Synthetic biology approaches to improve the production of fructooligossacharides using Zymomonas mobilis as chassis

Abstract

Fructooligosaccharides (FOS) are promising prebiotics in the increasing market of functional food. Industrially, FOS are produced from sucrose using fructosyltransferase (FTase) or -fructofuranosidase enzymes (FFase). To increase its economic competitiveness, cost-effective FOS production processes with high titers, yields and productivities must be designed. Zymomonas mobilis (ZM) is a well-known ethanologenic bacterium with outstanding characteristics which makes it a promising chassis for the biotechnological production of relevant industrial compounds. This bacterium does not possess a FTase/FFase enzyme to perform the transfructosylation of sucrose. Nevertheless, the overexpression of these enzymes from well-recognized FOS producing microorganisms using metabolic engineering and synthetic biology tools could be an excellent approach to increase the production of FOS in ZM. Hence, ZM was genetically modified to produce FOS using FTase/FFase genes from Schwanniomyces occidentalis, Aspergillus sydowii and A. fijiensis. Since this bacterium does not contain a sucrose transporter, a native signal sequence from ZM acid phosphatase gene (phoC) was inserted before the FTase/FFase genes for their expression in ZM. Moreover, synthetic ribosome binding sites with high translation initiation rate (TIRs) were designed using the RBS Calculator (v2.1) tool developed by Salis Lab (Penn State University) to improve protein production. This study demonstrates the potential of use ZM as chassis to produce significant amounts of prebiotics with relevant health benefits.