Process and Strain Development for Reduction of Broth Viscosity with Improved Yield in Coenzyme Q10 Fermentation by Agrobacterium tumefaciens ATCC 4452

Abstract

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.

Cellular factories for coenzyme Q10 production

“…Several native producers of CoQ 10 have been identified or optimized as candidates for CoQ 10 production, including S. pombe , S. johnsonii , Rhodobacter sphaeroides and Agrobacterium tumefaciens [ 78 , 83 , 87 , 88 ]. Several other organisms, including Pseudomonas , Paracoccus bacteria, Candida and Saitoella yeasts also produce CoQ 10 natively but have not been sufficiently characterized as producing hosts, and many require the inclusion of expensive constituents in the growth media for proper function.…”

“…Later efforts involved targeting the overexpression of IPP pathway genes, especially DXS [ 60 ]. A. tumefaciens , however, produces unwanted exopolysaccharides, which increases the viscosity of the sample and affects CoQ extraction [ 88 , 104 ].…”

Influence of Substrate Feeding and Process Parameters on Production of Coenzyme Q<sub>10</sub> Using <i>Paracoccus denitrificans</i> ATCC 19367 Mutant Strain P-87

“…The cheaper carbon source cane molasses was introduced to reduce the cost of medium in order to make the process economically viable. The organism was able to tolerate higher amount of cane molasses in PM-D medium very efficiently than pure sucrose based PM-A medium as mentioned earlier with Agrobacterium tumefaciens [30]. other two media namely PM-B and PM-C showed relatively less titer as well as specific CoQ 10 content.…”

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

“…It was thought that the variant tolerating higher concentration of cane molasses may be able to assimilate higher quantity of carbon source during fermentation as mentioned in the earlier studies with A. tumefaciens [17]. In this process a single colony was found to be survived on the agar plate containing 13 % cane molasses.…”

“…The hexane layer was separated, concentrated till dryness and finally reconstituted with 1 ml of hexane [17].…”