Flavin is a name given to a group of biologically valuable metabolites. Riboflavin, Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) are of major importance amongst flavins. Riboflavin (vitamin B2) is an obligatory diet component. Riboflavin is synthesized by all plants and microorganisms but not by humans. Industrially riboflavin is mainly utilized as food and feed additive and as a component of multi-vitamin mixture with annual production of >8000 tons. FAD is having pharmaceutical applications, used to treat certain genetic disorders and as ophthalmic agent to treat keratitis and blepharitis. Flavins are involved in several metabolic reactions due to the presence of flavin ring which can carry one or two electrons.
The present study focuses on de novo fermentative synthesis of flavins with filamentous natural flavogenic fungi Ashbya gossypii and Eremothecium ashbyi. Our objective was to increase flavins production by implementing oxidative stress and metabolic engineering approaches. Oxidative stress was reported to improve riboflavin in A.gossypii but similar studies were lacking in E.ashbyi. Oxidative stress was studied in E.ashbyi with optimization of different stress modulators. Design of experiments (DoE) and Central composite design (CCD) was employed to standardize a combination of oxidative stress supplements. Particularly, FAD production in response oxidative stress was studied for the first time and it was found to be increased by 2.18-fold. FAD role in glutathione metabolism and increased activity of FAD synthetase along with glutathione reductase, placed FAD in center as a putative link for increased flavins production to oxidative stress.
To further improve more valuable FAD production, genetic engineering of A.gossypii was planned. FMN1 gene expression was identified as a potential bottleneck from gene expression and HPLC metabolite analysis. A.gossypii TEF promoter was cloned in a vector adjacent to GEN3 marker gene for designing FMN1 gene promoter replacement cassette (PRC). Integrative transformation in A.gossypii was successfully achieved with 14.02-fold increased FAD production with a concurrent increase in riboflavin kinase activity.
Further, replicative transformation of both fungi was planned to improve riboflavin production by RIB gene overexpression. Since RIB1 and RIB3 genes control initial committed steps in riboflavin biosynthesis, overexpression of these genes was planned. Moreover, both the genes expression was increased in flavin production phase compared to growth phase. Constructed expression vectors had constitutively expressed TEF2 strong promoter under which A.gossypii RIB1 and RIB3 genes were cloned. Transformation with E.ashbyi could not be achieved. Stable transformants of A.gossypii were obtained with significantly improved riboflavin production (1.95-fold with RIB1 clone and 1.37-fold with RIB3 clone).
This is the first study to demonstrate improved FAD production in A.gossypii. This is also the first study demonstrating improved FAD production amongst flavins, in response to oxidative stress in E.ashbyi.

Publications:

Manan P., Chandra T.S. (2020). Metabolic engineering of Ashbya gossypii for enhanced FAD production through promoter replacement of FMN1 gene. Enzyme and Microbial Technology 133: 109455. doi.org/10.1016/j.enzmictec.2019.109455

Manan P., Chandra T.S. (2020) Enhanced FAD Production in Eremothecium ashbyi with Statistically Optimized Oxidative Stress Modulators. International Journal of Microbiology and Biotechnology 5(1): 7-15. doi:10.11648/j.ijmb.20200501.12