Non-natural Aldol Reactions Enable the Design and Construction of Novel One-Carbon Assimilation Pathways in vitro

Yufeng Mao; Qianqian Yuan; Xue Yang; Pi Liu; Ying Cheng; Jiahao Luo; Huanhuan Liu; Yonghong Yao; Hongbing Sun; Tao Cai; Hongwu Ma

doi:10.3389/fmicb.2021.677596

Non-natural Aldol Reactions Enable the Design and Construction of Novel One-Carbon Assimilation Pathways in vitro

Front Microbiol. 2021 Jun 2:12:677596. doi: 10.3389/fmicb.2021.677596. eCollection 2021.

Authors

Yufeng Mao^{1

2}, Qianqian Yuan^{1

2}, Xue Yang^{1

2}, Pi Liu^{1

2}, Ying Cheng³, Jiahao Luo⁴, Huanhuan Liu³, Yonghong Yao², Hongbing Sun², Tao Cai², Hongwu Ma^{1

2}

Affiliations

¹ Biodesign Center, Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
² Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
³ State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin, China.
⁴ Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.

Abstract

Methylotrophs utilizes cheap, abundant one-carbon compounds, offering a promising green, sustainable and economical alternative to current sugar-based biomanufacturing. However, natural one-carbon assimilation pathways come with many disadvantages, such as complicated reaction steps, the need for additional energy and/or reducing power, or loss of CO₂, resulting in unsatisfactory biomanufacturing performance. Here, we predicted eight simple, novel and carbon-conserving formaldehyde (FALD) assimilation pathways based on the extended metabolic network with non-natural aldol reactions using the comb-flux balance analysis (FBA) algorithm. Three of these pathways were found to be independent of energy/reducing equivalents, and thus chosen for further experimental verification. Then, two novel aldol reactions, condensing D-erythrose 4-phosphate and glycolaldehyde (GALD) into 2R,3R-stereo allose 6-phosphate by DeoC or 2S,3R-stereo altrose 6-phosphate by TalB^F178Y/Fsa, were identified for the first time. Finally, a novel FALD assimilation pathway proceeding via allose 6-phosphate, named as the glycolaldehyde-allose 6-phosphate assimilation (GAPA) pathway, was constructed in vitro with a high carbon yield of 94%. This work provides an elegant paradigm for systematic design of one-carbon assimilation pathways based on artificial aldolase (ALS) reactions, which could also be feasibly adapted for the mining of other metabolic pathways.

Keywords: In vitro pathway construction; aldolase reaction; allose 6-phosphate; computational pathway design; glycolaldehyde-allose 6-phosphate assimilation pathway; synthetic methylotrophy.