Morphological and Molecular Differentiation of Sporidiobolus johnsonii ATCC 20490 and Its Coenzyme Q10 Overproducing Mutant Strain UF16

Abstract

Coenzyme Q 10 (CoQ 10 ) is an industrially important molecule having nutraceutical and cosmeceutical applications. CoQ 10 is mainly produced by microbial fermentation and the process demands the use of strains with high productivity and yields of CoQ 10 . During strain improvement program consisting of sequential induced mutagenesis, rational selection and screening process, a mutant strain UF16 was generated from Sporidiobolus johnsonii ATCC 20490 with 2.3-fold improvements in CoQ 10 content. EMS and UV rays were used as mutagenic agents for generating UF16 and it was rationally selected based on atorvastatin resistance as well as survival at free radicals exposure. We investigated the genotypic and phenotypic changes in UF16 in order to differentiate it from wild type strain. Morphologically it was distinct due to reduced pigmentation of colony, reduced cell size and significant reduction in mycelial growth forms with abundance of yeast forms. At molecular level, UF16 was differentiated based on PCR fingerprinting method of RAPD as well as large and small-subunit rRNA gene sequences.Rapid molecular technique of RAPD analysis using six primers showed 34 % polymorphic fragments with mean genetic distance of 0.235. The partial sequences of rRNAgene revealed few mutation sites on nucleotide base pairs. However, the mutations detected on rRNA gene of UF16 were less than 1 % of total base pairs and its sequence showed 99 % homology with the wild type strain. These mutations in UF16 could not be linked to phenotypic or genotypic changes on CoQ 10 biosynthetic pathway that resulted in improved yield. Hence, investigating the mutations responsible for deregulation of CoQ 10 pathway is essential to understand the cause of overproduction in UF16. Phylogenetic analysis based on RAPD bands and rRNA gene sequences coupled with morphological variations, exhibited the novelty of mutant UF16 having potential for improved CoQ 10 production.

Cellular factories for coenzyme Q10 production

“…Efforts to use S. johnsonii as a production host at an industrial level have achieved 10 mg/g DCW; albeit, this yield involved exogenous PHB in the media [ 78 ]. Other mutagenesis attempts led to a mutant UF16 strain with 7.4 mg/g DCW [ 96 ].…”

Molecular, Physiological and Phenotypic Characterization of Paracoccus denitrificans ATCC 19367 Mutant Strain P-87 Producing Improved Coenzyme Q10

“…Hence, the strain improvement study consisting of sequential mutagenesis, rational selection and screening process was undertaken for this strain to bring improvement in CoQ 10 content. The strains that are developed by induced mutations are genotypically different from their respective parent strains, hence investigation of these changes are essential to characterize them as a novel strains [9][10][11]. Physiological and phenotypic characteristics as well as molecular differentiation methods like rRNA gene sequences, RAPD, RFLP or AFLP are being used to discriminate the strains of same genera or species and mutants [12][13][14].…”