Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers

Abstract

Riboflavin [7,8-dimethyl-10-(1'-d-ribityl)isoalloxazine, vitamin B₂] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

Fig. 1.

Chemical structures of flavins.

Fig. 2.

Biosynthesis of riboflavin and flavocoenzymes. (Reproduced from reference with permission of the Royal Society of Chemistry.)

Fig. 3.

Computer-generated model of a heavy riboflavin synthase complex. The capsid was generated using the coordinates from the lumazine synthase 60-mer of B. subtilis (Protein Data Bank [PDB] entry 1RVV) and from the riboflavin synthase trimer of E. coli (PDB entry 1I8D). (Reproduced from reference with permission of Elsevier.)

Fig. 4.

Biosynthesis of the F₀ cofactor. (Reproduced from reference with permission of the Royal Society of Chemistry.)

Fig. 5.

FMN causes transcription termination of the ribD RNA in vitro. (A) Sequence and secondary structure of the leader sequence ribD RNA. Shaded regions identify nucleotides that are complementary and might serve as an antiterminator structure. Nucleotides denoted with an asterisk have been altered from the wild-type sequence to generate a restriction site. The nucleotide sequence comprising the 62- and 30-nucleotide regions (not shown) is presented in references and . (B) Model for the FMN-dependent riboswitch. Shaded regions identify the putative antiterminator structure that is disrupted after binding of FMN and formation of the P1 structure. (Reproduced from reference with permission of the publisher. Copyright 2002 National Academy of Sciences, U.S.A.)

Fig. 6.

Overall riboswitch structure in a ribbon representation. P1 to P6, domains; L1 to L6, loops. The J1-2 segment participates in antiterminator formation in the absence of FMN, whereas in the FMN-bound state, it is locked up in the junction. (Reproduced from reference with permission of Macmillan Publishers Ltd., copyright 2009.)

Fig. 7.

Evaluation of the improved C. famata riboflavin overproducers isolated by metabolic engineering. RF synthesis was assayed after 5 days of cultivation in yeast extract-peptone-dextrose (YPD) medium. (Reproduced from reference with permission of Elsevier.)

Fig. 8.

RF kinase activity (A) and FMN accumulation in the culture medium (B) by C. famata strains overexpressing the FMN1 gene. wt, wild type. (Reproduced from reference with permission of Elsevier.)