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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (10): 65-75    DOI: 10.13523/j.cb.2006048
综述     
甲醇的生物利用与转化*
孙青1,2,刘德华1,2,陈振1,2,**()
1 清华大学化学工程系 工业生物催化教育部重点实验室 北京 100084
2 清华大学合成与系统生物学中心 北京 100084
Research Progress of Methanol Utilization and Bioconversion
SUN Qing1,2,LIU De-hua1,2,CHEN Zhen1,2,**()
1 Key Laboratory of Industrial Biocatalysis (Ministry of Education), Department of Chemical Engineering,Tsinghua University, Beijing 100084, China
2 Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China
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摘要：

甲醇作为一种来源广泛、价格低廉、还原度高的非粮原料有望成为下一代生物制造的关键原料。利用合成生物学技术构建能够高效利用甲醇的重组微生物以实现从甲醇到高值化学品的生物转化已成国内外研究热点,但由于甲醇代谢过程的特殊性及复杂性,目前人工设计的甲基营养菌还难以实现以甲醇为唯一碳源进行生长及产物合成。基于对天然甲基营养菌甲醇代谢过程的分析,从甲醇脱氢酶的筛选与改造、甲醛同化途径的重构与优化、甲醇到化学品的生物转化几个方面对合成型甲基营养菌的构建策略及面临的挑战进行总结与分析,以期为今后合成型甲基营养菌的人工设计和利用提供一定的借鉴。
关键词： 甲醇;  合成型甲基营养菌;  甲醇脱氢酶;  甲醛同化途径;  生物转化;  合成生物学    
Abstract:

Methanol is an abundant raw material with low price and high reduction degree, which is expected to be a promising feedstock for the next generation biomanufacturing. To convert methanol into value-added chemicals, constructing recombinant microorganisms using synthetic biology has received broad attention in recent years. However, due to the complex regulation of methanol metabolism, the current engineered synthetic methylotrophy still cannot use methanol as the sole carbon source for growth and chemical production. Based on the analysis of methanol metabolic mechanism in natural methylotrophy, this review summarized the main challenges for designing and constructing synthetic methylotrophy and proposed potential strategies to overcome these barriers. Especially, it focused on the following aspects, including: screening and engineering methanol dehydrogenase; optimizing and balancing formaldehyde assimilation pathways; bioconversion of methanol to chemicals.
Key words: Methanol    Methylotrophy    Methanol dehydrogenase    Formaldehyde assimilation    Bioconversion    Synthetic biology
收稿日期: 2020-06-24 出版日期: 2020-11-10
ZTFLH:  Q819  
基金资助: * 国家重点研发计划(2018YFA0901500)
通讯作者: 陈振     E-mail: zhenchen2013@mail.tsinghua.edu.cn
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引用本文:

孙青,刘德华,陈振. 甲醇的生物利用与转化*[J]. 中国生物工程杂志, 2020, 40(10): 65-75.

SUN Qing,LIU De-hua,CHEN Zhen. Research Progress of Methanol Utilization and Bioconversion. China Biotechnology, 2020, 40(10): 65-75.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2006048    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I10/65

Name EC
number		 Representative
microorganisms		 Location Methanol redox
reaction		 ΔG①
(kJ/mol)		 Energy
generation
PQQ-dependent
MDH		 1.1.2.7 Methylophilus
methylotrophus,
Methylobacterium extorquens		 Periplasm CH3OH+PQQ→
HCHO+PQQH2		 -24.8 ATP
NAD-dependent
MDH		 1.1.1.244 Bacillus methanolicus,
Bacillus stearothermophilus		 Cytoplasm CH3OH+NAD?
HCHO+NADH+H+		 34.2 NADH
O2-dependent
AOD		 1.1.3.13 Pichia pastoris Peroxisome CH3OH+O2→
HCHO+H2O2		 -99.2 None
表1  不同甲醇脱氢酶的分类及比较
图1  甲醛同化路径图
Pathway Representative
microorganisms		 Pathway stoichiometry Replenish
pathway		 Key enzyme
RuMP Bacillus methanolicus,
Methylomonas methanolica		 3HCHO+NAD++ADP→
Pyruvate+NADH+ATP (TA)		 Non-oxidative
PPP		 HPS, PHI
XuMP Pichia pastoris 3HCHO+NAD++ATP→
Pyruvate+NADH+ADP		 Non-oxidative
PPP		 AOD, DAS
Serine cycle Methylobacterium extorquens CO2+2HCHO+2NADH+2ATP→
Pyruvate+2NAD++2ADP+FPH2		 EMC pathway SHMT
表2  甲醇同化途径的分类及比较
Substance
class		 Product Titer 13C-labeled
carbon		 Strain/Pathway/Genetic
modifications		 Medium and carbon
source		 Refs
C2 Ethanol 4.6g/L 43% E. coli/RuMP/ΔrpiAB, pdc, adhB
expressed		 MOPS medium,
13C- methanol,xylose		 [32]
Glycolate 1.2g/L - E. coli/Artificial 2-hydroxyacyl
CoA lyase (HACL) expressed		 Designed minimal
medium, formaldehyde		 [43]
C3 Acetone 45.0mmol/L 3.6% E. coli/RuMP/ΔfrmA, Δpgithl,
ctfAB, adc expressed		 M9 minimal medium,
13C-methanol,
glucose, yeast extract		 [41]
Pyruvate 0.26g/L Not proved S. cerevisiae/XuMP/aod, cat
expressed		 Synthetic medium,
methanol		 [44]
C4 1-Butanol 2.0g/L 71% E. coli/RuMP/ΔrpiAB, pdc, adhB
expressed		 MOPS medium,
13C- methanol,xylose		 [32]
Succinic acid 68.75g/L 1.45% E. coli/RuMP/based on
previously constructed succinic
acid producer		 Chemically defined
medium, glucose,
citric acid, 13C-methanol		 [40]
Amino
acids		 L- Glutamate 90mg/L 63% C. glutamicum/RuMP/ΔfrmA,
ΔrpiB, Δald/aldH		 CGXII minimal medium,
methanol, xylose		 [45]
69g/L - B. methanolicus MGA3 MeOH200 medium, 50℃ [22]
L-Lysine 11g/L - B. methanolicus MGA3 MeOH200 medium, 50℃ [22]
Cadaverine 1.5g/L Max 15% C. glutamicum/RuMP/
Δald, ΔfadH		 CGXII minimal medium,
13C- methanol, glucose/ribose		 [39]
11.3g/L - B. methanolicus MeOH200 medium [46]
Others Naringenin 3.5mg/L 4.7% E. coli/RuMP/ΔfrmA M9 minimal medium,
13C-methanol, yeast extract,
p-coumaric acid		 [14]
GABA 9g/L - B. methanolicus MGA3 MVcM minimal medium, methanol [37]
表3  天然和合成甲基营养菌在合成化学品中的应用
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