Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol

Abstract

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L −1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanolbased overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.

Riboflavin overproduction from diverse feedstocks with engineered Corynebacterium glutamicum

“…RF is industrially produced via fermentation with genetically modified microbial strains of A. gossypii, C. famata, and B. subtilis, being the RF biosynthetic operon one of the main metabolic targets (Averianova et al 2020). Enhancing RF formation has been achieved by increasing rib operon expression through operon duplication, promoter exchange, or plasmid-based expression of endogenous or exogenous rib operons (Perkins et al 1999, Lin et al 2014, Ledesma-Amaro et al 2015, Pérez-García et al 2022b, Klein et al 2023. For example, overexpressing E. coli's RF biosynthesis genes ribABDEC under the inducible Ptrc promoter yielded 0.2 g l −1 of RF in sugarbased growth (Lin et al 2014).…”

“…For example, overexpressing E. coli's RF biosynthesis genes ribABDEC under the inducible Ptrc promoter yielded 0.2 g l −1 of RF in sugarbased growth (Lin et al 2014). In B. methanolicus, methanol-based RF production reached 0.5 g l −1 through heterologous overexpression of rib operons from Bacillus licheniformis, B. methanolicus, and B. subtilis in methanol fed-batch fermentation (Klein et al 2023). Additionally, in B. subtilis, significant efforts have focused on regulating the rib operon and overexpressing its structural genes ribDGEABH (Averianova et al 2020).…”

“…In B. methanolicus, genes prs, guaA, and guaB from the purine pathway, along with rpe and tkt from the RuMP cycle, were found to boost RF titers by enhancing GTP production. (Klein et al 2023). Moreover, Engineering of the purine pathway for RF production in A. gossypii has been broadly applied (Jiménez et al 2005, Ledesma-Amaro et al 2015.…”

“…This suggests a need for improved carbon source utilization and medium optimization in future strategies. Most RF production processes rely on single carbon sources with additives like yeast extract (Wu et al 2007, Man et al 2014, Klein et al 2023 or complex substrates like corn steep liquor or rapeseed oil (Park et al 2007(Park et al , 2011, making yield comparisons challenging. Medium optimization, such as the two-level Plackett-Burman design used for B. subtilis (Wu et al 2007, Li et al 2013, has proven effective in enhancing RF production (Li et al 2013).…”

“…One of the most common organisms used for the large production of RF is the bacterium Bacillus subtilis (Liu et al 2020). Other bacteria such as Corynebacterium ammoniagenes, and Bacillus methanolicus have also been engineered for this purpose (Koizumi et al 2000, Klein et al 2023. In addition to bacteria, several species of fungi and yeasts are also used to produce RF which include Ashbya gossypii, Candida famata, and Candida guilliermondii (Ledesma-Amaro et al 2015, Averianova et al 2020, Tsyrulnyk et al 2021.…”