Developing a Synthetic Biology Toolkit for Comamonas testosteroni, an Emerging Cellular Chassis for Bioremediation

Abstract

Synthetic biology is rapidly evolving into a new phase that emphasizes real-world applications such as environmental remediation. Recently, Comamonas testosteroni has become a promising chassis for bioremediation due to its natural pollutant-degrading capacity; however, its application is hindered by the lack of fundamental gene expression tools. Here, we present a synthetic biology toolkit that enables rapid creation of functional gene circuits in C. testosteroni. We first built a shuttle system that allows efficient circuit construction in E. coli and necessary phenotypic testing in C. testosteroni. Then, we tested a set of wildtype inducible promoters, and further used a hybrid strategy to create engineered promoters to expand expression strength and dynamics. Additionally, we tested the T7 RNA Polymerase-P promoter system and reduced its leaky expression through promoter mutation for gene expression. By coupling random library construction with FACS screening, we further developed a synthetic T7 promoter library to confer a wider range of expression strength and dynamic characteristics. This study provides a set of valuable tools to engineer gene circuits in C. testosteroni, facilitating the establishment of the organism as a useful microbial chassis for bioremediation purposes.

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria

“…Since the Redγ has been reported to increase the HR efficiency of the RecET system by protecting linear dsDNA substrates (Fu et al ., 2012), it was also included in iEDB. For both architectures, the strong IPTG inducible hybrid P CNO promoter (Tang et al ., 2018) was utilized for their efficient expression.…”

Production of Biogenic Nanoparticles for the Reduction of 4-Nitrophenol and Oxidative Laccase-Like Reactions

“…Hidden in the genomes and the physiology of the organism lies the ability for the reprogramming and repurposing of these pathways that make these small highly sought after particles. In recent years, the emergence of synthetic biology and its potential to be a powerful genetic engineering platform has made it possible to convert non-model organisms into novel chasses for the bioremediation of different metals and chemicals into valuable products (Tang et al, 2018). Both Morganella and Desulfovibrio sp.…”

Alternative Strategies for Microbial Remediation of Pollutants via Synthetic Biology

“…Nowadays, the new insights toward CRISPR tool kits and designing gRNA for expression of function-specific genes related to remediation in nonmodel organisms (i.e., Rhodococcus ruber TH, Comamonas testosteroni and Achromobacter sp. HZ01) are also suggested in the field of bioremediation (Mougiakos et al, 2016;Jusiak et al, 2016;Hong et al, 2017;Stein et al, 2018;Tang et al, 2018;Liang et al, 2020). For gene editing and metabolic engineering, the contaminant-inhabited bacteria are the most suitable candidates because they are used to survive and harbor in stress conditions of various toxic, recalcitrant and non-degradable xenobiotics, as discussed above.…”