Application of the CRISPRi system to repress sepF expression in Mycobacterium smegmatis

Xiao, Jing; Jia, Hongyan; Pan, Liping; Li, Zihui; Lv, Lingna; Du, Boping; Zhang, Lanyue; Du, Fengjiao; Huang, Yinxia; Cao, Tingming; Sun, Qi; Wei, Rongrong; Xing, Aiying; Zhang, Zongde

doi:10.1016/j.meegid.2018.06.033

Cited by 14 publications

(6 citation statements)

References 27 publications

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“…In addition, compared with knocking out the ERG6 gene directly in the previous studies, CRISPRi technology provided a simple, efficient, and cost‐effective tool to repress the expression of essential genes dynamically (Xiao et al, 2019). Because the ERG6 gene showed a significant role on regulating lipid metabolism balance and cell growth, four dcas9 binding sites were randomly selected on the ERG6 promoter to inhibit GFP expression dynamically.…”

Section: Discussionmentioning

confidence: 99%

Engineered yeast for efficient de novo synthesis of 7‐dehydrocholesterol

Xiu

Sun

et al. 2022

Biotech & Bioengineering

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The synthesis of vitamin D3 precursor 7-dehydrocholesterol (7-DHC) by microbial fermentation has much attracted attention owing to its advantages of environmental protection. In this study, Saccharomyces cerevisiae was engineered for a de novo biosynthesis of 7-DHC. First, seven essential genes (six endogenous genes and one heterologous gene) were overexpressed, and the ROX1 gene (heme-dependent repressor of hypoxic genes) was knocked out. The resulting strain produced 82.6 mg/L 7-DHC from glucose. Then, we predicted five gene knockout targets for 7-DHC overproduction by the reconstruction of genome-scale metabolic model. GDH1 gene knockout increased the 7-DHC titer from 82.6 to 101.5 mg/L, and the specific growth rate of the ΔGDH1 mutant was also increased by 28%. Next, Ty1 transposon in S. cerevisiae was applied to increase the copies of the ERG1 gene and DHCR24 gene, resulting in a 120% increase in 7-DHC titer to 223.3 mg/L. Besides, to optimize the metabolic flux distribution, Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was used to dynamically inhibit the competitive pathway, and the best binding site of ERG6 (delta (24)-sterol C-methyltransferase) promoter was screened out. The OD 600 value of ERG6 regulated cells increased by 43% than knocking out ERG6 directly, and 7-DHC titer increased to 365.5 mg/L in a shake flask. Finally, the 7-DHC titer reached 1328 mg/L in 3-L bioreactor and the specific titer of 7-DHC reached up to 114.7 mg/g dry cell weight). Overall, this study constructed a yeast chassis for the highly efficient production of 7-DHC by systems metabolic engineering.

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Section: Discussionmentioning

confidence: 99%

Engineered yeast for efficient de novo synthesis of 7‐dehydrocholesterol

Xiu

Sun

et al. 2022

Biotech & Bioengineering

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“…1). CRISPRi has been developed in a diverse group of bacteria, including E. coli [5] , Corynebacterium glutamicum [7] , Lactococcus lactis [18] , Rhodococcus opacus [19] , Burkholderia [20] , Clostridia [21–25] , Bacillus subtilis [26–29] , Bacillus methanolicus [30] , Streptomyces [31,32], and Mycobacteria [33–36], and applied for gene repression to interrogate their physiology or to identify gene targets for biotech applications (see Sections 3 and 4).…”

Section: Establishment Of Crisprimentioning

confidence: 99%

“…Furthermore, multiplexed CRISPRi was achieved in Streptomyces when reduced mRNA levels (up to 32% of the control) were observed by simultaneous targeting of four genes in S. coelicolor [79]. In Lactobacillus plantarum and Mycobacterium smegmatis , CRISPRi was used to screen different knockdown strains in order to gain more insights into the physiology of these bacteria [34,80]. For instance, targeting sepF in M. smegmatis changed growth and morphology leading to elongated, filamentous, and branched bacterial cells [34].…”

Section: Gaining Physiological Insights From Crispri Screensmentioning

confidence: 99%

“…In Lactobacillus plantarum and Mycobacterium smegmatis , CRISPRi was used to screen different knockdown strains in order to gain more insights into the physiology of these bacteria [34,80]. For instance, targeting sepF in M. smegmatis changed growth and morphology leading to elongated, filamentous, and branched bacterial cells [34]. A knockdown sgRNA library of 37 predicted essential genes inhibited viability in Vibrio cholerae , validating these genes as potential drug target candidates [81].…”

Section: Gaining Physiological Insights From Crispri Screensmentioning

confidence: 99%

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Impact of CRISPR interference on strain development in biotechnology

Schultenkämper

Brito

Wendisch

2020

Biotech and App Biochem

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Genetic perturbation systems are of great interest to redirect metabolic fluxes for value‐added production, as well as genetic screening for the development of new drugs, or to identify new targets for biotechnological applications. Here, we review CRISPR interference (CRISPRi), a method for gene expression using a catalytically inactive version of the CRISPR‐associated protein 9 (dCas9) of the widely applied CRISPR‐Cas9 genome editing system. In combination with the appropriate sgRNA, dCas9 binds to specific DNA sequences without causing double‐stranded DNA breakage but interfering with transcription initiation or elongation. Besides manifold uses to interrogate the physiology of a bacterial cell, CRISPRi is used in applications for metabolic engineering and strain development in industrial biotechnology. Albeit in its infancy, CRISPRi has already delivered the first success stories; however, we also analyze limitations of the CRISPRi system and give future perspectives.

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“…Electrocompetent M. smegmatis cells were prepared as previously described. 20 Approximately 500 ng of plasmid was added to 200 µL of cell suspension and mixed by gentle pipetting then dispensed into a prechilled electroporation cuvette (with a 0.2 cm electrode gap) followed by incubation on ice. The Bio-Rad Gene Pulser used for electroporation was set at 2.5 kV, 1000 Ω and 25 μF.…”

Section: Electroporationmentioning

confidence: 99%

<p>Comparison of in vitro Susceptibility of Mycobacteria Against PA-824 to Identify Key Residues of Ddn, the Deazoflavin-Dependent Nitroreductase from <em>Mycobacterium tuberculosis</em></p>

Zhang¹,

Li²,

Wen³

et al. 2020

IDR

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Objective: PA-824 (Pretomanid), a bicyclic nitroimidazole drug, exhibits significant bactericidal activity toward Mycobacterium tuberculosis (MTB) in vitro and in vivo, but not against Mycobacterium smegmatis. Through catalytic bioreduction, deazaflavin-dependent nitroreductase (Ddn) within MTB directly converts PA-824 to potent bactericidal products. This study aimed to identify key MTB Ddn residues involved in PA-824 conversion toward development of in vitro surrogate markers for detection of mycobacterial resistance to PA-824. Methods: We evaluated in vitro activity of PA-824 toward MTB and nontuberculous mycobacterial species using antimicrobial susceptibility testing. Ddn amino acid sequence alignments and phylogenetic analysis revealed putative key enzyme active site residues. Candidate MTB Ddn residues required for PA-824 conversion activity were evaluated for loss-of-function using recombinantly cloned Ddn mutant proteins expressed in Mycobacterium smegmatis. Results: PA-824 minimum inhibitory concentrations of 90% of bacterial growth (MIC 90 s) against MTB and Mycobacterium kansasii were 0.12 mg/L and 8 mg/L, respectively, but >32 mg/L for Mycobacterium spp. M. avium, M. intracellulare, M. abscessus and M. fortuitum. MTB Ddn and M. kansasii Ddn homologous sequences shared the greatest similarity (89.3% amino acid identity). M. smegmatis expressing Ddn proteins with Y65L, A76V or Y133F substitutions (but not V75L, Q125K or V148I) were resistant to PA-824. Conclusion: Our data demonstrated that PA-824 exhibited excellent and moderate levels of in vitro activity against MTB and M. kansasii, respectively. Substitutions of Ddn residues Y65, A76 or Y133 conferred mycobacterial resistance to PA-824.

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Application of the CRISPRi system to repress sepF expression in Mycobacterium smegmatis

Cited by 14 publications

References 27 publications

Engineered yeast for efficient de novo synthesis of 7‐dehydrocholesterol

Engineered yeast for efficient de novo synthesis of 7‐dehydrocholesterol

Impact of CRISPR interference on strain development in biotechnology

<p>Comparison of in vitro Susceptibility of Mycobacteria Against PA-824 to Identify Key Residues of Ddn, the Deazoflavin-Dependent Nitroreductase from <em>Mycobacterium tuberculosis</em></p>

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