International Journal of Food Microbiology 133 (2009) 17

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Review

Bacterial vitamin B2, B11 and B12 overproduction: An overview

Catherine M. Burgess a,, Eddy J. Smid b,c, Douwe van Sinderen d
a

Department of Food Safety, Teagasc-Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland
NIZO food research, Kernhemseweg 2, P.O. Box 20, 6710 BA Ede, The Netherlands
c
TI Food & Nutrition. Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands
d
Department of Microbiology and Alimentary Pharmabiotic Centre, Biosciences Institute, National University of Ireland Cork, Western Road, Cork, Ireland
b

a r t i c l e

i n f o

Article history:
Received 25 December 2008
Received in revised form 12 April 2009
Accepted 14 April 2009
Keywords:
Riboavin
Folic acid
Cobalamin
Overproduction

a b s t r a c t
Consumers are becoming increasingly health conscious and therefore more discerning in their food choices.
The production of fermented food products with elevated levels of B-vitamins increase both their commercial
and nutritional value, and eliminate the need for subsequent fortication with these essential vitamins. Such
novel products could reduce the incidence of inadequate vitamin intake which is common in many parts of
the world, not only in developing countries, but also in many industrialised countries. Moreover, the concept
of in situ fortication by bacterial fermentation opens the way for development of food products targeted at
specic groups in society such as the elderly and adolescents. This review looks at how vitamin
overproduction strategies have been developed, some of which have successfully been tested in animal
models. Such innovative strategies could be relatively easily adapted by the food industry to develop novel
vitamin-enhanced functional foods with enhanced consumer appeal.
2009 Elsevier B.V. All rights reserved.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . . . .
Riboavin . . . . . . . . . . . . . . . . . . . . .
2.1.
Riboavin biosynthesis and overproduction . .
3.
Folate . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Folate biosynthesis and overproduction . . . .
4.
Vitamin B12 (cobalamin) . . . . . . . . . . . . . .
4.1.
Vitamin B12 biosynthesis and overproduction .
5.
Concluding comments . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
In the developed world consumers are increasingly health conscious and aware of their nutritional requirements. Meanwhile, in
the developing world malnutrition can result in dietary deciencies, particularly in vitamins, which may cause a variety of health
disorders.
Vitamins are generally classied into two groups, the fat-soluble
vitamins, which include vitamins A, D, E and K, and the water-soluble
vitamins, which include vitamin C, biotin (vitamin H or B7) and a
series of B vitamins thiamin (B1), riboavin (B2), niacin (B3),
pantothenic acid (B5), pyridoxine (B6), folic acid (B11) and cobalamin

Corresponding author.
E-mail address: kaye.burgess@teagasc.ie (C.M. Burgess).
0168-1605/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2009.04.012

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1
2
2
3
3
4
4
6
6

(B12). The fat-soluble vitamins appear to function as integral parts of

cell membranes while, generally speaking, the water-soluble vitamins
act as coenzymes, often as carriers of a particular chemical group
(Baku and Dickerson, 1996).
Although many prokaryotes have nutritional requirements for
the water-soluble vitamins (Snell, 1993), biosynthetic capabilities for
the various B vitamins are just as common among these microorganisms. This natural capacity for B vitamin production by certain
microorganisms has the potential to be exploited, either to replace
costly chemical synthesis of such vitamins for food fortication or
by harnessing this natural capacity and enhancing it for in situ
fortication of fermented foods. Much work has been done in recent
years to elucidate the biosynthetic pathways of these vitamins in a
number of microorganisms. The generated knowledge has also
allowed for different strategies to be employed to enhance vitamin
production. This review will focus on three of the water-soluble B

C.M. Burgess et al. / International Journal of Food Microbiology 133 (2009) 17

vitamins whose biosynthetic pathways are inextricably linked;

riboavin, folic acid and cobalamin, briey covering their physiological function and dietary source and focusing on novel overproduction strategies in prokaryotes.
2. Riboavin
Riboavin or vitamin B2 is a dietary necessity for humans as,
unlike many plants, fungi and bacteria, they are unable to synthesise
the vitamin. Two sources of riboavin are available to humans: a
dietary source and riboavin produced by microora of the large
intestine (Hill, 1997; Wrong et al., 1981). The recommended daily
intake for riboavin is 1.3 mg/day for men and 1.1 mg/day for women
(Food and Nutrition Board, 1998a). In Western diets milk and dairy
products contribute mostly to the daily intake of riboavin (Powers
et al., 1993). Other good sources of the vitamin include yeast, cereals,
meats, fatty sh and green leafy vegetables (Cooperman and Lopez,
1991). Grain products contain only low amounts of riboavin as
much of the vitamin is lost due to processing. However, fortication
practices make certain breads and cereals very good sources of
riboavin (Darnton-Hill and Nalubola, 2002; Powers, 2003).
Metabolically, riboavin is the precursor of avin mononucleotide
(FMN) and avin adenine dinucleotide (FAD), both of which act as
electron carriers in oxidation-reduction reactions, functioning as
coenzymes for hundreds of FMN- or FAD-dependent enzymes called
avoproteins. The majority of avin-enzyme interactions involve
the ribityl side chain, which represents the position where FMN
and FAD differ, thus explaining the specicity of enzymes for either of
these two cofactors (Massey, 2000). Flavoproteins are, among other
functions, essential for the metabolism of amino acids, energy production and activation of folate and pyridoxine to their respective
coenzyme forms (Food and Nutrition Board, 1998a; Massey, 2000).
Riboavin deciency is endemic in populations whose diet lack dairy
products and meat, thus conning it essentially to developing nations
(Combs, 1992; Rohner et al., 2007). Nevertheless, a high prevalence of
poor riboavin status has been observed among adolescent girls in
the United Kingdom and among the Irish population (O'Brien et al.,
2001; Powers, 2003).

2.1. Riboavin biosynthesis and overproduction

Riboavin is synthesised by many bacteria and its biosynthetic
pathway has been studied extensively in Bacillus (B.) subtilis and
Escherichia (E.) coli. This has been reviewed by Bacher et al. (2001)
and is summarised in Fig. 1. Riboavin biosynthesis requires the precursors guanosine 5'-triphosphate (GTP) and ribulose 5-phosphate.
The rst step of the GTP-dependent branch of the biosynthetic pathway is encoded by ribA in E. coli. In B. subtilis it is also encoded by ribA
but in this case RibA is a bifunctional enzyme which also catalyses the
formation of 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5phosphate (Richter et al., 1993). RibA overexpression in B. subtilis
increases riboavin yield up to 25%, suggesting that this enzyme is
rate-limiting in riboavin biosynthesis (Hmbelin et al., 1999).
However, in Lactococcus (L.) lactis, overexpression of ribA alone does
not lead to increased riboavin production (Burgess et al., 2004).
To be biologically active, riboavin must be converted to its
coenzyme forms, FMN and FAD. This is accomplished by a bifunctional
avokinase/FAD synthetase, which is encoded in B. subtilis by ribC or
ribF in E. coli (Bacher et al., 1996; Mack et al., 1998). Certain mutations
in the B. subtilis ribC gene have been found to result in riboavin
overproduction (Coquard et al., 1997; Kreneva and Perumov, 1990).
B. subtilis also encodes a separate monofunctional riboavin kinaseencoding gene, ribR, which can suppress the effect of ribC mutations,
restoring normal riboavin production (Solovieva et al., 1999).
Regulation of riboavin biosynthesis and transport in B. subtilis is
achieved by means of a conserved regulatory region, the so-called RFN
element, located upstream of both the rib operon, which encodes
the riboavin biosynthetic genes, and the riboavin transporter gene
ypaA/ribU. The RFN element can be found on the chromosome of
many, but not all bacterial species (Gelfand et al., 1999; Vitreschak
et al., 2002; Wels et al., 2006). Mutations in this regulatory region
result in riboavin overproduction (Kil et al., 1992).
In recent years a number of biotechnological processes have
been developed to replace the more costly chemical synthesis of the
vitamin (Hmbelin et al., 1999; Stahmann et al., 2000). Besides the
economic advantage, additional benets of the biotechnological
approach include the use of renewable sources, the fact that it is more

Fig. 1. Riboavin biosynthesis in prokaryotes. The E. coli genes encoding each enzymatic activity are indicated on the left while the corresponding B. subtilis genes are indicated to the
right. Adapted from Bacher et al. (1996) and Perkins and Pero (2002).

C.M. Burgess et al. / International Journal of Food Microbiology 133 (2009) 17

environmentally friendly and the yield of a product of equal or

superior quality (van Loon et al., 1996). Some bacteria and fungi
have been shown to be capable of riboavin overproduction and this
ability has been harnessed for industrial production. Such commercial producers include the ascomycetes Eremothecium (Er.) ashbyii
and Ashbya (A.) gossypii. However, advantages were perceived in
developing bacterial and yeast fermentations to avail of their high
growth rates, and less costly and complex growth media. Currently,
three microorganisms are exploited for riboavin production:
A. gossypii, Candida (C.) famata and B. subtilis with riboavin production levels reaching 15 g l 1, 20 g l 1 and 14 g l 1, respectively
(Perkins et al., 1999; Schallmey et al., 2004; Stahmann et al., 2000). In
A. gossypii riboavin production was increased almost ten-fold by
metabolic engineering (Jimenez et al., 2005). A. gossypii has also
been targeted as an microorganism to overproduce riboavin using
oil waste (Park et al., 2007). In the case of B. subtilis high levels of
riboavin production were achieved as a result of exposure to purine
analogues and the toxic riboavin analogue roseoavin, or by genetic
engineering (Perkins et al., 1999, 1991). In L. lactis both of these
approaches have been used with success (Burgess et al., 2004) and it
has been demonstrated that riboavin overproduced by these strains
can be used to reverse an induced riboavin deciency in rats
(LeBlanc et al., 2005a,b). The toxic analogue approach has also been
successfully employed for Lactobacillus (Lb.) plantarum, Leuconostoc
(Lc.) mesenteroides and Propionibacterium (P.) freudenreichii (Burgess
et al., 2006) and a fermented dairy product made with the latter
strain was shown to counteract riboavin deciency in an animal
model (LeBlanc et al., 2006). Such strategies do not attempt to generate alternative production strains, but rather replacing riboavinconsuming strains used in traditional food fermentation processes
with riboavin-producing counterparts, thereby increasing riboavin bioavailability in the food product and introducing an added
health benet. The elevated levels of the vitamin which would be
produced in such foods would not have any negative health
implications as no upper limit of intake has been set for riboavin
due to a lack of evidence on adverse effect in humans (Flynn et al.,
2003).

fortied with folic acid (Food & Drug Administration, 1996) and over
forty countries worldwide have introduced mandatory fortication
of foods. Despite the observed benecial effects of folic acid supplementation, fortication may have adverse affects in subpopulations,
such as masking vitamin B12 deciency, primarily in the elderly
(Cuskelly et al., 2007). Furthermore, there is conicting evidence in
the relationship of folate and cancer and for these reasons many
European countries have thus far not adopted mandatory folic acid
fortication (Bailey et al., 2003, Food Safety Authority of Ireland,
2008). The recommended daily intake of folic acid is 400 mg of dietary
folate equivalents (DFEs). DFEs adjust for the nearly 50% lower
bioavailability of naturally occurring food-derived folate compared
with that of synthetic folic acid (Food and Nutrition Board, 1998c).
A shortage of folate compromises DNA replication and cell division,
and therefore one of the noticeable effects of folate deciency is the
occurrence of abnormalities of cells which have the highest turnover
rates in tissues such as the intestinal mucosa, regenerating liver and
bone marrow. The most obvious manifestations of folate deciency
are seen in white and red blood cells. Dietary folate deciency is the
leading cause of megaloblastic anaemia in the world (Gallagher and
Ehrenkranz, 1995). The folic acid status of expectant mothers is also
known to affect the incidence of neural tube defects such as spina
bida and anencephaly in the unborn infant, where many studies have
shown consistent protective effects of folates (recently reviewed by
Pitkin (2007)). Folates have also been shown to reduce homocysteine
concentrations in the blood, thereby possibly playing a role in the
prevention of heart disease (Verhoef et al., 1998).
As mentioned above, unlike mammals many microorganisms and
plants possess the ability to synthesise folic acid derivatives de novo in
which the end product is tetrahydrofolate. Because of this exclusive
ability, the biosynthetic pathway has been used as a drug target in the
treatment of diseases such as malaria or Pneumocystis (Pn.) carinii
infections (Anderson, 2005; Fulton et al., 1995), although in the case of
malaria the causative agent, Plasmodium (Pl.) falciparum, has been
reported to develop resistance to these drugs (Gatton et al., 2004;
Sibley et al., 2001).
3.1. Folate biosynthesis and overproduction

3. Folate
Folate, also known as vitamin B11, is the generic descriptor for
folic acid (pteroylmonoglutamic acid) and related compounds
exhibiting the biological activity of folic acid. Folates can consist of
a mono- or polyglutamyl conjugate and these compounds are named
after the number of glutamyl residues (PteGlun), where n denotes
the total number of glutamyl residues. The folates function as enzyme
co-substrates in one-carbon (C1) metabolism of amino acids and
nucleotides where the fully reduced (tetrahydro-) form serves as an
acceptor or donor of a single carbon unit (Combs, 1992). The most
signicant role of folic acid appears to be its involvement in the synthesis of purines and pyrimidines, and therefore in DNA synthesis.
Methionine synthase uses 5-methyltetrahydrofolate in the conversion of l-homocysteine to l-methionine (Brody, 1991). Much of the
methionine formed is converted to S-adenosylmethionine, a universal donor of methyl groups for DNA, RNA, hormones, neurotransmitters, membrane lipids and proteins (For a comprehensive
overview see Selhub (2002)).
Folic acid is synthesised by both microorganisms and plants. Folate
biosynthesis requires the coupling of the pteridine molecule to paminobenzoic acid (pABA) to form pteroic acid. Mammals, which
synthesise large amounts of pteridines, cannot catalyse this coupling
and are therefore auxotrophic for the vitamin (Baku and Dickerson,
1996). The main dietary sources of folate are leafy, green vegetables,
dairy products and cereal products, especially fortied breads and
breakfast cereals (Finglas et al., 2003). In the USA the FDA ruled that it
is mandatory for all our and uncooked cereal grain products to be

Dihydrofolate is synthesised from the precursors GTP, p-aminobenzoate (pABA) and glutamate, in seven enzymatic steps (Fig. 2).
The ATP-consuming conversion step of 6-hydromethylneopterin
to its pyrophosphate ester is catalysed by FolK in B. subtilis and has
been shown to represent the rate-limiting step in folate (over)production (Perkins and Pero, 2002). In L. lactis this step is catalysed by a
bifunctional enzyme whose overexpression was shown to result in
a more than two fold increase in total folate production (Sybesma
et al., 2003a).
The second part of the folate biosynthetic pathway is the production of pABA. Exogenously added pABA can support growth of
certain folic acidauxotrophic strains (Green et al., 1996), while pABA
supplementation of the growth medium of L. lactis was shown to
result in a two-fold increase in folate production (Sybesma et al.,
2003b), pointing to an as of yet unidentied uptake system for this
compound. However, while overexpression of the pABA genes alone
does not increase folate production levels in L. lactis, folate overproduction is observed when pabBC overexpression is combined with
overexpression of the folate biosynthetic genes (Wegkamp et al.,
2007).
Industrially, folate is primarily synthesised via chemical synthesis
due to the low yields of folic acid produced by current bacterial strains.
However, there may be technological advantages to microbial
production of folate. These include the fact that microbially produced
folate does not require reduction prior to use by humans and the more
environmental friendly character of biological production as compared to chemical synthesis (Zhu et al., 2005).

C.M. Burgess et al. / International Journal of Food Microbiology 133 (2009) 17

Fig. 2. Folic acid biosynthetic pathway in prokaryotes. The genes known to encode the enzymes in E. coli are shown on the left while those in known to encode the enzymes in B.
subtilis are shown to the right. The enzyme names indicate that the activity has been shown but the corresponding gene encoding it has not been identied. The asterisk denotes that
the gene encoding this enzyme, folQ, has been identied in L. lactis. Adapted from Green et al. (1996), Perkins and Pero (2002) and Sybesma et al. (2004).

The use of folate-producing lactic acid bacteria as a fermented

functional food has previously been suggested (Lin and Young, 2000).
As mentioned above metabolic engineering allows increased folate
production in L. lactis (Sybesma et al., 2003a; Wegkamp et al., 2007),
and this overproduction has also been combined with riboavin
overproduction in a single strain (Sybesma et al., 2004). This strategy
has also been successfully employed in Lb. plantarum (Wegkamp,
2008). Overexpression of the Lb. plantarum folate gene cluster
resulted in enhanced folate production in the vitamin B12 producer
Lb. reuteri (Santos et al., 2008b). In B. subtilis a targeted mutagenesis
strategy resulted in an eight fold increase in folic acid (Zhu et al.,
2005). Random mutagenesis and exposure to the folate antagonist
methotrexate were both shown to be successful in obtaining folateoverproducing strains of Lb. plantarum, although this phenotype was
shown to be rather unstable (Wegkamp, 2008).
4. Vitamin B12 (cobalamin)
Vitamin B12 is the name used for all corrinoids exhibiting the
qualitative biological activity of cyanocobalamin. Cobalamin is unique
in the fact that its de novo synthesis appears to be restricted solely to
some bacteria and archaea. It forms a nutritional requirement for
animals and protists although they do not synthesise it, while plant
and fungi apparently neither require nor synthesise it (Martens et al.,
2002). These vitamin B12-producing microorganisms form the
original biological source of vitamin B12. The vitamin is therefore
found only in foods fermented by such bacteria, or those derived from
the tissues of animals which have ingested B12-containing foods or
which have obtained it from B12-producing elements of their
commensal microora. Certain bacteria of the microora in ruminant
animals synthesise the vitamin and the liver of such animals is
therefore a rich source of the vitamin (Baku and Dickerson, 1996). In
carnivorous animals and humans intestinal synthesis is not sufcient
and so they must obtain it from their food, most often from the esh of
other animals (recommended daily intake is 2.4 mg (Food and
Nutrition Board, 1998b))(Ellenbogen and Cooper, 1991; Stabler and
Allen, 2004). Humans only require vitamin B12 for two enzymatic
activities, but it is nonetheless essential. (R)-methyl-malonyl-CoA

mutase is involved in the metabolism of propionyl-CoA which is

derived from the degradation of compounds such as thymine, valine,
methionine and odd-chain fatty acids. This ado-cobalamin-dependent
enzyme catalyses the rearrangement of propionyl-CoA subsequent to
its carboxylation and epimerisation to succinyl-CoA, which then
enters the citric acid cycle. Methionine synthase requires vitamin B12
in the form methylcobalamin. This enzyme methylates homocysteine
to form methionine using 5-methyltetrahydrofolate as a methyl donor
(Ellenbogen and Cooper, 1991).
Shellsh are a good source of the vitamin as they scavenge
microorganisms from their surroundings thereby concentrating the
vitamin. Foods of plant origin contain no detectable vitamin B12
unless contaminated with microorganisms. Cobalamin stores in the
body exceed the daily requirement by about one thousand-fold and
this, coupled to the microbial synthesis of the vitamin in the small
intestine, probably indicates why clinical vitamin B12 deciency is
rarely due to nutritional deciency and also why clinical symptoms
take so long to manifest themselves (Ellenbogen and Cooper, 1991).
It would take 3 to 4 years of zero cobalamin intake to cause depletion
of this vitamin, due to the large storage capacity combined with an
efcient enterohepatic circulation that recycles the vitamin from bile
and other intestinal secretions (Fishman et al., 2000). That said, the
fact that vitamin B12 is almost exclusively found in foods of animal
origin poses a risk for people on a strict vegetarian or vegan diet
(Dagnelie et al., 1989). B12 deciency occurs in India due to lactovegetarianism, coupled to the expense and consequent scarcity of
meat ingestion (Antony, 2001). A comprehensive review on vitamin
B12 deciency is provided by Stabler and Allen (2004). Vitamin B12
deciency causes delay or failure of normal cell division, particularly
in the bone marrow and intestinal mucosa where cell turnover rate is
high (Combs, 1992). The interrelationship between vitamin B12 and
folate deciency leads to very similar haematological changes, but
with the latter appearing much earlier (Baku and Dickerson, 1996).
4.1. Vitamin B12 biosynthesis and overproduction
Cobalamin has the most complex structure of all the vitamins
synthesised by bacteria with around thirty genes being required for its

C.M. Burgess et al. / International Journal of Food Microbiology 133 (2009) 17

biosynthesis. Many excellent reviews describe the biosynthetic pathways for the vitamin (Heldt et al., 2005; Martens et al., 2002; Roessner
et al., 2001; Roessner and Scott, 2006; Scott and Roessner, 2002; Scott,
2003). Most of the work in characterising cobalamin biosynthesis has been performed in Salmonella (S.) typhimurium and Pseudomonas (Ps.) denitricans. Two different pathways exist for adenosylcobalamin (ado-cobalamin) biosynthesis: (1) an oxygen-dependent
pathway, which is found in Ps. denitricans, and (2) an anaerobic
pathway, which has been identied in, among others, S. typhimurium,
P. freudenreichii subsp. shermanii and B. megaterium. All genes
necessary for anaerobic cobalamin biosynthesis have been identied
on the genome of St. sanguinis (Xu et al., 2007). Genes encoding
enzymes contributing to the oxygen-dependent pathway have been
given the prex cob, while those involved in the oxygen-independent

pathway have the prex cbi (Martens et al., 2002). The genes involved
and the reactions which they catalyse in each pathway are listed in
Table 1. Due to the early insertion of cobalt in the anaerobic pathway
the remaining intermediates are cobalto-complexes and therefore
require enzymes with different substrate specicities than the
intermediates in the aerobic pathway although many of the reactions
catalysed are similar. It is clear from Table 1 that most steps of the two
pathways have been elucidated, although the genes encoding some
enzymes remain to be identied. A relatively recent nding is the
identication of CobZ in Rhodobacter (R.) capsulatus, which catalyses
a reaction similar to that promoted by CobG, although it appears that it
mediates this reaction in a different manner, as the two proteins
display no primary sequence similarity. CobZ has also been shown to
contain a avin in the form of a non-covalently bound FAD, two FeS

Table 1
Reactions and genes required for the biosynthesis of adenosylcobalamin.

The genes listed for the oxygen-dependent pathway are from P. denitricans. The genes indicated for the oxygen-independent pathway are from S. typhimurium. The ? indicates that
enzymatic activity has been identied, but that the corresponding gene is still unknown. The major differences in the pathway are highlighted in discriminating colours. Adapted
from Roessner et al. (2001) and Roessner and Scott (2006).

C.M. Burgess et al. / International Journal of Food Microbiology 133 (2009) 17

centers, and a b-type heme which is unlike CobG (McGoldrick et al.,

2005). It has recently been suggested that the last step in the cobalamin biosynthetic pathway in S. typhimurium involves dephosphorylation of adenosylcobalamin-5'-phosphate, catalysed by CobC, which
is contradicting the pathway indicated in Table 1 where CobS catalyses
the condensation of a-ribazole and AdoGDPcobinamide (Zayas and
Escalante-Semerena, 2007). In the aerobic pathway the gene responsible for cobalt reduction has not yet been identied, although two
candidate genes have been identied to encode this enzyme (named
CobR) (Heldt et al., 2005).
Industrial vitamin B12 production is solely performed by fermentation processes as chemical synthesis of cobalamin is too expensive. Examples of industrially used bacteria include various species
of Propionibacterium, Pseudomonas and Nocardia (Bykhovsky et al.,
1998). A common method to improve B12 yields is random mutagenesis
and the use of genetic engineering (Martens et al., 2002; Pouwels et al.,
1999). It has also been reported that cobalt-resistant strains of
propionibacteria have enhanced vitamin B12 production (Seidametova
et al., 2004). Different metabolic engineering strategies have been
applied to increase vitamin B12 production in P. freudenreichii (Piao
et al., 2004a,b). A recombinant P. freudenreichii strain harbouring a
plasmid containing hemA, from Rhodobacter sphaeroides, and homologues of hemB and cobA, showed 2.2-fold overproduction of vitamin
B12 (Piao et al., 2004b). These studies show that multigene expression
systems improve the vitamin B12 production levels in propionibacteria.
The emphasis is now on strategies to boost the production of precursors
such as aminolevulinic acid or intermediates for improvement of
vitamin B12 production (Murooka et al., 2005).
Lactic acid bacterial strains isolated from fermented foods have
been found to be cobalamin producers which offers the advantage
of exploiting their generally regarded as safe (GRAS) status (Santos
et al., 2007). A limitation to this however is that the vitamin is not
secreted and therefore to increase bioavailability autolytic mutants
would be needed (Martens et al., 2002; Taranto et al., 2003). Lb.
reuteri, unlike other bacteria, has been shown to encode all the genes
necessary for the synthesis of vitamin B12 in one continuous stretch of
its chromosome. This provides a major advantage for consideration of
metabolic engineering strategies for transferring the B12 production
capability to other bacteria (Santos et al., 2008a). Subsequent studies
on this organism have indicated that alterations of the growth
medium such as supplementation with glycerol or omission of single
amino acids such as cysteine can signicantly enhance production of
the vitamin providing further avenues for investigation for industrial
production (Santos, 2008). Similarly to riboavin cobalamin toxicity
in humans has not been observed and upper limits for intake have not
been dened (Flynn et al., 2003).
5. Concluding comments
Fermentation with food grade bacteria offers unique opportunities to
improve the nutritional value of food products. Fermentation can
be viewed as a second chance to add value to primary products.
The development of novel functional foods with an enhanced vitamin
content due to fermentation has been suggested and would contribute to an ever growing market for these products (Stanton et al., 2005).
Different strategies have been applied to improve microbial production of
vitamins during fermentation. These strategies include (i) metabolic
engineering, (ii) strain selection by mining for diversity in culture
collections and (iii) selection of natural overproducers using chemical
analogues of vitamins. The latter two strategies have an important
advantage over the rst because the selected strains are readily applicable
in fermented food products without the need to pass legislative hurdles
(Sybesma et al., 2006). The production of fermented food products with
elevated levels of B-vitamins increase their commercial and nutritional
value and eliminate the need for fortication. Such novel products could
serve the goal of reducing the number of persons with vitamin deciency

which is common in many parts of the world, not only in developing

countries, but also in many industrialised countries. Finally, the concept of
in situ fortication by fermentation with specially selected strains opens
the way for development of food products targeted at specic groups in
society like for instance the elderly, adolescents, pregnant women,
children, sportsmen and vegetarians.
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