Native xylose-inducible promoter expands the genetic tools for the biomass-degrading, extremely thermophilic bacterium Caldicellulosiruptor bescii

Abstract

Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

“…Coupled with the previously established genomics, transcriptomics, and proteomics data available for the genus, the stage is set for further advancements towards targeted strain development. Initial reports of an inducible promoter [143] and identification of a redox-responsive regulon [144] in C. bescii are supporting the nascent field of Caldicellulosiruptor synthetic biology. Moving forward, the use of established '-omics' tools to identify additional regulated promoters, optimal ribosomal binding sites, and other 5 untranslated sequences will be critical for continued advancements in genetic manipulation of the genus Caldicellulosiruptor (see Figure 2).…”

Construction of lactic acid overproducing Clostridium thermocellum through enhancement of lactate dehydrogenase expression

“…Although this has not been confirmed in C. thermocellum yet, ldh promoter engineering obtained in this study may have altered this regulation. Recently, a similar approach was used to improve the expression of the ldh gene from Caldicellulosyruptor bescii [33]. In this case the original promoter was replaced with the xylose-inducible promoter P xi but improvement of the specific LDH activity with respect to the wild type strain (about 3 fold) was lower than that obtained in the present study.…”

Novel multidomain, multifunctional glycoside hydrolases from highly lignocellulolytic Caldicellulosiruptor species

“…Now that genetic tools have been established for C. bescii , it could be engineered to produce an optimized set of multidomain GHs that relate to the plant biomass substrate. This would require strategic design of inducible promoters driving expression to create maximum carbohydrate hydrolysis and conversion to bio‐based fuels and chemicals. This will be the next step in developing C. bescii or other Caldicellulosiruptor species for industrial biotechnology applications.…”