Overexpressing atpXF enhanced photo-fermentative hydrogen production performance of Rhodobacter sphaeroides

Zhang, Yang; Hu, Jun; Ma, Hongyu; Yang, Honghui; Guo, Liejin

doi:10.1016/j.ijhydene.2017.01.076

Cited by 14 publications

(6 citation statements)

References 46 publications

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“…To the best of our knowledge, CRISPRi using DNase‐deactivated Cas9 (dCas9) or dCas12a has not been reported in any Rhodobacter ‐related species. Previous findings suggested that dCas12a exhibits better repression efficiency with crRNAs targeting at the template strand over the non‐template strand, and the closer crRNA near to the translation start site, the higher repression efficiency is obtained (Zhang et al ., 2017a , 2017b , 2017c , 2017a , 2017b , 2017c , 2017a , 2017b , 2017c ; Liu et al., 2019 ; Zhao et al., 2019 ). In this study, similar results were observed when targeting at egfp or lacZ reporter genes in R. capsulatus using CRISPR/dCas12a.…”

Section: Discussionmentioning

confidence: 99%

“…Fluorescence intensities were normalized to culture OD 630 for comparing the relative expression level of eGFP. The β‐galactosidase activity assay was performed as previously described (Zhang et al ., 2017a , 2017b , 2017c , 2017a , 2017b , 2017c , 2017a , 2017b , 2017c ). In brief, the active protein exacted from R. capsulatus reacted with o ‐nitrophenyl‐β‐ d ‐galactopyranoside (ONPG) at 35°C.…”

Section: Methodsmentioning

confidence: 99%

“…To survive under such diverse living conditions, PNSB have evolved a complex metabolic network with highly specialized enzyme complexes and regulatory mechanisms. Recently, PNSB have attracted special attention as a platform microorganism for biotechnological applications such as H 2 production as an alternative renewable energy to fossil fuels (Zhang et al ., 2017a , 2017b , 2017c , 2016 , 2017a , 2017b , 2017c , 2016 , 2017a , 2017b , 2017c , 2016 ) and polyhydroxybutyrate as a biodegradable plastic (Kranz et al., 1997 ; Merugu et al., 2012 ), bioremediation of wastewater (Idi et al., 2014 ) and heavy metal (Feng et al., 2007 ), fixation of CO 2 and N 2 (Wahlund et al., 1996 ; Atsumi et al., 2009 ), expression of membrane proteins (Berry et al., 2004 ; Han and Perner, 2016 ) and metalloenzymes (Vignais et al., 2000 ; Kappler and McEwan, 2002 ). As the synthesis of photopigments in PNSB relies on the isoprenoid biosynthetic pathway, it provides a robust isoprenoid metabolism that makes PNSB as a promising host for isoprenoid biosynthesis (Heck and Drepper, 2017 ; Loeschcke et al., 2017 ; Troost et al., 2019 ).…”

Section: Introductionmentioning

confidence: 99%

“…Currently, the genetic tools in PNSB heavily rely on traditional homologous recombination using suicide plasmids (Zhang et al ., 2017a , 2017b , 2017c , 2017a , 2017b , 2017c , 2017a , 2017b , 2017c ), interposon‐mutagenesis using gene transfer agent (GTA) that leads to gene knockout via the insertion of a kanamycin cassette (Daldal et al., 1986 ), and gene mutation using transposon (Zhang et al., 2016 ). The synthetic biology tools for engineering PNSB are still limited, which significantly slows the progress in PNSB‐related biotechnological applications.…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas12a‐mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus

Zhang

2021

Microbial Biotechnology

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Purple non-sulfur photosynthetic bacteria (PNSB) such as Rhodobacter capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for genome editing and transcriptional regulation in R. capsulatus. Templatefree disruption method mediated by CRISPR/Cas12a reached ~90% editing efficiency when targeting ccoO or nifH gene. When both genes were simultaneously edited, the multiplex editing efficiency reached > 63%. In addition, CRISPR interference (CRISPRi) using deactivated Cas12a was also evaluated using reporter genes egfp and lacZ, and the transcriptional repression efficiency reached ~80%. In summary, our work represents the first report to develop CRISPR/ Cas12a-mediated genome editing and transcriptional regulation in R. capsulatus, which would greatly accelerate PNSB-related researches.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas12a‐mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus

Zhang

2021

Microbial Biotechnology

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“…Fluorescence intensities were normalized to culture OD630 for comparing the relative expression level of eGFP. The β-galactosidase activity assay was performed as previously described (48). In brief, the active protein exacted from R. capsulatus reacted with o-nitrophenyl-β-Dgalactopyranoside (ONPG) at 35°C.…”

Section: Fluorescence Intensities and β-Galactosidase Activity Assaysmentioning

confidence: 99%

CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

Zhang

2020

Preprint

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Purple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for both genome editing and gene down-regulation in R. capsulatus. About 90% editing efficiency was achieved by using CRISPR/Cas12a driven by a strong promoter Ppuc when targeting ccoO or nifH gene. When both genes were simultaneously targeted, the multiplex gene editing efficiency reached >63%. In addition, CRISPR interference using deactivated Cas12a was also evaluated using reporter genes gfp and lacZ, and the repression efficiency reached >80%. In summary, our work represents the first report to develop CRISPR/Cas12a mediated genome editing/transcriptional repression in R. capsulatus, which would greatly accelerate PNSB-related researches.IMPORTANCEPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. However, lack of efficient gene editing tools remains a main obstacle for progressing in PNSB-related researches. Here, we developed CRISPR/Cas12a for genome editing via the non-homologous end joining (NHEJ) repair machinery in R. capsulatus. In addition, DNase-deactivated Cas12a was found to simultaneously suppress multiple targeted genes. Taken together, our work offers a new set of tools for efficient genome engineering in PNSB such as R. capsulatus.

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Metabolic Engineering and Synthetic Biology in Biohydrogen Production

Ramasamy,

Pakshirajan

2024

Biofuel and Biorefinery Technologies

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Overexpressing atpXF enhanced photo-fermentative hydrogen production performance of Rhodobacter sphaeroides

Cited by 14 publications

References 46 publications

CRISPR/Cas12a‐mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus

CRISPR/Cas12a‐mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus

CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

Metabolic Engineering and Synthetic Biology in Biohydrogen Production

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