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

• Published in: Frontiers in microbiology Vol. 12; p. 677596
• Main Authors: Mao, Yufeng, Yuan, Qianqian, Yang, Xue, Liu, Pi, Cheng, Ying, Luo, Jiahao, Liu, Huanhuan, Yao, Yonghong, Sun, Hongbing, Cai, Tao, Ma, Hongwu
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 02.06.2021
• Subjects: aldolase reaction; allose 6-phosphate; computational pathway design; glycolaldehyde-allose 6-phosphate assimilation pathway; In vitro pathway construction; Microbiology; synthetic methylotrophy
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2021.677596

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 CO 2 , 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 2 R ,3 R -stereo allose 6-phosphate by DeoC or 2 S ,3 R -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.