Production of Vitamin B2 (Riboflavin) by Microorganisms: An Overview

Abstract

Riboflavin is a crucial micronutrient that is a precursor to coenzymes flavin mononucleotide and flavin adenine dinucleotide, and it is required for biochemical reactions in all living cells. For decades, one of the most important applications of riboflavin has been its global use as an animal and human nutritional supplement. Being well-informed of the latest research on riboflavin production via the fermentation process is necessary for the development of new and improved microbial strains using biotechnology and metabolic engineering techniques to increase vitamin B2 yield. In this review, we describe well-known industrial microbial producers, namely, Ashbya gossypii, Bacillus subtilis, and Candida spp. and summarize their biosynthetic pathway optimizations through genetic and metabolic engineering, combined with random chemical mutagenesis and rational medium components to increase riboflavin production.

Keywords: food/feed additive; genetically modified microorganisms; metabolic engineering; riboflavin; vitamin B2.

FIGURE 1

Schematic diagram of the riboflavin pathways in Ashbya gossypii and Bacillus subtilis. B. subtilis enzymes (in blue color): RibAB, bifunctional enzyme GTP cyclohydrolase II (A)/3,4-DHBP synthase (B); RibDG, bifunctional deaminase (D)/reductase (G); RibH, lumazine synthase; RibE, riboflavin synthase; RibFC, bifunctional flavokinase (F)/FAD synthetase (C). The phosphatase converting ArPP to ArP is unknown. RibU (originally known as ypaA) – riboflavin transmembrane transporter, its substrates are FMN and riboflavin analogue roseoflavin; RibT, N-acetyltransferase GCN5 (earlier predicted as transporter), which transfers the acetyl group from acetyl coenzyme A (AcCoA) to a variety of substrates (unknown). Produced or consumed cofactors are shown with curved arrows near the Rib enzymes. A. gossypii enzymes (in red color): RIB1, GTP cyclohydrolase II; RIB7, DARPP reductase; RIB2, DArPP deaminase; RIB3, DHBP synthase; RIB4, lumazine synthase; RIB5, riboflavin synthase. Precursors (metabolites): GTP, guanosine triphosphate; DARPP, 2,5-diamino-6-ribosyl-amino-4(3H)pyrimidinedione 5′-phosphate; ARPP, 5-amino-6-ribosyl-amino-2,4(1H,3H)pyrimidinedione 5′-phosphate; DArPP, 2,5-diamino-6-ribityl-amino-4(3H)pyrimidinedione 5′-phosphate; ArPP, 5-amino-6-ribityl-amino-2,4(1H,3H)pyrimidinedione 5′-phosphate; ArP, 5-amino-6-ribityl-amino-2,4(1H,3H)pyrimidinedione; DHBP, 3,4-dihydroxy-2-butanone-4-phosphate; DRL, 6,7-dimethyl-8-ribityllumazine; Ribu5P, ribulose 5-phosphate; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide.

FIGURE 2

Scheme of the riboflavin biosynthesis regulation in B. subtilis. RFN, chromosomal FMN-specific element; FMN riboswitch, coding sequence for FMN binding; ribDG-E-AB-H-T, rib operon; ribU, gene encoding riboflavin transporter; ribU FMN riboswitch, the second chromosomal FMN-specific element (RFN); ribFC, gene encoding bifunctional flavokinase/FAD synthetase RibFC; ribR, gene encoding monofunctional flavokinase RibR (the part of a transcription unit encoding proteins for sulfur uptake and degradation), possibly involved in the regulation of the riboflavin biosynthesis genes (indicated by dashed arrows); P1, P2, and P3 denote confirmed promoters; P – predicted promoter (indicated by arrows). The hairpins symbols denote confirmed transcription terminators.

FIGURE 3

Chemical versus microbial riboflavin synthesis.