可降解塑料.目前3-HP的生物合成过程均以糖为原料,且需求不断增长,亟需寻找不依赖耕地的替代原料.碳一资源来源广泛,是理想的... 详细信息

3-羟基丙酸(3-HP)是公认的高附加值化学品之一,可用于合成多种化学品,例如丙烯酰胺、丙烯酸、1,3-丙二醇以及可降解塑料.目前3-HP的生物合成过程均以糖为原料,且需求不断增长,亟需寻找不依赖耕地的替代原料.碳一资源来源广泛,是理想的生物制造原料.其中,甲醇可由煤、天然气制得,也可由CO_(2)加氢制备.多形汉逊酵母作为典型的甲基营养型酵母,能够以甲醇为唯一碳源和能源进行生长,并且能够耐受高温、高渗透压和低pH等条件,是非常优秀的工业菌株.近年来基因编辑技术的发展,大大加快了多形汉逊酵母代谢工程改造,有望构建高效细胞工厂,实现甲醇生物转化.本文以多形汉逊酵母为宿主,系统改造其细胞代谢实现甲醇高效转化为3-HP.由于汉逊酵母自身不能合成3-HP,因此需要表达来源于Chloroflexus aurantiacus的3-HP合成途径关键基因MCR,该途径以乙酰-CoA和丙二酰-CoA为前体,以还原力NADPH为辅因子.首先优化MCR表达,采用融合蛋白并结合甲醇诱导型启动子PAOX基因组整合MCR表达, 3-HP合成效率最高;其次,强化供应前体乙酰-CoA以及丙二酰-CoA,使3-HP产量提高26%;进一步改造氧化磷酸戊糖途径以增强辅因子NADPH的供应,使3-HP产量提高30%;最后,将增强前体和辅因子供应的有效策略结合,工程菌HP15相比出发菌HP07摇瓶发酵3-HP产量提高135%,达到1.45 g/L.通过摇瓶补料分批发酵, 3-HP产量达到7.10 g/L,得率达到0.14 g/g甲醇,为目前报道碳一资源合成3-HP的较高产量,表明多形汉逊酵母作为甲醇细胞工厂合成化学品具有很好的潜力.预期结合“液态阳光”CO_(2)制备甲醇技术以及甲醇生物转化过程,有望将CO_(2)制备成高附加值化学品,有助于实现碳中和.

关键词：多形汉逊酵母甲醇生物转化代谢工程 3-羟基丙酸碳中和

蛋白质组学和代谢组学在微生物代谢工程中的应用

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色谱 2019年第8期37卷 798-805页

作者：禹伟高教琪周雍进中国科学院大连化学物理研究所中国科学院分离分析化学重点实验室

构建微生物细胞工厂是化学品、生物能源以及药物分子可持续生产的可行性策略。然而,微生物的代谢复杂、调控严谨,制约着目标产物高效合成。蛋白质组学和代谢组学可以从系统生物学角度分析酶和代谢物组分,从而理解复杂的生物系统,为微生... 详细信息

构建微生物细胞工厂是化学品、生物能源以及药物分子可持续生产的可行性策略。然而,微生物的代谢复杂、调控严谨,制约着目标产物高效合成。蛋白质组学和代谢组学可以从系统生物学角度分析酶和代谢物组分,从而理解复杂的生物系统,为微生物代谢工程改造提供重要线索。该文介绍了蛋白质组学和代谢组学在微生物代谢工程中的应用,包括基因组尺度代谢模型构建、菌株生物合成优化、指导菌株耐受性改造、限速步骤预测、植物次级代谢途径挖掘,从而为微生物合成天然产物提供新的基因或途径。在此基础上,该文还展望了生物大数据未来的发展方向。

关键词：蛋白质组学代谢组学基因组尺度代谢模型植物次级代谢微生物代谢工程综述

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一碳生物转化合成有机酸的研究进展

合成生物学 2024年第5期5卷 1169-1188页

作者：禹伟高教琪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

有机酸在食品、医药、化工、农业等领域有着广泛的应用。目前有机酸的生产主要以微生物发酵法为主,采用糖类为原料,然而长此以往可能面临“与人争粮”的困境。CO、CO_(2)、甲烷、甲醇和甲酸等含有一个碳原子的物质被称为一碳(one carbon... 详细信息

有机酸在食品、医药、化工、农业等领域有着广泛的应用。目前有机酸的生产主要以微生物发酵法为主,采用糖类为原料,然而长此以往可能面临“与人争粮”的困境。CO、CO_(2)、甲烷、甲醇和甲酸等含有一个碳原子的物质被称为一碳(one carbon,C_(1))资源,其来源广泛且价格低廉,有望成为生物制造的替代原料,且C_(1)原料生物转化有助于缓解温室效应、助力“碳中和”目标。本文总结了近年来CO_(2)、甲烷和甲醇生物合成3种重要有机酸(3-羟基丙酸、乳酸、琥珀酸)的研究进展,主要论述了C_(1)生物利用途径、有机酸的生物合成途径以及代谢工程策略,也讨论了C_(1)合成有机酸的挑战及应对措施,并展望了有机酸产业化新路线,尤其是化学催化与生物转化耦合以CO_(2)为原料合成有机酸。本综述对于C_(1)生物炼制以及有机酸产业升级具有一定的参考意义。

关键词： C_(1)生物炼制代谢工程 3-羟基丙酸乳酸琥珀酸

多形汉逊酵母代谢改造生产脂肪酸及发酵条件优化

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生物工程学报 2022年第2期38卷 760-771页

作者：冯叨高教琪龚志伟周雍进武汉科技大学化学与化工学院湖北武汉430080 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

脂肪酸作为一种化工原料,在生物能源、化妆品、个人护理产品和工业润滑剂等领域具有广泛应用。多形汉逊酵母因其能够利用甲醇、耐高温、底物谱广等优点,被认为是微生物细胞工厂的理想底盘宿主。本研究首先通过代谢工程构建了产脂肪酸的... 详细信息

脂肪酸作为一种化工原料,在生物能源、化妆品、个人护理产品和工业润滑剂等领域具有广泛应用。多形汉逊酵母因其能够利用甲醇、耐高温、底物谱广等优点,被认为是微生物细胞工厂的理想底盘宿主。本研究首先通过代谢工程构建了产脂肪酸的汉逊酵母细胞工厂。在此基础上,通过发酵条件优化进一步提升了工程菌株生产性能。在温度37℃、pH 6.4、培养基碳氮摩尔比为120、种子液OD_(600)在6–8之间时,摇瓶中工程菌脂肪酸产量达到1.86 g/L。在发酵罐中,采用溶氧(DO)关联法控制补料速度,初始培养基碳氮摩尔比为17.5,在DO高于30%时,补料碳氮摩尔比为120的葡萄糖培养基,脂肪酸产量达到18.0 g/L,显示了汉逊酵母作为脂肪酸合成细胞工厂的潜力,为实现工业化奠定了坚实的理论与应用基础。

关键词：多形汉逊酵母代谢工程脂肪酸限氮发酵发酵条件优化