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Advances in microbial engineering for the production of value‑added products in a biorefinery

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Systems Microbiology and Biomanufacturing 2023年第2期3卷 246-261页

作者： Cong Gao Liang Guo Wei Song Jing Wu xiulai chen Liming Liu State Key Laboratory of Food Science and Technology Jiangnan UniversityWuxi 214122China International Joint Laboratory on Food Safety Jiangnan UniversityWuxi 214122China School of Life Sciences and Health Engineering Jiangnan UniversityWuxi 214122China

Microbial biorefineries to produce chemicals from renewable feedstock provides attractive advantages,including mild reaction conditions and sustainable manufacturing.However,low-efficiency biorefineries always result in an uncompetitive biological process compared to the current petrochemical process.Thus,improving microbial capacity to maximize product yield,productivity,and titer has been recognized as a central goal for bioengineers and biochemists.The knowledge of cellular biochemistry has enabled the regulation of microbial physiology to couple with chemical production.The rapid development in metabolic engineering provides diverse strategies to enhance the efficiency of chemical biosynthesis pathways.New synthetic biology tools as well as novel regulatory targets also offer the opportunity to improve biorefinery environmental adaptivity.In this review,the recent advances in building efficient biorefineries were showcased.In addition,the challenges and future perspectives of microbial host engineering for increased microbial capacity of a biorefinery were discussed.

关键词： Microbial cell factory Metabolic engineering Synthetic biology Biorefinery

Efficient synthesis of tyrosol from L-tyrosine via heterologous Ehrlich pathway in Escherichia coli

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Chinese Journal of Chemical Engineering 2022年第7期35卷 18-30页

作者： Xiaobo Ruan Sheng Zhang Wei Song Jia Liu xiulai chen Liming Liu Jing Wu School of Pharmaceutical Science Jiangnan UniversityWuxi 214122China State Key Laboratory of Food Science and Technology Jiangnan UniversityWuxi 214122China Tianrui Chemical Co. LtdDepartment of ChemistryQuzhou 324400China

For the efficient conversion of L-tyrosine(L-Tyr)to tyrosol,which is an aromatic compound widely used in the pharmaceutical and chemical industries,a novel four-enzyme cascade pathway based on the Ehrlich pathway of Saccharomyces cerevisiae was designed and reconstructed in Escherichia coli.Then,the expression levels of the relevant enzymes were coordinated using a modular approach and gene duplication after the identification of the pyruvate decarboxylase from Candida tropicalis(CtPDC)as the rate-limiting enzymatic step.In situ product removal(ISPR)strategy with XAD4 resins was explored to avoid product inhibition and further improve tyrosol yield.As a result,the titer and conversion rate of tyrosol obtained were 35.7 g·L^(-1) and 93.6%,respectively,in a 3-L bioreactor.Results presented here provide a potential enzymatic process for industrial production of tyrosol from cheap amino acids.

关键词： Tyrosol L-tyrosine Ehrlich pathway Enzyme cascade In situ product removal

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Improving succinate production by engineering oxygen‑dependent dynamic pathway regulation in Escherichia coli

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Systems Microbiology and Biomanufacturing 2022年第2期2卷 331-344页

作者： Cong Gao Wenxiu Tang Liang Guo Guipeng Hu Jia Liu Liming Liu xiulai chen State Key Laboratory of Food Science and Technology Jiangnan University1800 Lihu RoadWuxi 214122China International Joint Laboratory on Food Safety Jiangnan UniversityWuxi 214122China School of Pharmaceutical Science Jiangnan UniversityWuxi 214122China

Succinate is an important building block for chemical synthesis.However,during the fermentation process,excessive osmotic stress and byproduct accumulation substantively impair the performance of the microbial cell factory.To this end,two strategies were proposed.First,an osmo-tolerant mutant,Escherichia coli FMME-N-2,was screened by combined mutagenesis(ARTP and^(60)Co-γirradiation)to produce 51.8 g L^(−1)succinate with a productivity of 0.81 g L^(−1)h^(−1).Second,an oxygen-dependent bifunctional switch(OBS)was developed with promoter PfnrF8-based activation and tobacco etch virus protease-based inhibition functions.With ribosomal binding site(RBS)and degron optimization of OBS,the optimal strain E.coli FMME-N-30 achieved a succinate titer and productivity of 119 g L^(−1)and 1.65 g L^(−1) h^(−1),respectively,in a 30-L fermentor,while only 7.1 g L^(−1)acetate and no formate or lactate were detected.Compared to the wild-type strain E.coli FMME-N,the succinate titer was increased by 3.3-fold.These results highlight the applicability of OBS for the large-scale production of value-added chemicals.

关键词： Dynamic regulation Oxygen-dependent switch Succinate Protease Escherichia coli

Metabolic engineering strategies for microbial utilization of C1 feedstocks

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Systems Microbiology and Biomanufacturing 2023年第1期3卷 122-136页

作者： Jian Zhang Liang Guo Cong Gao Wei Song Jing Wu Liming Liu xiulai chen State Key Laboratory of Food Science and Technology Jiangnan University1800 Lihu RoadWuxi 214122China International Joint Laboratory On Food Safety Jiangnan UniversityWuxi 214122China School of Life Sciences and Health Engineering Jiangnan UniversityWuxi 214122China

The use of abundant and cheap one carbon(C1)feedstocks to produce value-added chemicals is an important approach for achieving carbon neutrality and tackling environmental problems.The conversion of C1 feedstocks to high-value chemicals is dependent on efficient C1 assimilation pathways and microbial chassis adapted for efficient incorporation.Here,we opted to summarize the natural and synthetic C1 assimilation pathways and their key factors for metabolizing C1 feedstock.Accordingly,we discussed the metabolic engineering strategies for enabling the microbial utilization of C1 feedstocks for the bioproduction of value-added chemicals.In addition,we highlighted future perspectives of C1-based biomanufacturing for achieving a low-carbon footprint for the biosynthesis of chemicals.

关键词： C1-based biomanufacturing C1 assimilation pathways Metabolic engineering strategies C1 feedstocks Chemicals biosynthesis

Current state and future perspectives of cytochrome P450 enzymes for C-H and C=C oxygenation

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Synthetic and Systems Biotechnology 2022年第3期7卷 887-899页

作者： Yu Yan Jing Wu Guipeng Hu Cong Gao Liang Guo xiulai chen Liming Liu Wei Song State Key Laboratory of Food Science and Technology Jiangnan UniversityWuxi214122China School of Life Sciences and Health Engineering Jiangnan UniversityWuxi214122China

Cytochrome P450 enzymes(CYPs)catalyze a series of C-H and C=C oxygenation reactions,including hydroxylation,epoxidation,and ketonization.They are attractive biocatalysts because of their ability to selectively introduce oxygen into inert molecules under mild conditions.This review provides a comprehensive overview of the C-H and C=C oxygenation reactions catalyzed by CYPs and the various strategies for achieving higher selectivity and enzymatic activity.Furthermore,we discuss the application of C-H and C=C oxygenation catalyzed by CYPs to obtain the desired chemicals or pharmaceutical intermediates in practical production.The rapid development of protein engineering for CYPs provides excellent biocatalysts for selective C-H and C=C oxygenation reactions,thereby promoting the development of environmentally friendly and sustainable production processes.

关键词： Biocatalysis P450 enzymes C-H and C=C oxygenation

Metabolic engineering strategies for microbial utilization of methanol

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Engineering Microbiology 2023年第3期3卷 37-50页

作者： Yamei Gan Xin Meng Cong Gao Wei Song Liming Liu xiulai chen State Key Laboratory of Food Science and Technology Jiangnan UniversityWuxi 214122China International Joint Laboratory on Food Safety Jiangnan UniversityWuxi 214122China School of Life Sciences and Health Engineering Jiangnan UniversityWuxi 214122China

The increasing shortage of fossil resources and environmental pollution has renewed interest in the synthesis of value-added biochemicals from methanol.However,most of native or synthetic methylotrophs are unable to assimilate methanol at a sufficient rate to produce biochemicals.Thus,the performance of methylotrophs still needs to be optimized to meet the demands of industrial applications.In this review,we provide an in-depth discussion on the properties of natural and synthetic methylotrophs,and summarize the natural and synthetic methanol assimilation pathways.Further,we discuss metabolic engineering strategies for enabling microbial utilization of methanol for the bioproduction of value-added chemicals.Finally,we highlight the potential of microbial engineering for methanol assimilation and offer guidance for achieving a low-carbon footprint for the biosynthesis of chemicals.

关键词： Methanol Methylotrophs Methanol assimilation pathways Metabolic engineering strategies

Enhancement of α-ketoisovalerate production by relieving the product inhibition of L-amino acid deaminase from Proteus mirabilis

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Chinese Journal of Chemical Engineering 2020年第8期28卷 2190-2199页

作者： Shanshan Pei Xiaobo Ruan Jia Liu Wei Song xiulai chen Qiuling Luo Liming Liu Jing Wu School of Pharmaceutical Science Jiangnan UniversityWuxi 214122China Key Laboratory of Industrial Biotechnology Ministry of EducationJiangnan UniversityWuxi 214122China State Key Laboratory of Food Science and Technology Jiangnan UniversityWuxi 214122China National Engineering Laboratory for Cereal Fermentation Technology Jiangnan UniversityWuxi 214122China

L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.

关键词： Product inhibition L-Amino acid deaminases α-Ketoisovalerate Bio-catalysis Protein engineering

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元宇宙赋能图书馆智慧健康知识服务研究

医学信息学杂志 2024年第4期45卷 97-102页

作者：王修来陈梁马宁玲南京信息工程大学管理工程学院南京210044 东部战区总医院南京210002 南京航空航天大学经济与管理学院南京210016

目的/意义基于元宇宙视角完善图书馆智慧健康知识服务理论体系。方法/过程采用文献调研和内容分析法,在阐述图书馆智慧健康知识服务开展动因的基础上,分析元宇宙赋能图书馆智慧健康知识服务的内在逻辑与应用场景。结果/结论元宇宙可以... 详细信息

目的/意义基于元宇宙视角完善图书馆智慧健康知识服务理论体系。方法/过程采用文献调研和内容分析法,在阐述图书馆智慧健康知识服务开展动因的基础上,分析元宇宙赋能图书馆智慧健康知识服务的内在逻辑与应用场景。结果/结论元宇宙可以赋能图书馆提供智能化、个性化、沉浸式和虚拟式的智慧健康知识服务,能够更好地满足用户需求,促进知识的传播和应用。

关键词：元宇宙智慧健康知识服务图书馆

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利用光驱动的生物杂合系统实现CO_(2)转化生产化学品

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Chinese Journal of Catalysis 2024年第5期60卷 294-303页

作者：干雅梅柴甜甜张健高聪宋伟吴静刘立明陈修来江南大学生物工程学院工业生物技术教育部重点实验室江苏无锡214122 江南大学生命科学与健康工程学院江苏无锡214122

太阳能作为清洁能源之一,为缓解化石燃料枯竭和温室气体排放等问题提供了一种高效、经济、可持续的解决方案.自然界中的植物和光合微生物通过自身的光合系统收集并转化太阳能,从而生产生物燃料和生物化学品.然而,由于自然光合系统存在... 详细信息

太阳能作为清洁能源之一,为缓解化石燃料枯竭和温室气体排放等问题提供了一种高效、经济、可持续的解决方案.自然界中的植物和光合微生物通过自身的光合系统收集并转化太阳能,从而生产生物燃料和生物化学品.然而,由于自然光合系统存在光吸收范围相对较窄、光生电子在传输过程中易损耗等问题,限制了太阳能到化学品的转化效率.为了解决上述难题,科研人员模仿自然光合作用中的关键部分,探索构建人工光合系统,相关研究引起了广泛关注.本文通过将碲化镉量子点(CdTe QDs)与大肠杆菌(E.coli)相耦合,构建了一种光驱动无机-生物杂合系统(IBPHS),用于捕获太阳能并驱动CO_(2)转化合成高价值化学品.该系统主要由光催化模块和生物催化模块组成.在光催化模块中,通过生物合成CdTe QDs进行光能捕获,并将其转化为电子.通过敲除E.coli的Cd2+外排蛋白(ZNTA)编码基因,实现了E.coli胞内Cd2+过量积累.通过“时空耦合”方式,并借助共聚焦显微镜、高分辨率透射电镜和X射线能谱分析,确认了CdTe QDs在E.coli胞内的组装合成.利用紫外-可见分光光度计研究了光催化模块对光子的吸收能力.结果表明,光催化模块的吸收峰位于400-420 nm.利用瞬时光电流,评估了光催化模块的光生电子能力.实验发现,该模块可以产生0.07μA光电流,表明完成了光催化模块的构建.在生物催化模块中,将光催化模块产生的电子用于还原NAD+再生NADH.采用NADH生物传感器,分析了E.coli胞内NADH含量,结果表明,在蓝光照射下E.coli胞内NADH含量比黑暗条件下提高了5.1倍.在此基础上,通过表达NADH依赖型乳酸脱氢酶(LDH)将丙酮酸还原为乳酸,在蓝光光照下乳酸积累量达到了0.44 g/L,而黑暗条件下无乳酸积累,从而验证了生物催化模块的有效性.基于光催化模块和生物催化模块,进一步组装构建了IBPSH,用于驱动CO_(2)还原合成甲酸和丙酮酸.在蓝光照射下,IBPHS能够合成0.65 g/L甲酸和0.18 g/L丙酮酸,其CO_(2)利用速率分别达到51.98 mg/gDCW/h和21.92 mg/gDCW/h,超过了光合细菌.综上所述,本文利用光催化模块与生物催化模块相耦合的方式,组装构建了一种新型的人工光合系统,实现了光驱动CO_(2)还原合成高附加值化学品,为理性设计材料-生物杂合系统提供了借鉴,同时也为挖掘绿色生物制造潜力、开发太阳能化学制造平台提供参考.

关键词：人工光合系统 CO_(2)利用光能转换 CdTe生物合成 NADH再生