Creation of non-natural cofactor-dependent methanol dehydrogenase

WANG Junting; GUO Xiaojia; LI Qing; WAN Li; ZHAO Zongbao

doi:10.12211/2096-8280.2021-016

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Creation of non-natural cofactor-dependent methanol dehydrogenase

更新时间：2026-03-05

- Creation of non-natural cofactor-dependent methanol dehydrogenase
- Synthetic Biology Journal Vol. 2, Issue 4, Pages: 651-661(2021)
- 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：
- 基金信息：
- DOI：10.12211/2096-8280.2021-016
  CLC： Q55
- Received：04 February 2021，
  
  Revised：2021-03-16，
  
  Published：31 August 2021
- 稿件说明：
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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

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