Metabolic engineering of streptomyces sp. for ethanol production

Abstract

Streptomyces strains have a wide range of substrate specificity and produce enzymes such as cellulase, xylanase, amylase, β-glucosidase etc, and this makes them good candidates for bioproducts production using biomass hydrolysate containing many different monosaccharides and disaccharides. A pet operon containing two genes from Zymomonas mobilis that are responsible for ethanol production, pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhII), were constructed. They were heterologously expressed in Streptomyces lividans TK24. An examination of carbon distribution revealed that a significant portion of carbon metabolism was switched from cell mass and organic acid biosynthesis to ethanol production upon their expression. As a molecular strategy to increase the expression level of AdhII, a thiostrepton-inducible tipA promoter was inserted in front of the adhII gene, which resulted in the construction of Tpet module. The strain transformed with Tpet module was named as S. lividans TK24/Tpet. The activity of AdhII with Tpet module was 2.75-fold higher than that with pet operon promoter with a higher ethanol yield. S. lividans TK24/Tpet was able to produce ethanol from glucose with a yield of 23.7% (46.7 % of the theoretical yield). It produced ethanol from xylose, L-arabinose, mannose, L-rhamnose, galactose, ribose, and cellobiose with yields of 16.0%, 25.6%, 21.5%, 33.6%, 30.6%, 14.6% and 33.3% based on the consumed substrates, respectively. It also showed an ethanologenic capability on polymeric substances such as starch and xylan, with yields of 18.9% and 13.0%, respectively. A careful examination on substrate uptake rates and ethanol production rates among the carbon sources mentioned above reveals that both rates are closely related each other. A carbon source with a higher uptake showed an ethanol production rate. This implies that excess carbon flux over the oxidation capacity of S. lividans TK24/Tpet was diverted to ethanol biosynthesis. Existence of catabolic repression by glucose and thus its effects on the utilization of each carbon source were investigated. Glucose of 10 g/L was added to the fermentation medium containing 10 g/L of galactose, xylose, L-arabinose, mannose, L-rhamnose, ribose, or cellobiose. All of the above-mentioned carbon sources were utilized simultaneously with glucose, implying that the catabolic repression by glucose was not significant. Knowing that a hydrolysate of biomass in general is a mixture of glucose and many other carbon substrates, simultaneous utilization of glucose and other sugar is a huge advantage in using biomass hydrolysates as a feedstock for fermentation. Providing a microbial platform having a broad range of substrate specificity is valuable for efficient utilization of biomass which comprises of a variety of carbon sources, such as lignocellulosic or microalgal biomass. Noticeably, this is the first incident of using a genetically engineered Streptomyces strain for bioethanol production and demonstrating its broad substrate specificity with significant ethanol yields.