Institute of Chemical Technology, Mumbai

Department of Chemical Engineering

Metabolic pathway engineering for the production of L-ascorbic acid

Project II Report submitted in Partial Fulfillment

of the Requirements for the Award of the Degree of

Master of Chemical Engineering

By

Santosh G. Jadhav

Department of Chemical Engineering

Institute of Chemical Technology
Mumbai-400019
Maharashtra, India

November 2010

AVP
 Index

Serial number Title Page number

1 Introduction 1

2 Objective and scope 2

3 Pathways for production Ascorbic acid 3

3.1 Microbial Pathway 3
3.1.1 Bacterial Pathway 3
3.1.2 Algae Pathway 6
3.2 Pathway for Plants 7
3.3 Pathway for Animals 9
3.4 Reichstein Process 12

4 Conclusion 16

5 References 17
 Metabolic pathway engineering for the production of L-ascorbic acid

1. Introduction

Ascorbic acid or L-Ascorbic acid, L-xylo-Ascorbic acid, L-threo-hex-2-enoic acid γ-

Lactone is IUPAC name for Vitamin C (ULLMANN’S Encyclopedia vol. 38).

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).

Later on many peoples were working on Ascorbic acid. In which, Szent and Gyorgyi
(1928) first isolated ascorbic acid form adrenal gland and from plant tissue by Herbert et al.
(1933). Chemical synthesis of L-ascorbic acid from L-xylosone was achieved. This work is
also done by Haworth and Hirst (1933). They also propose the chemical structure for
Ascorbic acid, Reichstein et al. (1933). Biochemical reaction for preparation of Ascorbic
from D-Glucose was introduced by Reichstein and Grössner (1934) and this sequence is
adapted for manufacturing of Ascorbic acid industrially (Hancock and R. Viola, 2001).

This plays 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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2. Objective and Scope

Ascorbic acid is used by human as dietary purpose to fulfill their nutrients

requirement and fight against disease called Scurvy and some fatal disease known to be
cancer and cardiovascular disease. It also acts as antioxidant in human body which gives free
electron to reactive oxygen formed from hydrogen peroxide.

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 microbes are used as it is but yield is low so
for this to overcome genetically modified strains (Acetobacter suboxidans, Bacterium
xylinum, Erwinia sp., Corynebacterium sp.) 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.

To overcome this intermediate compounds are biosynthetically synthesize to increase

the yield of the ascorbic acid with low energy consumption.

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3. Pathways for production Ascorbic acid

3.1 Microbiological fermentation

In microbiological fermentation different species are used to produce Ascorbic acid

these are Bacteria, and Algae. Different pathway has been proposed to describe how they
produce Ascorbic acid by initial starting materials.

3.1.1 Metabolic Pathway for Bacteria

At present there are six bacterial fermentation processes for the production of 2-keto-
L-gulonic acid, a direct precursor of L-ascorbic acid.
These are:
a) Sorbitol pathway
b) L-idonic acid pathway
c) L-gulonic acid pathway
d) 2-keto-D-gluconic acid pathway
e) 2, 5-diketo-D-gluconic acid pathway
f) 2-keto-L-gulonic acid pathway

The pathway cycle for production of ascorbic acid is given below:

Figure 3.1 Bremus et al. (2006) gives different pathway cycle for production Ascorbic acid using Bacteria.

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The most commercially advanced methods are the oxidation of D-sorbitol or L-sorbose to 2-
keto-L-gulonic acid (2-KLG) and oxidation of D-glucose to 2-keto-L-gulonate (2-keto-D-
gluconic acid pathway) (Shrikant et al., 2006).

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).

3.1.1.2 2-Keto-D-Gluconic Acid Pathway

D-Glucose is converted to 2-KLG via D-Gluconic acid, 2-Keto-D-Gluconic acid and
2, 5-Diketo-D-Gluconic acid (2, 5-DKG). Until now there is no bacterial strains capable of
efficiently catalyzing the complete conversion of D-Glcose to 2-KLG have been isolated.
This is carried out in three main steps; each step is carried out by using different
microorganisms.
(i) Transformation of D-glucose to 2-keto-D-gluconic acid: the transformation of
glucose to 2-keto-D-gluconic acid is carried out by Acetobacter melanogenus and

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 Metabolic pathway engineering for the production of L-ascorbic acid

Pseudomonas albosesamae. Some Acetobacter strains also synthesize 2-keto-D-

gluconic acid;
(ii) Oxidation of 2-keto-D-gluconic acid: this oxidation is carried out by Bacterium
hoshigaki and Bacterium gluconicum with 2, 5-DKG as a product. In addition,
Acetomonas albosesamae can directly transform D-glucose to 2, 5-DKG;
(iii) Oxidation of 2, 5-DKG acid: conversion of 2, 5-DKG into 2-KLG. The
mentioned strain is of the genera Brevibacterium and Pseudomonas, and
maximum yield was obtained from Brevibacterium ketosoreductum. The use of
Corynebacterium has also been suggested.
A mutant Erwinia strain convert D-glucose into 2, 5-DKG. The whole culture broth is
then treated with sodium dodecyl sulphate to reduce cell viability, mixed with glucose and
then supplied to a mutant strain of Corynebacterium for conversion to 2-KLG. The whole
process achieved yields of 2-KLG of up to 85 % from D-glucose with final 2-KLG
concentrations of 10.5 g/L. bacterial strain via genetic engineering. A cloned cytosolic 2, 5-
DKG reductase gene from Corynebacterium species is expressed in Erwinia herbicola. The
recombinant organism was capable of synthesizing 2-KLG from D-glucose but the yields
attained were very poor (1 g/L of 2-KLG from 20 g/L of D-glucose). Recombinant E. citreus
expressing cytoplasmic 2, 5-DKG reductase from Corynebacterium species could accumulate
19.8 g/L of 2-KLG with a 49 % conversion efficiency from glucose (Hancock and R. Viola
2001). Such improvements in yield were attained by optimization of the fermentation
conditions, careful selection of the promoter controlling the expression of the 2, 5-DKG
reductase gene and by the use of mutant strains of E. citreus which were unable to use 2, 5-
DKG or 2-KLG for growth.

3.1.1.3 Bioconversion Of 2-KLG to L-Ascorbic Acid (Hancock and R. Viola 2001),

(Shrikant et al. 2006)

2-KLG is converted to L-ascorbic acid chemically via two methods.

The first involves multiple steps including:
• Esterification of a 2-KLG derivative under strongly acidic conditions to produce
methyl 2-keto-L-gulonate (MeKLG);
• Reaction of MeKLG with a base to produce a metal ascorbate salt;
• Treatment of metal ascorbate salt with an acidulant to obtain ascorbic acid.
The second way is a one-step method comprising acid-catalyzed cyclization of KLG.

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Both methods are commercially undesirable due to the requirement for multiple chemical
steps for first method, or the use of large amounts of gaseous hydrogen chloride or
requirement for very expensive process equipment for second method.
To overcome this situation hydrolase enzyme is used to convert ester of 2-KLG to L-
Ascorbic acid. In similar way lactonases is extracted from Zymomonas mobilis, Escherichia
coli and Fusarium oxysporium are capable to convert 2-KLG to L-Ascorbic acid.

3.1.2 Metabolic Pathway for Algae

The pathway for the production of ascorbic acid from D-Glucose using Algae is
shown below.

Figure 3.2 Bremus et al. (2006) gives 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 Chlorella pyrenoidosa produces
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 Prototheca moriformis. 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.

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 Metabolic pathway engineering for the production of L-ascorbic
L acid

For strain-improvement
improvement Prototheca used for many mutant strains with increased and
reduced abilities to accumulate L-Ascorbic acid. These mutants are used to identify the
pathway for L-Ascorbic
scorbic acid biosynthesis that involves mannose-containing
containing intermediates.
intermediates
Similarly in plants, l-galactono
galactono-1, 4,-lactone is produced from d-glucose
glucose through GDP-d-
GDP
mannose, GDP-l-galactose
galactose and l-galactose.
l Finally l-galactono-1,, 4,-lactone
4 is converted
into l-AA by l-GL-DH.
DH. This is shown by Wheeler et al. (1998) in higher plants.

3.2 Metabolic Pathway for Plants (Hancock

Hancock and R. Viola 2002),
2002) (Ishikawa1 et al.,
2006)

The pathway cycle for production of Ascorbic acid from D-Glucose

ose is
i given below:

Figure 3.3 Hancock and R Viola (2002) gives Pathway cycle for production of Ascorbic acid in Plants.

It was proposed that ascorbate is synthesized in plants by oxidation of L-galactose

L
(Wheeler et al. 1998).
The enzymatic steps are summarized below.
In this pathway, four chemical events are minimally required for the non inverted
conversion of glucose to ascorbic acid are present:
(1) Oxidation of the C1 of glucose;
(2) Oxidation at C2 or C3;

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(3) Epimerization at C5; and

(4) Lactonization between C1 and C4.
Phosphomannose isomerase (PMI) catalyses the first step in directing hexose
phosphates into D-Mannose metabolism. PMI from Escherichia coli has been used as a
selectable marker for plant transformation, because it renders plants mannose resistant.
However, A. thaliana plants expressing E. coli PMI do not have increased ascorbate.
Conversion of D-Man 6-P to D-Man 1-P is catalyzed by Phosphomannose mutase (PMM).
GDP-D-Man synthesis from D-Man 1-P and GTP is catalyses by GDPD- Man
pyrophosphorylase (GMP). GDP-D Man is converted to GDP-L-Gal by a reversible double
epimerization, catalyzed 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. 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 hydrolyzed to L-Gal.
Enzymes catalyzing 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.
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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3.3 Metabolic Pathway for Animals

The pathway cycle for production of ascorbic acid from D-glucose is given below:

Figure 3.4 Linster and E V Schaftingen (2006) gives Pathway cycle for production of Ascorbic acid in Animals.

Ascorbic acid is synthesized by many vertebrates. The occurrence of ascorbic acid

biosynthesis in sea lamprey suggests that this feature appeared early in the evolutionary
history of fishes (590–500 million years ago), i.e. prior to terrestrial vertebrate emergence.
The biosynthetic capacity has, however, subsequently been lost in a number of species, such
as teleost fishes, passeriform birds, bats, guinea pigs, and primates including humans, for
whom ascorbate has thus become a vitamin. Fish, amphibians and reptiles synthesize
ascorbate in the kidney, whereas mammals produce it in the liver.
In animals, d-glucuronate, derived from UDP-glucuronate, is reduced to l-gulonate,
which leads to inversion of the numbering of the carbon chain (‘inversion of configuration’)
since the aldehyde function of d-glucuronate (C1) becomes a hydroxymethyl group in the
resulting l-gulonate. In plants, the pathway starts with GDP-d-mannose, which is converted

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(without change in carbon numbering) to l-galactonolactone, the substrate for the plant
homologue of GLO, l-galactonolactone dehydrogenase.

3.3.1 Formation of glucuronate from UDP-glucuronate (Linster and E. V.

Schaftingen 2006)

The formation of glucuronate from UDP-glucuronate could hypothetically involve

following three mechanisms to convert glucuronate from UDP-glucuronate.
(a) The cleavage of UDPglucuronate to glucuronate 1-phosphate, followed by
dephosphorylation of the latter by a glucuronate-1- phosphatase;
(b) The formation of a glucuronidated intermediate followed by its hydrolysis by b-
glucuronidase or esterases (which could hydrolyze acyl-glucuronides); or
(c) Direct hydrolysis of UDP-glucuronate to UDP and glucuronate.

Mechanism (a): It is show that a series of nonglucuronidable xenobiotics rapidly

stimulate the formation of glucuronate in isolated hepatocytes, the same xenobiotics also
stimulated the formation of glucuronate from UDP-glucuronate in liver cell-free extracts
enriched with ATP or in liver microsomes supplemented with ATP and a heat-stable cofactor
identified as coenzyme A. Quantitatively, the synthesis of glucuronate observed under these
conditions accounted for the formation of glucuronate observed in intact cells, indicating that
glucuronate is formed from UDP-glucuronate by a microsomal enzyme. These enzymes are
present in the endoplasmic reticulum as, similarly to UGTs, it is stimulated by UDP-N-
acetylglucosamine, which enhances the transport of UDP-glucuronate into vesicles derived
from the endoplasmic reticulum
Mechanism (b): It is ruled out by the fact that glucuronate formation from UDP-
glucuronate occurs in rat liver microsomes in the absence of UGT substrates and is actually
inhibited by such substrates. Furthermore, inhibitors of β-glucuronidase and esterases do not
affect the formation of glucuronate from UDP-glucuronate by microsomal preparations.
Mechanism (c): Here both (a) and (b) are taken together, which lead to the conclusion
that glucuronate is formed by direct hydrolysis of UDP-glucuronate by a UDP-glucuronidase.
It is likely that the ability of UGTs to hydrolyze UDP-glucuronate varies among isoforms and
depends on the phospholipidic environment. This last point may explain the observation that
UDPglucuronidase is inhibited in rat liver microsomes by the addition of ATP and coenzyme
A, the latter combination of cofactors could allow the reesterification of lipids in a subcellular
fraction known to contain free fatty acids, acyl-CoA synthetase and acyltransferases.

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3.3.2 Glucuronate reductase (aldehyde reductase) (Linster and E. V. Schaftingen

2006)

The reduction of d-glucuronate to L-gulonate is catalyzed by an NADPH-dependent

reductase, with broad specificity, known as aldehyde reductase or TPN L-hexonate
dehydrogenase and now referred to as aldo-keto reductase for the human enzyme.
The compound D-glucuronate and D-glucuronolactoneare converted by aldehyde reductase to
L-gulonate and L-gulonolactone, respectively. Aldehyde reductase belongs to the large group
of monomeric NADPH-dependent oxidoreductases, known as aldo-keto reductase, which
comprise many members in the human genome, including aldose reductase and
hydroxysteroid dehydrogenases. These enzymes show broad substrate specificities and it
would therefore not be surprising that, besides aldehyde reductase, other members of the
aldo-keto reductase super family participate in the reduction of d-glucuronate. Aldose
reductase appears to be much less efficient than aldehyde reductase in this respect.

3.3.3 Urono- and gulonolactonase

The conversion of D-glucuronate to L-gulonolactone requires the action of two
enzymes, a reductase and a lactonase, proceeding either via D-glucuronolactone if the
lactonization is the first step, or via l-gulonate if the first reaction is the reduction. Three
different types of lactonases acting on sugar derivatives have been characterized in
mammalian tissues: 6-phosphogluconolactonase, uronolactonase and aldonolactonase. The
first one is an enzyme of the pentose phosphate pathway, which belongs, in mammals, to the
same family of proteins as glucosamine 6-phosphate isomerase and has no direct role to play
in the formation of vitamin C. Uronolactonase, a microsomal enzyme, hydrolyzes D-
glucurono-3,6-lactone, but is inactive against aldonolactones. Aldonolactonase
(gulonolactonase) is also a metal dependent enzyme, acting best with Mn2+, which is present
in the cytosol and hydrolyzes a number of C- and D-lactones of a variety of 5-, 6-, and 7-
carbon aldonates, including L-gulono-1, 4-lactone and D-glucono-1, 5- lactone. It also
catalyzes the lactonization of aldonates, e.g. of L-gulonate, and can therefore participate in
the formation of vitamin C.

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3.3.4 L-Gulonolactone oxidase (Linster and E. V. Schaftingen 2006)

GLO, a microsomal enzyme, catalyzes aerobically the conversion of L-gulonolactone

to L-ascorbate with production of H2O2. The immediate oxidation product of GLO is 2-keto-
l-gulonolactone, an intermediate that spontaneously isomerizes to L-ascorbate. The preferred
substrate of the enzyme is L-gulono- 1, 4-lactone, but it also acts on L-galactono-, D-
mannono- and d-altrono-1, 4-lactone. Other C-lactones, including L-idono- and D-
gluconolactone, were not oxidized by the enzyme, indicating it’s configurationally specificity
for the hydroxyl group at C2. The production of H2O2 is unusual for an enzyme of the
endoplasmic reticulum, and one may wonder if this membrane-bound oxidoreductase does
not transfer its electrons to another acceptor in intact cells, most particularly because its plant
homologues do so. The latter share 30% sequence identity with mammalian GLO and differ
from this enzyme in three main aspects:
(1) They act specifically on l-galactono-1, 4-lactone, which is their physiological substrate;
(2) They are bound to the inner mitochondrial membrane; and
(3) They do not transfer electrons directly to O2, but to cytochrome c.

3.4 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).

a. D-Sorbitol

The catalytic hydrogenation of D-glucose to D-sorbitol is accomplished at high

pressures and elevated temperatures in the presence of a Raney nickel catalyst. The
hydrogenation is carried out in batch or continuous operations and affords almost quantitative
yields with minimal formation of D-mannitol and L-iditol. After removal of the catalyst, the
sorbitol solution is employed in fermentation without any further purification. Therefore,
manufacturers of ascorbic acid make high demands on the quality of D-glucose; crystalline
dextrose or purified starch hydrolysates are used (ULLMANN’S Encyclopedia vol. 38).

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b. L-Sorbose

L-sorbose is obtained in the pyranose form by microbiological oxidation of sterile aqueous

solutions of D-sorbitol in batch or continuous operations in the presence of air. The most
frequently used strains belong to the Gluconobacter oxydans family, which converts d-
sorbitol to L-sorbose with more than 90% efficiency. Large-scale fermentations have to be
carried out at pH 4–6 and at 30 – 35oC under sterile conditions to avoid loss of product during
oxidation and work-up by filtration and crystallization (ULLMANN’S Encyclopedia vol. 38).

Figure 3.5 Hancock and R Viola (2002) gives Pathway cycle for production of Ascorbic acid by Reichstein
Process.

c. 2, 3:4, 6-Di-O-isopropylidene-α-l-sorbofuranose

The protection of the 2, 3-and 4, 6-hydroxyl groups by forming cyclic ketals is

achieved in acetone with excess sulfuric acid as catalyst and dehydrating agent, generally at
low temperatures (e.g., 4oC). In the final reaction mixture 2, 3:4, 6-di-O-isopropylidene-α-
lsorbofuranose is obtained as main product. Byproducts are 2, 3-O-isopropylidene-α-l-
sorbofuranose and 1, 2-O-isopropylidene-α- l-sorbopyranose. After neutralization, excess
acetone is recovered by distillation. 2, 3:4, 6-di-O-isopropylidene-α-l-sorbofuranose is
extracted from the aqueous solution with aromatic solvents (e.g., toluene). The remaining

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monoisopropylidenesorboses are also recovered from the aqueous solution and returned to
the Process.

d. 2, 3:4, 6-Di-O-isopropylidene-2-ketol-gulonic acid

Originally, the oxidation of 2,3 : 4,6-di-O-isopropylidene-α-lsorbofuranose to 2,3 :

4,6-di-O-isopropylidene- 2-keto-l-gulonic acid was performed at elevated temperatures in
dilute sodium hydroxide withKMnO4, yielding ca. 90% of product. Much less expensive
oxidation methods are applied in modern continuous processes: sodium hypochlorite,
electrochemical oxidation, or catalytic air oxidation. Oxidation of 2,3:4,6-di-O-
isopropylidene-α-lsorbofuranose with hypochlorite in the presence of catalytic amounts of
nickel chloride or sulfate at 60oC yields >93% of 2, 3:4, 6-di-O-isopropylidene-2-keto-l-
gulonic acid. The active oxidant is presumably nickel peroxide. Electrochemical oxidation is
performed with nickel or nickel oxide electrodes in alkaline solution. Hydrogen, evolved at
the cathodes, can be used for hydrogenation of D-glucose. It can be advantageous to stop
electrochemical conversion to 2, 3:4, 6-di-O-isopropylidene-2-keto-l-gulonic acid at 90% to
avoid a drop in selectivity at the end of electrolysis. Oxidation can be completed with sodium
hypochlorite solution. At the end of the electro oxidation, selectivity can be improved by
electrolytic cells with high electrode surfaces, such as Swiss roll cells or cells with three-
dimensional electrodes. Alternatively, the oxidation has been performed by using air or
oxygen and a metal catalyst in alkaline solution. Good yields are achieved with palladium or
platinum on carbon. The reaction product 2, 3:4, 6-di-O-isopropylidene-2-keto-l-gulonic acid
is isolated by acidification and precipitation as the monohydrate.

e. L-Ascorbic acid.

The conversion of 2, 3:4, 6-di-O-isopropylidene-2-keto-l-gulonic acid to l-ascorbic

acid is achieved by two different procedures:

1) Deprotection to give 2-keto-l-gulonic acid, followed by esterification with

methanol and base catalyzed cyclization.

2) Acid-catalyzed cyclization to ascorbic acid directly from the protected or released

2-keto-l-gulonic acid.

The starting material for base-catalyzed reactions is methyl 2-keto-l-gulonate,

prepared by treatment of the 2, 3:4, 6-di-O-isopropylidene-2-keto-l-gulonic acid with acidic

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methanol. Finally, reaction of the methyl ester with sodium hydrogencarbonate or sodium
acetate affords sodium ascorbate in high yield. The first acid-catalyzed route to ascorbic acid
was published only few years after discovery of the Reichstein process. Today, the industrial
process is performed with an inert solvent (e.g., trichloromethane or toluene) in the presence
of hydrochloric acid. The advantage of this method is that ascorbic acid precipitates from the
mixture as it is formed, minimizing decomposition during reaction. The crude product is
obtained by filtration in high yield and high purity. After dissolution of crude ascorbic acid in
water, impurities are removed by refining with activated carbon, decolorizing resins, or ion
exchange resins, followed by crystallization.

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4. Conclusion

Ascorbic acid is synthesized by different pathways using Bacteria, Algae and by

means of chemical reactions called Reichstein process. They all have their merits as well
demerits. In Bacterial biosynthesis the overall yield is 60 g/l. of 2-KLG from L-Sorbose or D-
sorbitol with 60% conversion in D-Sorbitol pathway (Shrikant et al., 2006).while that of 2-
Keto-D-Gluconic Acid Pathway have 85% yield using genetically modified strain
(Glucanobacter Oxydans)(Shrikant et al., 2006) and 10.5 g/l concentration of 2-KLG from D-
Glucose. Using genetically modified strain erwina citerus gives 19.8 g/l of 2-KLG with 49%
conversion (Hancock and R. Viola, 2001). So the D-Sorbitol pathway is more economical
pathway in bacterial biosynthesis of Ascorbic acid.

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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Institute Of Chemical Engineering, Mumbai
 Metabolic pathway engineering for the production of L-ascorbic acid

5. References

Anderson, D.B., Gomez, M.L., Mesquita, C.H., Lajolo, F.M., 2004. Ascorbic acid
biosynthesis: a precursor study on plants. Brazilian Journal of Plant Physiology 16(3),
147-154.

Bremus, C., Herrmann, U., Bringer, S., Sahm, H., 2006. The use of microorganisms in l-
ascorbic acid production. Journal of Biotechnology 124, 196–205.

Grindley, J.F., Payton, T.A., Pol, T.H., Hardyt K.G., 1988. Conversion of Glucose to 2-Keto-
L-Gulonate, an Intermediate in L-Ascorbate Synthesis, by a Recombinant Strain of
Erwinia citreus. Applied And Environmental Microbiology 54(7), 1770-1775.

Hancock, R.D., Violak, R., 2001. The use of micro-organisms for L-ascorbic acid production:
current status and future perspectives, Applied Microbiology Biotechnology, 56:567–576.

Hancock, R.D., Viola, R., 2002. Biotechnological approaches for L-ascorbic acid production.
Trends In Biotechnology 20(7), 299-305.

Ishikawa, T., Dowdle J., Smirnoff, N. 2006. Progress in manipulating ascorbic acid
biosynthesis and accumulation in plants. Physiologia Plantarum 126, 343–355.

Linster, C.L., Schaftingen, E.V., 2006. Biosynthesis, recycling and degradation in mammals.
FEBS Journal 274, 1–22.

Oster, B., Fechtel, U., Merck, E., 2003. ULLMANN’S Encyclopedia of Industrial Chemistry
(Wiley- VCH), sixth edn. 38, 218-231.

Survase, S.A., Bajaj, I.B., Singhal, R.S., 2006. Biotechnological Production of Vitamins.
Food Technology Biotechnology 44 (3), 381–396.

Wheeler, G.L., Jones, M.A., Smirnoff N., 1998. The Biosynthetic Pathway of Vitamin C in
Higher Plants. Nature 393, 365-369.

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Institute Of Chemical Engineering, Mumbai