Riboflavin Production in Lactococcus lactis : Potential for In Situ Production of Vitamin-Enriched Foods

Abstract

This study describes the genetic analysis of the riboflavin (vitamin B 2 ) biosynthetic (rib) operon in the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NZ9000. Functional analysis of the genes of the L. lactis rib operon was performed by using complementation studies, as well as by deletion analysis. In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction. Transcriptional regulation of the L. lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L. lactis isolates. The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon. The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification.Riboflavin (vitamin B 2 ) is an essential component of basic cellular metabolism since it is the precursor of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The latter two biomolecules play a central role in metabolism acting as hydrogen carriers in biological redox reactions involving enzymes such as NADH dehydrogenase (for a review of this topic, see reference 32). Many microorganisms, plants, and fungi possess the biosynthetic ability to produce riboflavin. However, vertebrates, including humans, lack this ability and must therefore obtain this vitamin from their diet.Dietary riboflavin is present in liver, egg yolk, milk, and meat, whereas the vitamin is commercially synthesized for nutritional use in the fortification of various food products such as bread and breakfast cereals. Because of its intense yellow color it is also used in small amounts as a coloring agent in foods such as ice cream and sauces, and as a medical identification aid. The recommended daily requirement of riboflavin is set at 1.3 mg (14) and sufficient amounts of riboflavin need to be ingested regularly since the body is unable to store the vitamin. Symptoms of riboflavin deficiency (ariboflavinosis) in humans, which still occurs in both developing and developed countries (6, 34), include sore throat, hyperemia, edema of oral and mucous membranes, cheilosis, and glossitis (48). Furthermore, riboflavin is used as a treatment for nucleoside analogue-induced type B lactic acidosis, which can occur as a result of AIDS treatment (9), for migraine (23), and for malaria (1). Commercially available riboflavin has traditionally been produced by chemical processes, but in recent times this has been replaced by biotechnological and more economical processes with Ashbya gossypii, Candida famata, or Bacillus subtilis (43).Riboflavin biosynthesis has been studied in both gram-positive and gram-negative bacteria, in most detail in B. subtilis (36) and Escherichia coli (4). Th...

Biosynthesis of Vitamins by Probiotic Bacteria

“…The overexpression of RibA in B. subtilis produces 25% more riboflavin, indicating that this enzyme is rate-limiting in riboflavin biosynthesis [18]. However, in Lactococcus lactis, the overexpression of ribA did not lead to increased riboflavin production [12].…”

“…Several lactic acid bacteria (LAB) species (e.g., Lactococcus lactis, Lactobacillus gasseri, and Lactobacillus reuteri) and Bifidobacterium (e.g., B. adolescentis) produce these vitamins, often in large quantities, and are, therefore, often found in fermented foods [9,10]. Moreover, increased vitamin biosynthesis has been obtained by metabolic engineering [11,12]. Folate biosynthetic genes and riboflavin biosynthetic operon have been overexpressed in L. lactis, resulting in types that produce folate [12] or riboflavin [12] at higher rates.…”

“…Moreover, increased vitamin biosynthesis has been obtained by metabolic engineering [11,12]. Folate biosynthetic genes and riboflavin biosynthetic operon have been overexpressed in L. lactis, resulting in types that produce folate [12] or riboflavin [12] at higher rates. Sybesma et al [13] modified the biosynthetic pathways of folate and riboflavin in L. lactis, resulting in the simultaneous overproduction of both vitamins, through directed mutagenesis and selection and metabolic engineering.…”

Excess of threonine compared with serine promotes threonine aldolase activity in Lactococcus lactis IL1403

“…Lastly, low amounts of produced glycine might have been directed to the biosynthesis of purines, which are precursors in the production of riboflavin (Burgess et al, 2004). This claim is based on proteome results, which show that enzymes involved in riboflavin biosynthesis (RibAB) were upregulated at higher L-threonine availability.…”

Engineering Complex Phenotypes in Industrial Strains

“…To highlight a few, amino acids such L-phenylalanine, glutamate, and lysine in E. coli and Corynebacterium glutamicum have been successfully produced in excess of 50-80 g/L (28)(29)(30)(31)(32). Food additives, such as vitamins and glucosamine, in Bacillus subtilis and E. coli have been produced in excess of 30-100 g/L, and similar titers for organic acids such as citric, lactic, and succinic acid are observed (33)(34)(35)(36)(37)(38)(39). The success of these metabolic engineering targets was predominantly due to the availability of detailed knowledge about the metabolism of the hosts and the ability to draw phenotype to genotype correlations.…”