Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides

Abstract

The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides , has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides . In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q 10 (Q 10 ) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, P rsp _ 7571 and P rsp _ 6124 , showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a P rsp _ 7571 -derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides . Finally, as a demonstration, the synthetic pathway of Q 10 was modulated by the selected promoter T334* in JDW-710; the Q 10 yield in shake-flasks increased 28% and the production reached 226 mg/L . These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides -derived MCFs.

Harnessing photosynthetic bacterium for light-powered biocatalysis

“…The untranslated regions of approximately 500 bp upstream from puc , puf, bch and crt operons were amplified from the genomic DNA of R. palustris CGA009 with primers Ppuc-F/Ppuc-R, Ppuf-F/Ppuf-R, PbchP-F/PbchP-R, and PcrtE-F/PcrtE-R, and then cloned into pBRT between Eco RI and Kpn I sites to obtain pBRPpuc, pBRPpuf, pBRPbchP, and pBRPcrtE, respectively. The P T334-6 promoter 18 was synthesized by GenScript (Nanjing, China) then cloned into pBRT between Eco RI and Kpn I sites to yield pBRPt334-6, and the DNA fragments containing the P T334-6 promoter and the oxygen-regulatory protein binding site of P puc from R. sphaeroides attained by PCR with primers Pt334O-F/Pt334O-R was digested with Eco RI and Kpn I and then cloned into pBRT to produce pBRPt334O. Besides, P lac and P tac promoters amplified from the vector pBBR1MCS-2 and pBBR-tacGFP 30 with primers Plac-F/Plac-R and Ptac-F/Ptac-R were respectively cloned into pBRT digested by Eco RI and Kpn I to produce pBRPlac and pBRPtac.…”

“…In this study, serval promoters to drive the reporter gene of egfp were examined in R. palustris, including promoters of puc operon (Ppuc), puf operon (Ppuc), bch operon (PbchP) and crt operon (PcrtE) from R. palustris, PT334-6 from Rhodobacter sphaeroides, lac operon (Plac) and its variant Ptac from E. coli. Specifically, the expression levels of puc and puf operon encoding the light-harvesting complex II and complex I are relatively high under light-anaerobic conditions; bch operon takes charge of the bacteriochlorophyll synthesis and crt operon is responsible for the carotenoid synthesis; PT334-6, a Prsp_7571-derived synthetic promoter, showed 32-fold higher activity than that of Ptac under the testing condition in R. sphaeroides 18 . As depicted in Figure 2a, five endogenous promoters displayed much higher activity than Plac but lower than Ptac, and the synthetic promoter of PT334-6 exhibited approximately 14-fold higher activity than that of Ptac under light-anaerobic conditions.…”

“…The copyright holder for this this version posted December 20, 2022. ; https://doi.org/10.1101/2022.12.20.521182 doi: bioRxiv preprint promoter 18 was synthesized by GenScript (Nanjing, China) then cloned into pBRT between EcoRI and KpnI sites to yield pBRPt334-6, and the DNA fragments containing the PT334-6 promoter and the oxygen-regulatory protein binding site of Ppuc from R. sphaeroides attained by PCR with primers Pt334O-F/Pt334O-R was digested with EcoRI and KpnI and then cloned into pBRT to produce pBRPt334O. Besides, Plac and Ptac promoters amplified from the vector pBBR1MCS-2 and pBBR-tacGFP 30 with primers Plac-F/Plac-R and Ptac-F/Ptac-R were respectively cloned into pBRT digested by EcoRI and KpnI to produce pBRPlac and pBRPtac.…”

Screening and Constructing a Library of Promoter-5′-UTR Complexes with Gradient Strength in Pediococcus acidilactici

“…17 RNA-sequencing (RNA-seq) can precisely identify an enormous number of promoter activities and transcriptional start sites. This strategy has been proven to be efficient and accurate in many cases, such as with Rhodobacter sphaeroides, 18 Burkholderiales, 19 Bacillus thuringiensis, 20 Bacillus anthracis, 21 Streptomyces albus, 22 and Gluconobacter oxydans. 23 In this regard, based on transcriptomic analysis, a large number of native PUTRs can be screened from the genome of P. acidilactici, which could provide gene regulatory elements for metabolic engineering modification of P. acidilactici.…”

Determination of Nucleotide Sequences within Promoter Regions Affecting Promoter Compatibility between Zymomonas mobilis and Escherichia coli

“…A promoter is a range of DNA sequences required for transcription initiation, which is usually located upstream of the 5′ end of a functional gene that includes conserved sequences to specifically bind RNA polymerase and transcriptional regulatory factors such as regulatory proteins and small RNA . Promoter sequences determine the expression intensity and expression patterns (constitutive or inducible) of downstream genes and thus become a key component of synthetic biology practices for driving gene expression, regulating gene circuits, and obtaining highly efficient cell factories. − Significant efforts have been made to achieve highly efficient and fine-tuned expression and regulation of target genes with diverse strengths through promoter engineering strategies. − …”