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 TalBF178Y/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.

Bioelectrochemical systems (BESs) based on Shewanella oneidensis MR-1 offer great promise for sustainable energy/chemical production, but the low rate of electron generation remains a crucial bottleneck preventing their industrial application. Here, we reconstructed a genome-scale metabolic model of MR-1 to provide a strong theoretical basis for novel BES applications. The model iLJ1162, comprising 1,162 genes, 1,818 metabolites and 2,084 reactions, accurately predicted cellular growth using a variety of substrates with 86.9% agreement with experimental results, which is significantly higher than the previously published models iMR1_799 and iSO783. The simulation of microbial fuel cells indicated that expanding the substrate spectrum of MR-1 to highly reduced feedstocks, such as glucose and glycerol, would be beneficial for electron generation. In addition, 31 metabolic engineering targets were predicted to improve electricity production, three of which have been experimentally demonstrated, while the remainder are potential targets for modification. Two potential electron transfer pathways were identified, which could be new engineering targets for increasing the electricity production capacity of MR-1. Finally, the iLJ1162 model was used to simulate the optimal biosynthetic pathways for six platform chemicals based on the MR-1 chassis in microbial electrosynthesis systems. These results offer guidance for rational design of novel BESs.