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All known disease called scurvy was known to human from ancient (Egyptians) age
occurred due to deficiency of Vitamin C. But this is not known up to 1753 when James Lind
described it to be cured by dietary mean (ULLMANN¶S Encyclopedia vol. 38).
This play very important role in the human as conjunctive tissue formation, ion
transportation, and cell protection against free radicals. In plants, act against reactive oxygen
species that are formed from Photosynthesis and Respiratory processes. It also linked with
cell growth, cell cycle and cofactor for many enzymes (Anderson et al. 2004).
Wide range of reactive oxygen species, such as singlet oxygen, superoxide anion and
Hydroxyl radicals which have been implicated with many chronic disorders including cancer
and cardiovascular disease.
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ccccccccccccccccccccccccccccccccccccccccccc cccccccccccccccccccccccccccccccccc Metabolic pathway engineering for the production of L-ascorbic acid
For this purpose we synthesize ascorbic acid on natural way by means of plant which
gives direct feed to humans. This was also synthesizing by many methods using microbes
such as bacteria and algae by biosynthesis. These microbe used as it is but yield is low so for
this to overcome genetically modified strains (×
,
,
,
are used. Using algae we also synthesize Ascorbic acid.
The more well known and commercial adapted pathway is Reichstein process. This is
chemical oriented method have a single fermentation step. Due to its energy consumption,
and required high temperature, pressure for many steps in Ascorbic acid synthesis.
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3.1Microbiological fermentation
Figure 3.1 Bremus et al. gives different pathway cycle for production Ascorbic acid using Bacteria.
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3.1.1.1 D-Sorbitol pathway
Sorbitol is transformed by fermentation of 2-KLG (2-Keto-L-Gluconic acid) via
intermediate L-Sorbosone. This is done by many strains of bacteria namely Pseudomonas and
Acetobacter, which are catalyze the oxidation of D-Sorbitol to 2-KLG via the series of
membrane bound hydrogenase to produce L-Sorbosone. The final oxidation to 2-KLG is
catalyzed by either membrane bound or cystolic Sorbosone dehydorgenase. Using the strain
Glucano Oxydans that produce up to 60g/l. of 2-KLG from L-Sorbosone or D-Sorbitol with
60% conversion (Hancock and R. Viola, 2001).
Genetically modified strains are also used to carry out above steps. The location of
dehydrogenase required for conversion of D-Sorbitol to 2-KLG varies from strain to strain.
The transfer of D-Sorbitol pathway intermediate into cytoplasm of these strain determined by
the presence of cystolic reductase which give path to the intermediate in the pentose cycle. To
overcome this problem membrane bound dehydrogenase recombinant with Glucanobacter
Oxydans is replace this or substitute cystolic enzymes. For example Acetobacter liquifaciens
a membrane bound Sorbosone dehydrogenase is expressed in Glucanobacter Oxydans which
is membrane bound sorbitol dehydrogenase and sorbose dehydrogenase but not cystolic
dehydrogenase. A significant yield is observed from L-Sorbose (68-81%) and L-Sorbosone
(23-83%). But there is no yield improvement under fermentation condition (Shrikant et al.,
2006).
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The pathway for the production of ascorbic acid from D-Glucose using Algae is shown
below.
Figure 3.2 Bremus et al. gives (2006) Pathway cycle for production of Ascorbic acid using Algae.
Many attempts are employed to use microalgae for the direct production of L-
Ascorbic acid from inexpensive feedstock. One-step fermentation process for the production
of L-Ascorbic acid using the heterotrophic green microalga
produce
low quantity up to 40 mg/l L-Ascorbic acid.. After continuous chemical mutagenesis and
fermentation optimization, an improved amount of up to 2 g/l L-Ascorbic acid is obtained.
Accumulation of L-Ascorbic acid in the fermentation medium was obtained by the use of the
colorless microalgae . Using this alga it is shown that a pH reduction
could stabilize L-Ascorbic acid in the fermentation reactor, so most of the L-Ascorbic acid
became harvestable from the medium.
For strain-improvement used formany mutant strains with increased and
reduced abilities to accumulate L-Ascorbic acid. These mutants are used to identify the
pathway for L-Ascorbic acid biosynthesis that involves mannose-containing intermediates.
Similarly in plants, l-galactono-1, 4,-lactone is produced from d-glucose through GDP-d-
mannose, GDP-l-galactose and l-galactose. Finally l-galactono-1,4,-lactone is converted into
l-AA by l-GL-DH. This is shown by Wheeler et al. (1998) in higher plants.
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P and GTP is catalysed by GDPD- Man pyrophosphorylase (GMP). GDP-D Man is converted
to GDP-L-Gal by a reversible double epimerization, catalysed by GDP-D-Man-3,5-epimerase
(GME) that was first identified in Chlorella pea This enzyme has recently been purified and
cloned from A. thaliana and purified from the alga Prototheca (Running et al. 2004). As well
as being involved in ascorbate synthesis, GDP-D-Man and GDP-L-Gal are substrates for
polysaccharide synthesis and protein glycoslyation. The steps subsequent to GDP-L-Gal are
likely to be dedicated to ascorbate synthesis. GDP-L-Gal is initially broken down to L-Gal 1-
P, which is subsequently hydrolysed to L-Gal. Enzymes catalysing these steps have been
recently purified and characterized. GDP-L-Gal is converted to L-Gal 1-P and GDP by a
novel and highly specific phosphate-dependent GDP-L-Gal phosphorylase. The released L-
Gal is then oxidized in two steps, first by a cytosolic NAD-dependent L-Gal dehydrogenase
(L-GalDH) at C1 to form L-galactono-1,4-lactone (L-GalL) (Wheeler et al. 1998) and then by
L-GalL dehydrogenase (L-GalLDH) at C2/C3 resulting in the production of ascorbate.
The antisense suppression of LGalDH and L-GalLDH decreases ascorbate concentration
Although the D-Man/L-Gal pathway appears to be the predominant pathway, there is some
suggestion that other biosynthetic pathways via uronic acid intermediates contribute to the
ascorbate content of plant tissues and that these may be developmentally regulated.
The authors proposed that the hydrolysis of GDP-L-gulose would result in the production of
L-gulose which could be converted to L-gulono-1,4-lactone by L-GalDH and subsequently
into ascorbate by the L-gulono-1,4-lactone dehydrogenase activity known to exist in plants.
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The pathway cycle for production of ascorbic acid from D-glucose is given below:
Figure 2.4 Linster and E V Schaftingen (2006) gives Pathway cycle for production of Ascorbic acid in Animals.
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L-Gulonolactone oxidase (Linster and E. V. Schaftingen 2006)
Reichstein Process
The use of Reichstein¶s procedure into an industrial process is marked by great efforts
to improve each reaction step. As a result of many technical and chemical modifications each
step gives over 90% yields. The overall yield of ascorbic acid from d-glucose is now 60%
(ULLMANN¶S Encyclopedia vol. 38).
D-Sorbitol.
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L-Sorbose
Figure 2.5 Hancock and R Viola (2002) gives Pathway cycle for production of Ascorbic acid by Reichstein
Process.
,3:4,6-Di-O-isopropylidene-M-l-sorbofuranose.
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2, 3:4, 6-Di-O-isopropylidene-2-ketol-gulonic acid.
L-Ascorbic acid.
2) Acid-catalyzed cyclization to ascorbic acid directly from the protected or released 2-keto-l-
gulonic acid.
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r
The other pathway is Reichstein pathway in these process seven steps is included of
which one is fermentation and others are chemical oriented. The yield of each step is 90%
(ULLMANN¶S Encyclopedia vol. 38). Overall yield of the process is 50% (Hancock and R.
Viola, 2001). So that Reichstein process is more favorable for production of Ascorbic acid
industrially.
The intermediate steps in Reichstein process are energy consuming and required high
temperature , pressure condition make it non feasible for synthesis of ascorbic acid. For this
reason the intermediate are synthesize biotechnologically for higher yield and good
conversion.
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2.c Carole L. Linster and Emile Van Schaftingen (2006) Biosynthesis, recycling and
degradation in mammals, FEBS Journal, 274:1±22.
3.c Christoph Bremus, Ute Herrmann, Stephanie Bringer-Meyer, Hermann Sahm (2006)
The use of microorganisms in l-ascorbic acid production, Journal of Biotechnology,
124:196±205.
4.c Glen L. Wheeler, Mark A. Jones and Nicholas Smirnoff (1998) The Biosynthetic
Pathway of Vitamin C in Higher Plants, Nature, vol. 393:365-369.
6.c Robert D. Hancock and Roberto Viola (2002) Biotechnological approaches for L-
ascorbic acid production, Trends In Biotechnology Vol.20 No.7:299-305.
7.c Robert D. Hancock and Roberto Viola (2001) The use of micro-organisms for L-
ascorbic acid production: current status and future perspectives, Applied
Microbiology Biotechnology, 56:567±576.
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8.c Shrikant A. Survase, Ishwar B. Bajaj and Rekha S. Singhal (2006) Biotechnological
Production of Vitamins, Food Technology Biotechnology""(3): 381±396.
9.c Takahiro Ishikawa, John Dowdle and Nicholas Smirnoff (2006) Progress in
manipulating ascorbic acid biosynthesis and accumulation in plants, Physiologia
Plantarum, 126: 343±355.
10.cTeruhide Sugisawa, Taro Miyazaki, and Tatsuo Hoshino (2005) Microbial Production
Of L-Ascorbic Acid From D-sorbitol, L-sorbose, L-Glucose, and L-Sorbosone by
Ketogulonicigenium vulgare DSM 4025, Bioscience Biotechnology
Biochemicals,69(3):659-662.
11.cBernd Oster and Ulrich Fechtel, E. Merck, Darmstadt, Federal Republic of Germany,
(2003) ULLMANN¶S Encyclopedia of Industrial Chemistry (Wiley- VCH), sixth edn.
Vol. 38:218-231.
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