Challenges and Advances for Genetic Engineering of Non-model Bacteria and Uses in Consolidated Bioprocessing

Abstract

Metabolic diversity in microorganisms can provide the basis for creating novel biochemical products. However, most metabolic engineering projects utilize a handful of established model organisms and thus, a challenge for harnessing the potential of novel microbial functions is the ability to either heterologously express novel genes or directly utilize non-model organisms. Genetic manipulation of non-model microorganisms is still challenging due to organism-specific nuances that hinder universal molecular genetic tools and translatable knowledge of intracellular biochemical pathways and regulatory mechanisms. However, in the past several years, unprecedented progress has been made in synthetic biology, molecular genetics tools development, applications of omics data techniques, and computational tools that can aid in developing non-model hosts in a systematic manner. In this review, we focus on concerns and approaches related to working with non-model microorganisms including developing molecular genetics tools such as shuttle vectors, selectable markers, and expression systems. In addition, we will discuss: (1) current techniques in controlling gene expression (transcriptional/translational level), (2) advances in site-specific genome engineering tools [homologous recombination (HR) and clustered regularly interspaced short palindromic repeats (CRISPR)], and (3) advances in genome-scale metabolic models (GSMMs) in guiding design of non-model species. Application of these principles to metabolic engineering strategies for consolidated bioprocessing (CBP) will be discussed along with some brief comments on foreseeable future prospects.

Development and Application of CRISPR/Cas in Microbial Biotechnology

“…The CRISPR/Cas9 system has also been constructed with similar strategy for non-model microorganisms (Yan and Fong, 2017;Cho et al, 2018;Raschmanova et al, 2018;Wang and Coleman, 2019). Generally, species-specific strong promoters should be used for Cas9 expression, either constitutively or inducible expressed.…”

Insights into Thermophilic Plant Biomass Hydrolysis from Caldicellulosiruptor Systems Biology

“…In parallel with the intense enzymatic focus on the genus Caldicellulosiruptor, the development of genetic tools afforded the opportunity to develop C. bescii as a potential platform organism for consolidated bioprocessing (CBP) where the same organism would enzymatically attack and ferment plant biomass to usable products (CBP reviewed in [114,115]). Development of the Caldicellulosiruptor genetic system relied on the identification of restriction-modification systems [116] that were impeding transformation by improperly methylated DNA isolated from E. coli [116,117].…”

Metabolic engineering strategies for butanol production in Escherichia coli

“…The in situ application of some of these processes can help alleviating butanol toxicity issues (Dürre, 2007;Visioli et al, 2014) Lastly, the lack of efficient synthetic biology tools to edit microorganisms from genus Clostridium hinders the application of ME strategies. The main issues are the low efficiency of DNA transformation (below 10 CFU/μg), endonuclease activity, the requirement for a robust selective marker and the lack of a stable shuttle plasmid available (Joseph, Kim, & Sandoval, 2018;Yan & Fong, 2017). The advent of new genome-editing strategies, particularly of the CRISPR-Cas9 system, has facilitated strain engineering for several microorganisms and, simultaneously, enlarged the range of applications (Tian et al, 2017).…”