Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology

Huang, Jenq‐Kuen; Samassekou, Kadidia; Alhmadi, Hekmat B.; VanDerway, David; Diaz, Joshua D; Seiver, Jacob; McClenahan, Shawn; Holt, Scott M.; Wen, Lisa

doi:10.1371/journal.pone.0230915

Cited by 8 publications

(9 citation statements)

References 74 publications

(119 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This allows the UV-vis spectrophotometric monitoring of NADH production over time as a measure of Ohy activity under the right conditions. The 10-HSA oxidation activity was already reported in microbial whole cell reactions (Niehaus et al 1978), and a number of 10-HSA converting alcohol dehydrogenases have been identified (Huang et al 2020;Wu et al 2019). ADH010 and ADH020 out of a commercial screen of ten different NAD +dependent alcohol dehydrogenases (Evoxx, Monheim am Rhein, Germany) were found to convert 10-HSA efficiently (Busch et al 2020b).…”

Section: Enzyme Activity Assaysmentioning

confidence: 91%

Novel oleate hydratases and potential biotechnological applications

Hagedoorn

Hollmann

Hanefeld

2021

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Oleate hydratase catalyses the addition of water to the CC double bond of oleic acid to produce (R)-10-hydroxystearic acid. The enzyme requires an FAD cofactor that functions to optimise the active site structure. A wide range of unsaturated fatty acids can be hydrated at the C10 and in some cases the C13 position. The substrate scope can be expanded using ‘decoy’ small carboxylic acids to convert small chain alkenes to secondary alcohols, albeit at low conversion rates. Systematic protein engineering and directed evolution to widen the substrate scope and increase the conversion rate is possible, supported by new high throughput screening assays that have been developed. Multi-enzyme cascades allow the formation of a wide range of products including keto-fatty acids, secondary alcohols, secondary amines and α,ω-dicarboxylic acids. Key points • Phylogenetically distinct oleate hydratases may exhibit mechanistic differences. • Protein engineering to improve productivity and substrate scope is possible. • Multi-enzymatic cascades greatly widen the product portfolio.

show abstract

Section: Enzyme Activity Assaysmentioning

confidence: 91%

Novel oleate hydratases and potential biotechnological applications

Hagedoorn

Hollmann

Hanefeld

2021

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…There is a report of application of genome editing technology employing CRISPR/Cas9 technique to desirably knockout secondary alcohol dehydrogenase in N. cholesterolicum [73], if such technology is employed in case of rubber degrading microbes, we can expect positive outputs. To cope up with the threat of Nocardia contaminated wastes, lytic bacteriophages can be used, as a similar case has been reported for biocontrol of Nocardia-stabilized foams in activated sludge plants by lytic bacteriophage GTE2 [74].…”

Section: Presence Of Virulence Factors In the Genome Sequencesmentioning

confidence: 99%

Insights from the comparative genome analysis of natural rubber degrading Nocardia species

Sarkar¹

2021

Bioinformation

View full text Add to dashboard Cite

Nocardia are known to be a facultative human pathogen and can cause infection in immune compromised patients. Though the details research on the virulence factors of Nocardia are scanty but numerous genes that code such factors were reported from different species of Nocardia. Despite of the presence of several virulence factors, species of this genus have been shown to have role in remediation of many toxic and hazardous materials from the environment. In this study, genome sequences of rubber degrading Nocardia sp. BSTN01 and N. nova SH22a have been analyzed to locate the potential virulence genes. Also, the genomes of facultative pathogenic Nocardia like, N. africana, N. brasiliensis, N. kruczakiae, N. transvalensis and N. veterana have been analyzed to find the gene encoding latex clearing protein (Lcp), a rubber oxygenase enzyme of Gram-positive action bacteria. The study provides an insight about the potentiality of rubber-degrading Nocardia species to emerge as future human pathogens and also the probability of a serious concern if the studied facultative pathogens of Nocardia like N. africana, N. brasiliensis, N. kruczakiae, N. transvalensis and N. veteranaare capable of degrading rubber, a regularly used material in clinics. Moreover, use of such possible pathogenic strains for their known role in bioremediation of rubber waste from the environment might be deleterious.

show abstract

“…All of the 157 possible gRNAs sequences that can target PAM (Protospacer Adjacent Motif) sequences were analyzed based on efficiency and off-targets for mismatches. Four possible 24 nucleotides long protospacer sequences (4 nt 5' sticky end + 20 nt spacer sequence) with the sticky ends ACGC on the forward sequence and AAAC on the reverse sequence were chemically synthesized [19]. The protospacer sequence was generated by equimolar ligation as previously reported (Supplementary Figure S1) [19].…”

Section: Design and Construction Of Sgrnas Plasmidsmentioning

confidence: 99%

“…Four possible 24 nucleotides long protospacer sequences (4 nt 5' sticky end + 20 nt spacer sequence) with the sticky ends ACGC on the forward sequence and AAAC on the reverse sequence were chemically synthesized [19]. The protospacer sequence was generated by equimolar ligation as previously reported (Supplementary Figure S1) [19]. The gRNA sequence containing sticky end overhands was inserted into CRISPomyces-2 plasmid using golden-gate assembly protocol using Bbs1 (type IIs restriction enzyme) (Supplementary Figure S1) [20].…”

Section: Design and Construction Of Sgrnas Plasmidsmentioning

confidence: 99%

CRISPR-Cas9 mediated knockout of SagD gene for overexpression of streptokinase in Streptococcus equisimilis

Chaudhari

Vyas

Patel

et al. 2021

Preprint

View full text Add to dashboard Cite

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (Heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is high globally than the production due to increased incidences of various heart conditions. The main source of streptokinase is from various strains of Streptococcus. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD inhibitor gene for production of streptokinase that needs to be knocked out in order to increase it expression. However, FasX is a small RNA (sRNA) present in group A Streptococcus species which is responsible for post-transcriptional regulation of streptokinase (ska) gene by binding at the 5' end of ska mRNA. S. equisimilis is a β-hemolysin producing streptococcus bacterium (group C) containing the orthologue of FasX and natively expresses a clinically important thrombolytic streptokinase. In order to improve the stability of mRNA and increasing the expression of streptokinase which is inhibited by SagD. We used CRISPR-Cas9 to successfully knock- out of SagD gene and observed a 13.58-fold relative quantification of streptokinase expression in the mutant strain as compared to wild type. We have also demonstrated the successful target gene knockout using CRISPR-Cas9 in S. equisimilis that engineered strain can be used further for overexpression of streptokinase for therapeutic applications.

show abstract

Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology

Abstract: Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10

Cited by 8 publications

References 74 publications

Novel oleate hydratases and potential biotechnological applications

Novel oleate hydratases and potential biotechnological applications

Insights from the comparative genome analysis of natural rubber degrading Nocardia species

CRISPR-Cas9 mediated knockout of SagD gene for overexpression of streptokinase in Streptococcus equisimilis

Contact Info

Product

Resources

About