Abstract
Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5’ coding region and the 5’ upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.
Highlights
Hydroxy fatty acids (HFAs) have potential industrial applications as lubricants, waxes, resins, nylons, plastics, cosmetics, additives in coating and paintings, flavors, antimicrobial agent, or precursors for lactones and dicarboxylic acids [1,2,3,4,5]
Nocardia cholesterolicum NRRL 6767 (N. cholesterolicum NRRL5767) is the interest of our on-going research because it produces high yields and it has been proven to be a stable microbe with industrial applications [21,22,23]
It has been reported that resting cells of N. cholesterolicum NRRL 5767 are able to convert added oleic acid (OA) to ~88% of 10-hydroxysteatic acid (10-HSA) (w/w) and 11% of 10-ketostearic acid (10-KSA, w/w) [12]
Summary
Hydroxy fatty acids (HFAs) have potential industrial applications as lubricants, waxes, resins, nylons, plastics, cosmetics, additives in coating and paintings, flavors, antimicrobial agent, or precursors for lactones and dicarboxylic acids [1,2,3,4,5]. Three microbes have been reported to produce high yields of 10-hydroxysteatic acid (10-HSA) from oleic acid (OA) and 10-hydroxy-12(Z)octadecenoic acid (10OH12OD) from linoleic acid (LA) [12, 17, 20]. It has been reported that resting cells of N. cholesterolicum NRRL 5767 are able to convert added OA to ~88% of 10-HSA (w/w) and 11% of 10-ketostearic acid (10-KSA, w/w) [12]. The production of both 10-HSA and 10-KSA complicates downstream separation and purification of 10-HSA. The enzymes involved in OA metabolism to 10-HSA and 10-KSA have been established [24,25,26,27,28,29,30,31], Ohase converts OA to 10-HSA, which is subsequently converted to 10-KSA by a secondary alcohol dehydrogenase (2o-ADH)
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