Driving carbon flux through exogenous butyryl-CoA: Acetate CoA-transferase to produce butyric acid at high titer in Thermobifida fusca

Abstract

Butyric acid, a 4-carbon short chain fatty acid, is widely used in chemical, food, and pharmaceutical industries. The low activity of butyryl-CoA: acetate CoA-transferase in Thermobifida fusca muS, a thermophilic actinobacterium whose optimal temperature was 55°C, was found to hinder the accumulation of high yield of butyric acid. In order to solve this problem, an exogenous butyryl-CoA: acetate CoA-transferase gene (actA) from Thermoanaerobacterium thermosaccharolyticum DSM571 was integrated into the chromosome of T. fusca muS by replacing celR gene, forming T. fusca muS-1. We demonstrated that on 5g/L cellulose, the yield of butyric acid by the engineered muS-1 strain was increased by 42.9 % compared to the muS strain. On 100g/L of cellulose, the muS-1 strain could consume 90.5% of total cellulose in 144h, with 33.2g/L butyric acid produced. Furthermore, on the mix substrates including the major components of biomass: cellulose, xylose, mannose and galactose, 70.4g/L butyric acid was produced in 168h by fed-batch fermentation. To validate the ability of fermenting biomass, the muS-1 strain was grown on the milled corn stover ranging from 200 to 250μm. The muS-1 strain had the highest butyrate titer 17.1g/L on 90g/L corn stover.

Keywords: Butyric acid; Butyryl-CoAacetate CoA-transferase; Chromosome integration; Metabolic engineering.