PNNL

Abstract

Bioproduction from one-carbon compounds such as formate is an attractive prospect due to reduced energy requirements and potential for using CO2 as a sustainable feedstock. Formate-fixing pathways engineered using Escherichia coli lysate-based cell-free expression (CFE) biocatalysts have the potential to route 100% of feedstock carbon toward chemical synthesis, but are severely undermined by siphoning of in-pathway metabolites and cofactors by the endogenous CFE metabolism. To address this limitation, we engineer a CFE-based thermophilic multienzyme biocatalyst for the synthesis of serine from formate using genes from the moderate thermophile Moorella thermoacetica. After thermophilic formate-to-serine pathway gene expression, the mesophilic CFE machinery responsible for the CFE background metabolism is removed by simple heat denaturation, eliminating the diversion of cofactors and intermediates to other fates. After bioprocess optimization—including reaction temperatures, gene expression and chemical synthesis temperature and times—we achieve 97% yield of serine and glycine from formate. Importantly, the moderately thermophilic nature of the enzymes enables the chemical synthesis step to be performed at mesophilic temperatures, supporting cofactor stability. In fed-batch experiments, the biocatalyst shows steady chemical synthesis rates for 8 hours, paving the way toward a continuous bioprocess. Finally, toward reducing the cofactor cost in the CFE-based formate-to-serine biocatalyst, we performed a sensitivity analysis by reducing the concentration of individual cofactors and measuring its effect on process yields. To our knowledge, this is the first instance of expressing thermophilic genes in an E. coli-based CFE system to generate a thermophilic biocatalyst for use at mesophilic temperatures. Together, the thermophilic biocatalyst and heat denaturation strategy nearly triples product yields achieved by a previously developed mesophilic CFE-based formate-to-serine biocatalyst. To our knowledge, this is also the highest reported conversion of formate into chemicals using any biological process. Ultimately, this work opens the door to using E. coli CFE for thermophilic biocatalyst expression and rapid purification via heat denaturation. Further reductions in cofactor loading could enable high-yield and cost-effective amino acid synthesis.