Engineering dual-glycan responsive expression systems for tunable production of heterologous proteins in Bacteroides thetaiotaomicron

Sci Rep. 2019 Nov 22;9(1):17400. doi: 10.1038/s41598-019-53726-w.

Abstract

Genetically engineering intestinal bacteria, such as Bacteroides thetaiotaomicron (B. theta), holds potential for creating new classes of biological devices, such as diagnostics or therapeutic delivery systems. Here, we have developed a series of B. theta strains that produce functional transgenic enzymes in response to dextran and arabinogalactan, two chemically distinct glycans. Expression systems for single glycan induction, and a novel "dual-glycan" expression system, requiring the presence of both dextran and arabinogalactan, have been developed. In addition, we have created two different chromosomal integration systems and one episomal vector system, compatible with engineered recipient strains, to improve the throughput and flexibility of gene cloning, integration, and expression in B. theta. To monitor activity, we have demonstrated the functionality of two different transgenic enzymes: NanoLuc, a luciferase, and BuGH16C, an agarase from the human intestinal bacterium, Bacteroides uniforms NP1. Together this expression platform provides a new collection of glycan-responsive tools to improve the strength and fidelity of transgene expression in B. theta and provides proof-of-concept for engineering more complex multi-glycan expression systems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteroides thetaiotaomicron / genetics*
  • Bacteroides thetaiotaomicron / metabolism*
  • Chromosomes, Bacterial
  • Gene Expression Regulation, Bacterial
  • Gene Order
  • Gene Targeting
  • Genetic Engineering*
  • Genetic Vectors / chemistry
  • Glycoside Hydrolases / biosynthesis
  • Glycoside Hydrolases / genetics
  • Humans
  • Plasmids / genetics
  • Polysaccharides / metabolism*
  • Promoter Regions, Genetic
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / genetics*
  • Transgenes

Substances

  • Polysaccharides
  • Recombinant Proteins
  • Glycoside Hydrolases
  • agarase