Converging conversion – using promiscuous biocatalysts for the cell-free synthesis of chemicals from heterogeneous biomass

Abstract

Production of chemicals from lignocellulosic biomass has been proposed as a suitable replacement to petrochemicals. However, one inherent challenge of biomass utilization is the heterogeneity of the substrate resulting in the presence of mixed sugars after hydrolysis. Fermentation of mixed sugars often leads to poor yield and generation of multiple by-products, thus complicating the subsequent downstream processing. System biocatalysis has thus been developed in recent years to address this challenge. In this work, several novel enzymes with broad substrate promiscuity were identified using a sequence-based discovery approach as suitable biocatalysts in a conversion of D-xylose and L-arabinose, two major constituents of hemicellulose found in plant biomass. These promiscuous enzymes enabled simultaneous biotransformation of D-xylose and L-arabinose to yield 1,4-butanediol (BDO) with a maximum production rate of 3 g L⁻¹ h⁻¹ and a yield of >95%. This model system was further adapted toward the production of α-ketoglutarate (2-KG) from the pentoses using O₂ as a cosubstrate for cofactor recycling reaching a maximum production rate of 4.2 g L⁻¹ h⁻¹ and a yield of 99%. To verify the potential applicability of our system, we attempted to scale up the BDO and 2-KG production from D-xylose and L-arabinose. Simple optimization and reaction engineering allowed us to obtain BDO and 2-KG titers of 18 g L⁻¹ and 42 g L⁻¹, with theoretical yields of >75% and >99%, respectively. One of the promiscuous enzymes identified together with auxiliary promiscuous enzymes was also suitable for stereoconvergent synthesis from a mixture of D-glucose and D-galactose, predominant sugars found in food waste streams and microalgae biomass.