Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound.

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Viscous nature of the fermentation broth has phenomenal influence on process conditions and parameters in a fermentor. Though broth rheology has attracted significant influence in process research, still there is a challenge to modify fluid dynamics of fermentation broth. During the production of coenzymeQ 10 (CoQ 10 ) by Agrobacterium tumefaciens ATCC 4452, the culture broth becomes highly viscous due to excessive synthesis of exopolysaccharides. This hinders the CoQ 10 yield and complicates the downstream process. The present study describes how this problem was tackled by media modification and mutation. Induced mutants were generated using UV and EMS as mutagenic agents followed by rational selection based on antibiotic resistance. On screening of these mutants in sucrose based PM-2 medium, UV induced, vancomycin resistant mutant M-6, showed significant reduction (6.29 fold) in viscosity development in the broth. Mutant M-6(S), a natural variant of mutant M-6, resistant to high substrate concentration was further selected for the CoQ 10 production. Cane molasses as carbon source was found to be best suitable for CoQ 10 fermentation using mutant M-6(S). Replacing sucrose with cheaper cane molasses significantly reduced the broth viscosity with improved specific CoQ 10 content, thereby generating cost effective fermentation process. The newly developed mutant strain produced 48.89 mg/L of CoQ 10 with specific CoQ 10 content of 1.87 mg/g of DCW at 25°C, 500 rpm agitation and 0.2 vvm aeration using continuous fed batch fermentation and newly formulated cane molasses medium.

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.

Coenzyme Q10 (CoQ10) is a blockbuster nutraceutical molecule which is often used as an oral supplement in the supportive therapy for cardiovascular diseases, cancer and neurodegenerative diseases. It is commercially produced by fermentation process, hence constructing the high yielding CoQ10 producing strains is a pre-requisite for cost effective production. Paracoccus denitrificans ATCC 19367, a biochemically versatile organism was selected to carry out the studies on CoQ10 yield improvement. The wild type strain was subjected to iterative rounds of mutagenesis using gamma rays and NTG, followed by selection on various inhibitors like CoQ10 structural analogues and antibiotics. The screening of mutants were carried out using cane molasses based optimized medium with feeding strategies at shake flask level. In the course of study, the mutant P-87 having marked resistance to gentamicin showed 1.25-fold improvements in specific CoQ10 content which was highest among all tested mutant strains. P-87 was phenotypically differentiated from the wild type strain on the basis of carbohydrate assimilation and FAME profile. Molecular differentiation technique based on AFLP profile showed intra specific polymorphism between wild type strain and P-87. This study demonstrated the beneficial outcome of induced mutations leading to gentamicin resistance for improvement of CoQ10 production in P. denitrificans mutant strain P-87. To investigate the cause of gentamicin resistance, rpIF gene from P-87 and wild type was sequenced. No mutations were detected on the rpIF partial sequence of P-87; hence gentamicin resistance in P-87 could not be conferred with rpIF gene. However, detecting the mutations responsible for gentamicin resistance in P-87 and correlating its role in CoQ10 overproduction is essential. Although only 1.25-fold improvement in specific CoQ10 content was achieved through mutant P-87, this mutant showed very interesting characteristic, differentiating it from its wild type parent strain P. denitrificans ATCC 19367, which are presented in this paper.