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
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 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.