Synthetic Methanol and Formate Assimilation Via Modular Engineering and Selection Strategies.

One-carbon (C1) feedstocks can provide a vital link between cheap and sustainable abiotic resources and microbial bioproduction. Soluble C1 substrates, methanol and formate, could prove more suitable than gaseous feedstocks as they avoid mass transfer barriers. However, microorganisms that naturally assimilate methanol and formate are limited by a narrow product spectrum and a restricted genetic toolbox. Engineering biotechnological organisms to assimilate these soluble C1 substrates has therefore become an attractive goal. Here, we discuss the use of a step-wise, modular engineering approach for the implementation of C1-pathways. In this strategy, pathways are divided into metabolic modules, the activities of which are selected for in dedicated gene-deletion strains whose growth directly depends on module activity. This provides an easy way to identify and resolve metabolic barriers hampering pathway performance. Optimization of gene expression levels and adaptive laboratory evolution can be used to establish the desired activity if direct selection fails. We exemplify this approach using several pathways, focusing especially on the ribulose monophosphate cycle for methanol assimilation and the reductive glycine pathway for formate assimilation. We argue that such modular engineering and selection strategies will prove essential for rewiring microbial metabolism towards new growth phenotypes and sustainable bioproduction.