创制非天然辅酶偏好型甲醇脱氢酶

王俊婷; 郭潇佳; 李青; 万里; 赵宗保

doi:10.12211/2096-8280.2021-016

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创制非天然辅酶偏好型甲醇脱氢酶

研究论文 | 更新时间：2026-03-05

- 创制非天然辅酶偏好型甲醇脱氢酶
- Creation of non-natural cofactor-dependent methanol dehydrogenase
- 合成生物学 2021年2卷第4期页码：651-661
- 作者机构：
  
  1.中国科学院大连化学物理研究所，辽宁大连 116023
  2.中国科学院大学，北京 100049
- 作者简介：
  
  [ "王俊婷（1993—），女，博士研究生。研究方向为非天然辅酶偏好的酶催化反应。E-mail：wangjt@dicp.ac.cn" ]
  [ "赵宗保（1968—），男，研究员，博士生导师，研究组组长。研究方向为能源生物技术、合成微生物学和化学生物学等。E-mail：zhaozb@dicp.ac.cn" ]
- 基金信息：
  
  国家自然科学基金面上项目(21877112;21721004)
- DOI：10.12211/2096-8280.2021-016
  中图分类号： Q55
- 收稿：2021-02-04，
  
  修回：2021-03-16，
  
  纸质出版：2021-08-31
- 稿件说明：
移动端阅览
王俊婷，郭潇佳，李青，万里，赵宗保. 创制非天然辅酶偏好型甲醇脱氢酶［J］. 合成生物学， 2021， 2（4）： 651-661

WANG Junting， GUO Xiaojia， LI Qing， WAN Li， ZHAO Zongbao. Creation of non-natural cofactor-dependent methanol dehydrogenase［J］. Synthetic Biology Journal， 2021， 2（4）： 651-661
王俊婷，郭潇佳，李青，万里，赵宗保. 创制非天然辅酶偏好型甲醇脱氢酶［J］. 合成生物学， 2021， 2（4）： 651-661 DOI： 10.12211/2096-8280.2021-016.

WANG Junting， GUO Xiaojia， LI Qing， WAN Li， ZHAO Zongbao. Creation of non-natural cofactor-dependent methanol dehydrogenase［J］. Synthetic Biology Journal， 2021， 2（4）： 651-661 DOI： 10.12211/2096-8280.2021-016.

摘要

有机一碳化合物甲醇是未来生物炼制产业的重要原料。烟酰胺腺嘌呤二核苷酸（NAD）依赖型甲醇脱氢酶（MDH）可催化甲醇氧化合成甲醛为代谢提供碳源，同时生成化学计量的NADH，为代谢提供还原力。改造MDH的辅酶结合口袋，获得偏好非天然辅酶烟酰胺胞嘧啶二核苷酸（NCD）的突变体，可用甲醇作为碳源并产生NCDH，为特定代谢途径提供还原力。本文首先分析嗜热脂肪芽孢杆菌

Bacillus stearothermophilus

DSM2334来源NAD依赖型MDH的结构，通过虚拟筛选和单位点突变文库初筛，鉴别出辅酶结合敏感位点，进一步依据缩小辅酶结合空腔的预期，构建并筛选得到有效利用NCD的突变体。在大肠杆菌中过表达并纯化各突变体，进行酶催化动力学分析，结果表明突变体MDH 9D1以NCD为辅酶时催化效率达858 L/（mol·s），其NCD偏好性相对于野生型蛋白提高了13 000倍。研究结果为甲醇利用及新型氧化还原代谢途径构建提供了新的功能元件。

Abstract

The C1 organic carbon compound methanol is a potential raw material for biorefinery. Recently

intensive efforts have been devoted to engineer cell factories for direct conversion of methanol into valuable metabolites. The oxidation of methanol into formaldehyde is the first committed step to provide useful substance for metabolism. While some methylotrophic microorganisms oxidize methanol with hydrogen peroxide as a co-product

many engineered systems are designed to co-produce NADH

the reduced nicotinamide adenine dinucleotide (NAD). The later route

normally catalyzed by NAD-dependent methanol dehydrogenase (MDH)

is more attractive as NADH can be used as reducing power for cellular metabolism. However

NAD(H) are used by many redox enzymes and methanol oxidation-derived NADH can cause unpredictable biological

effects. We recently engineered the cofactor preference of several NAD-dependent redox enzymes to favor a non-natural cofactor nicotinamide cytosine dinucleotide (NCD). By coupling these enzymes we demonstrated the construction of NCD-linked redox systems orthogonal to the natural cofactor NAD(H)

which could be used for pathway-selective chemical energy transfer in

Escherichia coli

. By redesigning the cofactor binding pocket of MDH

it is possible to obtain mutants favoring NCD

and thus utilize methanol as a carbon source with co-producing reduced NCD for dedicated redox chemistry. In this paper

we first analyzed the cofactor-binding pocket of

Bacillus stearothermophilus

DSM2334 derived NAD-dependent MDH (UniProt: P42327.1). Through virtual screening and single-site mutation library screening

hot spots for cofactor binding were identified. More mutants were rationally generated based on insights into the volume of cofactor binding cavity and those were obtained with improved activities in the presence of NCD. The mutants were overexpressed in

E. coli

purified

and their catalytic performance were analyzed. The results showed that the catalytic efficiency of the mutant 9D1 (MDH Y171R/I196V/V237T/N240E/K241A) with NCD reached 858 L/(mol·s)

and its NCD preference was 13 000-fold higher than that of the wild-type protein. These MDH variants can be considered as new functional parts for the bioconversion of methanol and the construction of new redox metabolism.

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references

WHITAKER W B , SANDOVAL N R , BENNETT R K , et al . Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization [J ] . Current Opinion in Biotechnology , 2015 , 33 : 165 - 175 .

CHANG K , WANG T F , CHEN J G . Hydrogenation of CO 2 to methanol over CuCeTiO x catalysts [J ] . Applied Catalysis B-Environmental , 2017 , 206 : 704 - 711 .

DU X L , JIANG Z , SU D S , et al . Research progress on the indirect hydrogenation of carbon dioxide to methanol [J ] . ChemSusChem , 2016 , 9 ( 4 ): 322 - 332 .

COTTON C A R , CLAASSENS N J , BENITO-VAQUERIZO S , et al . Renewable methanol and formate as microbial feedstocks [J ] . Current Opinion in Biotechnology , 2020 , 62 : 168 - 180 .

ANTONIEWICZ M R . Synthetic methylotrophy: strategies to assimilate methanol for growth and chemicals production [J ] . Current Opinion in Biotechnology , 2019 , 59 : 165 - 174 .

ZHANG W M , ZHANG T , WU S H , et al . Guidance for engineering of synthetic methylotrophy based on methanol metabolism in methylotrophy [J ] . RSC Advances , 2017 , 7 ( 7 ): 4083 - 4091 .

SHEEHAN M C , BAILEY C J , DOWDS B C A , et al . A new alcohol dehydrogense, reactive towards methanol, from Bacillus stearothermophilus [J ] . Biochemical Journal , 1988 , 252 ( 3 ): 661 - 666 .

WHITAKER W B , JONES J A , BENNETT R K , et al . Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli [J ] . Metabolic Engineering , 2017 , 39 : 49 - 59 .

WU T-Y , CHEN C-T , LIU J T-J , et al . Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1 [J ] . Applied Microbiology and Biotechnology , 2016 , 100 ( 11 ): 4969 - 4983 .

凡立稳 , 王钰 , 郑平 , 等 . 一碳代谢关键酶——甲醇脱氢酶的研究进展与展望 [J ] . 生物工程学报 , 2021 , 37 ( 2 ): 530 - 540 .

FAN L W , WANG Y , ZHENG P , et al . Methanol dehydrogenase, a key enzyme of one-carbon metabolism: a review [J ] . Chinese Journal of Biotechnology , 2021 , 37 ( 2 ): 530 - 540 .

TUYISHIME P , WANG Y , FAN L W , et al . Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production [J ] . Metabolic Engineering , 2018 , 49 : 220 - 231 .

KIM S , LINDNER S N , ASLAN S , et al . Growth of E. coli on formate and methanol via the reductive glycine pathway [J ] . Nature Chemical Biology , 2020 , 16 ( 5 ): 538 - 545 .

KIM Y H , CAMPBELL E , YU J , et al . Complete oxidation of methanol in biobattery devices using a hydrogel created from three modified dehydrogenases [J ] . Angewandte Chemie International Edition , 2013 , 52 ( 5 ): 1437 - 1440 .

WANG Y P , SAN K Y , BENNETT G N . Cofactor engineering for advancing chemical biotechnology [J ] . Current Opinion in Biotechnology , 2013 , 24 ( 6 ): 994 - 999 .

KARA S , SCHRITTWIESER J H , HOLLMANN F , et al . Recent trends and novel concepts in cofactor-dependent biotransformations [J ] . Applied Microbiology and Biotechnology , 2014 , 98 ( 4 ): 1517 - 1529 .

XIAO W S , WANG R-S , HANDY D E , et al . NAD(H) and NADP(H) redox couples and cellular energy metabolism [J ] . Antioxidants & Redox Signaling , 2018 , 28 ( 3 ): 251 - 272 .

GRAY K A , RICHARDSON T H , KRETZ K , et al . Rapid evolution of reversible denaturation and elevated melting temperature in a microbial haloalkane dehalogenase [J ] . Advanced Synthesis & Catalysis , 2001 , 343 ( 6/7 ): 607 - 617 .

MAMPEL J , BUESCHER J M , MEURER G , et al . Coping with complexity in metabolic engineering [J ] . Trends in Biotechnology , 2013 , 31 ( 1 ): 52 - 60 .

PAUL C E , ARENDS I W C E , HOLLMANN F . Is simpler better? synthetic nicotinamide cofactor analogues for redox chemistry [J ] . ACS Catalysis , 2014 , 4 ( 3 ): 788 - 797 .

KNAUS T , PAUL C E , LEVY C W , et al . Better than nature: nicotinamide biomimetics that outperform natural coenzymes [J ] . Journal of the American Chemical Society , 2016 , 138 ( 3 ): 1033 - 1039 .

WEUSTHUIS R A , FOLCH P L , POZO-RODRÍGUEZ A , et al . Applying non-canonical redox cofactors in fermentation processes [J ] . iScience , 2020 , 23 ( 9 ): 101471 .

BLACK W B , ZHANG L Y , MAK W S , et al . Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis [J ] . Nature Chemical Biology , 2020 , 16 ( 1 ): 87 - 94 .

AGARWAL P K , WEBB S P , HAMMES-SCHIFFER S . Computational studies of the mechanism for proton and hydride transfer in liver alcohol dehydrogenase [J ] . Journal of the American Chemical Society , 2000 , 122 ( 19 ): 4803 - 4812 .

JI D B , WANG L , LIU W J , et al . Synthesis of NAD analogs to develop bioorthogonal redox system [J ] . Science China - Chemistry , 2013 , 56 ( 3 ): 296 - 300 .

HOU S H , LIU W J , JI D B , et al . Synthesis of 1,2,3-triazole moiety-containing NAD analogs and their potential as redox cofactors [J ] . Tetrahedron Letters , 2011 , 52 ( 44 ): 5855 - 5857 .

HOU S H , LIU W J , ZHAO Z B . Synthesis of novel nicotinamide adenine dinucleotide (NAD) analogs and their coenzyme activities [J ] . Chinese Journal of Organic Chemistry , 2012 , 32 ( 2 ): 349 - 353 .

JI D B , WANG L , HOU S H , et al . Creation of bioorthogonal redox systems depending on nicotinamide flucytosine dinucleotide [J ] . Journal of the American Chemical Society , 2011 , 133 ( 51 ): 20857 - 20862 .

WANG L , JI D B , LIU Y X , et al . Synthetic cofactor-linked metabolic circuits for selective energy transfer [J ] . ACS Catalysis , 2017 , 7 ( 3 ): 1977 - 1983 .

GUO X J , LIU Y X , WANG Q , et al . Non-natural cofactor and formate-driven reductive carboxylation of pyruvate [J ] . Angewandte Chemie International Edition , 2020 , 59 ( 8 ): 3143 - 3146 .

LIAO Z P , YANG X T , FU H X , et al . The significance of aspartate on NAD(H) biosynthesis and ABE fermentation in Clostridium acetobutylicum ATCC 824 [J ] . AMB Express , 2019 , 9 ( 1 ): 142 .

刘美霞 , 李强子 , 孟冬冬 , 等 . 烟酰胺类辅酶依赖型氧化还原酶的辅酶偏好性改造及其在合成生物学中的应用 [J ] . 合成生物学 , 2020 , 1 ( 5 ): 570 - 582 .

LIU M X , LI Q Z , MENG D D , et al . Protein engineering of nicotinamide coenzyme-dependent oxidoreductases for coenzyme preference and its application in synthetic biology [J ] . Synthetic Biology Journal , 2020 , 1 ( 5 ): 570 - 582 .

GUO X J , FENG Y B , WANG X Y , et al . Characterization of the substrate scope of an alcohol dehydrogenase commonly used as methanol dehydrogenase [J ] . Bioorganic & Medicinal Chemistry Letters , 2019 , 29 ( 12 ): 1446 - 1449 .

WATERHOUSE A , BERTONI M , BIENERT S , et al . SWISS-MODEL: homology modelling of protein structures and complexes [J ] . Nucleic Acids Research , 2018 , 46 ( W1 ): W296 - W303 .

LILL M A , DANIELSON M L . Computer-aided drug design platform using PyMOL [J ] . Journal of Computer-Aided Molecular Design , 2011 , 25 ( 1 ): 13 - 19 .

RUI L Y , CAO L , CHEN W , et al . Active site engineering of the epoxide hydrolase from Agrobacterium radiobacter AD1 to enhance aerobic mineralization of cis -1,2-dichloroethylene in cells expressing an evolved toluene ortho-monooxygenase [J ] . Journal of Biological Chemistry , 2004 , 279 ( 45 ): 46810 - 46817 .

VAN DEN ENT F , LÖWE J . RF cloning: A restriction-free method for inserting target genes into plasmids [J ] . Journal of Biochemical and Biophysical Methods , 2006 , 67 ( 1 ): 67 - 74 .

WU T K , LIU Y T , CHANG C H , et al . Site-saturated mutagenesis of histidine 234 of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase demonstrates dual functions in cyclization and rearrangement reactions [J ] . Journal of the American Chemical Society , 2006 , 128 ( 19 ): 6414 - 6419 .

GRAGEROV A , HORIE K , PAVLOVA M , et al . Large-scale, saturating insertional mutagenesis of the mouse genome [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2007 , 104 ( 36 ): 14406 - 14411 .

ELHAWRANI A S , SESSIONS R B , MORETON K M , et al . Guided evolution of enzymes with new substrate specificities [J ] . Journal of Molecular Biology , 1996 , 264 ( 1 ): 97 - 110 .

WANG L , ZHOU Y J , JI D B , et al . Identification of UshA as a major enzyme for NAD degradation in Escherichia coli [J ] . Enzyme and Microbial Technology , 2014 , 58/59 : 75 - 79 .

REETZ M T , CARBALLEIRA J D . Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes [J ] . Nature Protocols , 2007 , 2 ( 4 ): 891 - 903 .

LAMZIN V S , DAUTER Z , POPOV V O , et al . High resolution structures of holo and apo formate dehydrogense [J ] . Journal of Molecular Biology , 1994 , 236 ( 3 ): 759 - 785 .

REETZ M T , WANG L W , BOCOLA M . Directed evolution of enantioselective enzymes: Iterative cycles of CASTing for probing protein-sequence space [J ] . Angewandte Chemie International Edition , 2006 , 45 ( 8 ): 1236 - 1241 .

SUN Z T , LONSDALE R , ILIE A , et al . Catalytic asymmetric reduction of difficult-to-reduce ketones: triple-code saturation mutagenesis of an alcohol dehydrogenase [J ] . ACS Catalysis , 2016 , 6 ( 3 ): 1598 - 1605 .

LIU Y X , FENG Y B , WANG L , et al . Structural insights into phosphite dehydrogenase variants favoring a non-natural redox cofactor [J ] . ACS Catalysis , 2019 , 9 ( 3 ): 1883 - 1887 .

GUO X J , WANG X Y , LIU Y X , et al . Structure-guided design of formate dehydrogenase for regeneration of a non-natural redox cofactor [J ] . Chemistry a European Journal , 2020 , 26 ( 70 ): 16611 - 16615 .

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