Riboflavin microbial production

Several pigments, vitamins and coenzymes like carotenoids, riboflavin (B2), cobal-amine (B12), ascorbic acid (Q, ergosterol (D2), biotin (H), gibberellin, etc. are produced during the normal metabolism of microorganisms. Microbial production of some of... 

The first organism employed primarily for riboflavin production was Clostridium aceto-butylicum, the anaerobic bacterium used for the microbial production of acetone and butanol. Riboflavin was purely a byproduct and was found in the dried stillage residues in amounts ranging from 40 to 70 xg/g of dried fermentation solids. Later investigations disclosed that riboflavin could be produced by yeast such as Candida flareri or C. guillier-mondi, and the yield was as high as 200 mg/L. 

The aerobic microbial production of riboflavin in various microbes dates back to the 1930s [141] but was discontinued in the late 1960s because the chemical process described above was more profitable. Fermentation soon (Merck, 1974) made a comeback, however, and subsequently has steadily eroded the position of the Karrer-Tishler process. Around 1990, production levels of 15-20 g I. 1 had been achieved [136], up from 2 g I. 1 in 1940 [141], causing the product to crys-... 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

Photolytic. When propachlor in an aqueous ethanolic solution was irradiated with UV light (>, = 290 nm) for 5 h, 80% decomposed to the following cyclic photoproducts W-isopropyloxindole, W-isopropyl-3 hydroxyoxindole, and a spiro compound. Irradiation of propachlor in an aqueous solution containing riboflavin as a sensitizer resulted in completed degradation of the parent compound. 3-Hydroxypropachlor was the only compound identified in trace amounts which formed via ring hydroxylation (Rejtb et al, 1984). Hydrolyzes under alkaline conditions forming W-isopropylaniline (Sittig, 1985) which is also a product of microbial metabolism (Novick et al., 1986). 

There are already several examples of chemicals being produced by microbial fermentation of engineered cell factories, whose production through metabolic engineering has been boosted by the use of genomics tools, e.g., 1,3-propanediol used for polymer production, riboflavin used as a vitamin, and 7-aminodeacetoxy-cephalosporanic acid (7-ADCA) used as a precursor for antibiotics production. Furthermore, in the quest to develop a more sustainable society, the chemical industry is currently developing novel processes for many other fuels and chemicals, e.g., butanol, to be used for fuels, organic acids to be used for polymer production, and amino acids to be used as feed. 

Riboflavin (vitamin B2) is an essential nutritional factor for humans (0.3-1.8 mg d-1) and animals (1-4 mg (kg diet)-1), who need it as a precursor for fla-voproteins [135]. It is produced at a volume of approx. 3 kt a-1, mainly as an animal feed additive. Approx. 300 t a-1 is used as a food additive and food colorant (E-101) and the remainder (500 t a-1) is used in pharmaceutical applications. Major producers are Roche (Switzerland), BASF (Germany), Archer-Daniels-Midland (USA) and Takeda (Japan). Microbial and chemical production have coexisted for many years but the latter has recently been phased out [136]. 

Microbial riboflavin (vitamin B2) production processes converting commodity fermentation substrates like monomeric or oligomeric carbohydrates or vegetable oils to riboflavin are the focus of the present review. The current developmental status of the riboflavin production strains derived from metabolic pathway engineering... 

The present review is derived from publicly available academic and patent literature. Occasionally, unpublished observations are included where appropriate. The review incorporates mainly literature published in the English language during the last 10-15 years. This includes important contributions from Russian scientists. More recent literature from Chinese authors are incorporated as well. It is recognized that a vast body of Chinese literamre particularly on fungal riboflavin production strains exists. Certainly interesting aspects of microbial riboflavin production are communicated therein, but unfortunately, because of language barriers, this literature could not be included in the present review. 

Figure 3 Results of a cradle to grave life cycle comparison of five environmental impact categories of the traditional chemical (solid bars) and both the Bacillus subtilis and Ashbya gossypH-based microbial riboflavin production processes (checker board bars).

The intracellular C5 carbon sugar pools oiBacillus strains comprising transke-tolase knockout mutations, which are auxotroph for aromatic amino acids, can rise to concentrations by far exceeding the physiological requirements of the bacteria. After dephosphorylation, excess ribose is excreted into the fermentation broth. Highly efficient microbial processes based on Bacillus transketolase knockout mutants were used in the past to obtain D-ribose for chemical riboflavin production at industrial level. For a review on Bacillus ribose production strains, see [311]. 

Lastly, riboflavin is the paradigm vitamin whose industrial production process switched completely from chemistry to biotechnology. The latter is superior with regard to economic efficiency, but in addition, the advantages for the environment have been demonstrated in several ecological footprint studies. For more details on microbial riboflavin production, the reader is referred to a review by Hohmann and Stahmann [319]. 

Many products of industrial fermentation are added into food as flavors, vitamins, colors, preservatives, and antioxidants. These products are more desirable than food additives produced chemically. Many of the vitamins are made by microbial fermentations including thiamine (vitamin Bj), riboflavin (vitamin B ), cobalamin (vitamin Bj ), and vitamin G (ascorbic acid). Vitamin G is not only a vitamin but is also an important antioxidant that helps to prevent heart diseases. Garotenoids are another effective antioxidant. They are also used as a natural food color for butter and ice cream. Garotenoids are red, orange, and yellow pigments produced by bacteria, algae, and plants. 

Milk is not a rich source of dietary folate compared to other foods however, as is the case for riboflavin, folate concentrations can be significantly increased in many dairy products due to microbial fermentation. Among dairy products, fermented milks are considered a good potential matrix for folate fortification because folate-binding proteins present in milk improve folate stability and enhance the bioavailability of both 5-methyltetrahydrofolate (the most predominant natural form of the vitamin) and folic acid (Jones and Nixon 2002 Aryana 2003 Verwei et al. 2003). However, due to the potential risks of fortification with folic acid, the elaboration of fermented milks containing elevated levels of natural folates would be a better suited alternative. 

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