Discovery and Characterization of NativeDeinococcus radioduransPromoters for Tunable Gene Expression

Abstract

The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of Deinococcus radiodurans for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in D. radiodurans using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in D. radiodurans. Of these top eight, two promoter regions, PDR_1261 and PrpmB, were stronger and more consistent than the most widely used promoter sequence in D. radiodurans, PgroES. Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in Escherichia coli or were D. radiodurans specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in D. radiodurans. IMPORTANCE The tolerance of the extremophile, Deinococcus radiodurans, to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in D. radiodurans, very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for D. radiodurans via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, PgroES. This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in D. radiodurans. The promoters identified in this study will facilitate metabolic engineering of D. radiodurans and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.R ecent work regarding the biological engineering of extremophiles has increased interest in their use as a robust chassis for metabolic engineering. The appeal of using extremophiles in various applications is largely due to their ability to survive conditions toxic to traditional engineering strains. One extremophile considered attractive is Deinococcus radiodurans, a Gram-positive bacteria known for its tolerance to ionizing radiation, heavy metal exposure, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens (1-3). In the context of its applications in bioremediation,

SLICER: Seamless Loss of Integrated Cassettes Using Endonuclease Cleavage and Recombination inDeinococcus radiodurans

“…The I- Sce I enzyme was chosen because there are no recognition sites present in the wild-type genome of D. radiodurans . The I- Sce I endonuclease was designed under the regulation of the PDR_2508 promoter and terminator set as it was shown to have high expression in D. radiodurans but low expression in E. coli 18 . This plasmid was then transformed into an E. coli Δ dap A strain harbouring the conjugative plasmid pTA-Mob 19 .…”

CRISPRi inDeinococcus radiodurans

“…This study demonstrates application of Cascade-based system to knockdown an assayable gene, phoN as well as an essential gene, ssb . As promoters in this organism are not very well characterized and are poorly predicted by bioinformatics tools (Chen et al, 2019), we targeted the start of the ORF regions to ascertain silencing efficiency, a feature that will have to be necessarily used for functional studies of hypothetical, novel or poorly characterized genes. This is especially important in this organism considering the inability of in silico approaches to efficiently predict even strong deinococcal promoters (Chen et al, 2019).…”

“…As promoters in this organism are not very well characterized and are poorly predicted by bioinformatics tools (Chen et al, 2019), we targeted the start of the ORF regions to ascertain silencing efficiency, a feature that will have to be necessarily used for functional studies of hypothetical, novel or poorly characterized genes. This is especially important in this organism considering the inability of in silico approaches to efficiently predict even strong deinococcal promoters (Chen et al, 2019). The efficiency of knockdown of phoN as assayed by phosphatase activity was around 90%, despite the multipartite genome in this organism.…”

“…With the availability of defined promoters, for inducible expression of genes (Lecointe et al, 2004) (Chen et al, 2019), and selection markers (Smith et al, 1988) (Harris et al, 2004) (Bouthier de la Tour et al, 2009) and the development of shuttle plasmids (Smith et al, 1989) (Masters & Minton, 1992) (Meima & Lidstrom, 2000), recombineering system (Jeong et al, 2017) and conjugation system (Brumwell et al, 2022), the tool-kit for the genetic manipulation of this important organism has been expanding.…”

Conjugation-based genome engineering in Deinococcus radiodurans

“…Recent PacBio sequencing of this strain resulted in a high quality genome that identified both large insertions and single nucleotide polymorphisms (SNPs) when mapped to the original sequence 14 . Engineering in this organism is progressing with the available sequence data and expanding genetic toolkit including shuttle plasmids [15][16][17] , selection markers [18][19][20] , characterized promoters for inducible and tunable gene expression 21,22 , as well as a strategy for generating gene deletions using a Crelox system 23 . In order to employ engineering strategies, it is crucial to have a method of delivering DNA to the host which can currently be achieved in D. radiodurans by natural transformation, chemical transformation and electroporation 24 .…”