TRENDS in Biotechnology Vol.20 No.

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299 Review
The current worl d producti on of L-AA i s esti mated at
80 000 tons per annum wi th a gl obal market i n excess
of US$600 mi l l i on and an annual growth rate of 34%
[1,2]. Approxi matel y 50% of the syntheti c L-AA i s
used i n vi tami n suppl ements and pharmaceuti cal
preparati ons (e.g. oi ntments for the treatment of
burns). A rapi dl y expandi ng market i s the use of L-AA
as an addi ti ve to cosmeti c products, i n vi rtue of i ts
anti -oxi dant properti es and i ts potenti al to sti mul ate
col l agen producti on [3]. L-AA anti oxi dant properti es
are al so expl oi ted i n food processi ng (25% of total
producti on) and beverage manufacturi ng (15%) to
prevent pi gment di scol orati on and enzymi c browni ng,
to protect fl avour and aroma and to protect or
enhance nutri ent content [4]. Syntheti c L-AA i s al so
commonl y used i n ani mal feeds (10% of total ) even
though farm ani mal s synthesi ze the compound
i n thei r own bodi es. Farmers commonl y use
L-AA-suppl emented feeds for opti mum growth and
heal th i n chi cks, pi gl ets, cal ves and ani mal s under
stress [5]. L-AA i s al so frequentl y added to feeds i n
aquacul ture because several commerci al fi sh speci es
(e.g. sal mon) are unabl e to synthesi ze i t de novo[6].
At present, the majori ty of commerci al l y
manufactured L-AA i s synthesi zed vi a the seven-step
Rei chstei n process usi ng D-gl ucose (D-gl c) as a starti ng
poi nt (Fi g. 1). The process i nvol ves si x chemi cal steps
and one fermentati on step for the oxi dati on of
D-sorbi tol to L-sorbose. Overal l , the yi el d of L-AA from
D-gl c obtai ned by the Rei chstei n process i s ~50% [1].
Al though the Rei chstei n process has al l the advantages
that woul d be expected after >60 years devel opment,
i t i s sti l l hi ghl y energy consumi ng and requi res hi gh
temperatures and/or pressures for many steps.
I n addi ti on, most of the chemi cal transformati ons
i nvol ve consi derabl e quanti ti es of organi c and
i norgani c sol vents and reagents such as acetone,
sul phuri c aci d and sodi um hydroxi de. Al though
some of the compounds can be recycl ed, stri ngent
envi ronmental control i s requi red, resul ti ng i n
si gni fi cant waste di sposal costs. These and other
economi c factors have generated a substanti al i nterest
i n the expl oi tati on of mi crobi al bi otransformati ons i n
the manufacturi ng of L-AA. Recent i nnovati ons i n
technol ogi es associ ated wi th fermentati on processes
and cel l -free bi ocatal yti c systems have dri ven forward
the state-of-the-art. I n addi ti on, advances i n
bi ochemi stry and recombi nant DNA technol ogy,
together wi th the genomi c revol uti on, have wi dened
the opti ons avai l abl e for the expl oi tati on of
bi otechnol ogy i n L-AA manufacture and the producti on
of L-AA-enhanced bi omass. We present some of these
new opti ons here for an hi stori cal perspecti ve the
reader i s di rected towards earl i er revi ews [1,7,8].
Bacterial biotransformations for the synthesis of
Reichstein intermediates
The Rei chstei n process for L-AA synthesi s (Fi g. 1) was
devi sed soon after the di scovery of L-AA [9]. I t i s based
on chemi cal methods and bears no rel ati onshi p to the
bi ochemi cal pathways used by L-AA synthesi zi ng
organi sms. However, si nce the earl i est days a
consi derabl e effort was di rected towards usi ng
bi otransformati ons for the producti on of L-AA as
i ndi cated by the i ntroducti on of a mi crobi al step for
the oxi dati on of D-sorbi tol to L-sorbose [10]. Modern
bacterial strains of, for example, Gluconobacter oxydans
that gi ve excepti onal l y hi gh yi el ds of al most 100% for
thi s transformati on have been devel oped [11]. Gi ven
that i ndustri al l y useful prokaryotes do not synthesi ze
L-AA, attenti on has focused on thei r use for the
synthesi s of Rei chstei n i ntermedi ates. The two most
commerci al l y advanced methods are the oxi dati on of
D-gl c to 2-keto-L-gul onate (2-KLG) vi a D-gl uconate,
2-keto-D-gluconate and 2,5-diketo-D-gluconate (2,5-DKG
pathway) and the oxi dati on of D-sorbi tol or L-sorbose
to 2-KLG vi a the i ntermedi ate L-sorbosone (sorbi tol
pathway, Fig. 2). Given that individual bacterial strains
capabl e of catal yzi ng the effi ci ent conversi on of D-gl c to
2-KLG are not known, earl y approaches were based on
mi xed (e.g. [12]) or sequenti al (e.g. [13]) fermentati ons.
These efforts were l ater superseded by the extensi on of
the metabol i c capaci ty of bacteri al strai ns vi a geneti c
engi neeri ng. An earl y exampl e was the devel opment of
Over the past decade there has been increasing pressure to develop alternatives
to the Reichstein process,a largely chemical synthesis by which the vast majority
of world vitamin C (L-ascorbic acid,L-AA) is produced.The pressures include
increasing environmental concerns and legislation,and the need to increase
process efficiency and reduce capital costs.The development of efficient
fermentation processes in the past ten years has also represented a catalyst for
change.Here,we describe the development of biotechnological alternatives
for the synthesis of Reichstein intermediates by industrial microorganisms.
The recent elucidation of the plant biosynthetic pathway represents new
opportunities not only for the direct synthesis of L-AA by fermentation but also
for the production of human crop plants and animal fodder with enhanced
nutritional value.We discuss the potential for these developments in the light of
recent findings concerning L-AA biosynthesis in plants.
Published online: 17 May 2002
Biotechnological approaches for
L-ascorbic acid production
Robert D.Hancock and Roberto Viola
Robert D.Hancock
Roberto Viola*
Unit of Plant
Biochemistry, Scottish
Crop Research Institute,
Invergowrie, Dundee,
UK DD2 5DA.
*e-mail:
rviola@scri.sari.ac.uk
a strai n of Erwinia herbicolaengi neered to express
the gene that encodes 2,5-DKG reductase i n
Corynebacteriumsp., allowing the biosynthesis of 2-KLG
di rectl y from D-gl c [14]. However, poor yi el ds of 2-KLG
(~5%) were obtai ned owi ng l argel y to the di fferent
subcellular location of the three dehydrogenases required
for the producti on of 2,5-DKG (peri pl asm) and of the
2,5-DKG reductase required for its conversion to 2-KLG
(cytopl asm). Subsequent i mprovements resul ted i n the
devel opment of an E. citreusstrai n expressi ng the gene
encodi ng 2,5-DKG reductase i n Corynebacteriumsp.,
which produced 2-KLG from D-glc with a 49% yield [15].
Recombi nant Pantoea citreastrai ns capabl e of
produci ng i n excess of 120 g l
1
2-KLG from D-gl c i n
14l fermenters i n <120 h have now been reported
[2,16]. One unexpected fi ndi ng was that curi ng the
strai ns of a crypti c pl asmi d resul ted i n i mproved
growth characteri sti cs at hi gher temperatures thus
reduci ng the overal l fermentati on ti me [16].
Geneti c engi neeri ng has al so been used i n strai n
i mprovement to enhance yi el ds vi a the D-sorbi tol
pathway i n whi ch D-sorbi tol i s oxi di sed to 2-KLG by a
seri es of dehydrogenases (Fi g. 2) [17]. Gluconobacter
oxydansi s the speci es of choi ce for thi s purpose but the
subcel l ul ar l ocati on (cytosol i c or membrane-bound) of
the three dehydrogenases requi red for the conversi on
of D-sorbi tol to 2-KLG vari es from strai n to strai n
(Fi g. 3). Transfer of D-sorbi tol pathway i ntermedi ates
i nto the cytopl asm i n these strai ns i s detri mental
owi ng to the presence of cytosol i c reductases, whi ch
channel i ntermedi ates i nto the pentose phosphate
cycl e [18]. To overcome thi s probl em, membrane-bound
dehydrogenases from al ternati ve sources have been
expressed i n recombi nant G. oxydansto compl ement
or repl ace cytosol i c enzymes. For exampl e, strai n
I FO12258 of Acetobacter liquifaciensmembrane-
bound sorbosone dehydrogenase was expressed i n the
OX4 strai n of G. oxydans, whi ch had membrane-bound
sorbi tol and sorbose dehydrogenases but cytosol i c
sorbosone dehydrogenase. Si gni fi cant yi el d
i mprovements of 2-KLG from both L-sorbose (68% to
81%) and L-sorbosone (25% to 83%) were observed i n
resti ng cel l s but there were no yi el d i mprovements
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300 Review
TRENDS in Biotechnology
140150C
Ni
2+
conc H
2
SO
4
Fermentation
CH
3
COCH
3
CH
3
OH HCl
100C C
2
H
5
OH
D-Glucose
D-Sorbitol
L-Sorbose
2-Keto-L-gulonic acid
2-Keto-L-gulonic acid methylester
L-Ascorbic acid
Diacetone-L-sorbose
Pd
2+
80125 atm
Fig. 1. The Reichstein process for L-ascorbic acid(L-AA) manufacture.
In the widely used Reichstein process D-glucose is converted to L-AA
via a series of chemical steps and one bacterial fermentation for the
conversion of D-sorbitol to L-sorbose. Catalysts for the development
of alternative processes for L-AA synthesis include the use of
environmentally hazardous chemicals (orange boxes) or steps
requiring high-energy consumption (purple boxes). Abbreviations:
atm, atmospheric pressure; conc, concentrated.
TRENDS in Biotechnology
HO
OH
OH
OH
CH
2
OH
COOH
D-Gluconic acid
Glucose
dehydrogenase
Hydrogenation
D-Sorbitol
dehydrogenase
Gluconate
dehydrogenase
HO
OH
OH
OH
CH
2
OH
CHO
D-Glucose
HO
OH
OH
OH
CH
2
OH
CH
2
OH
D-Sorbitol
HO
OH
O
OH
CH
2
OH
COOH
2-Keto-D-gluconic acid
L-Sorbose
dehydrogenase
L-Sorbosone
dehydrogenase
2-Keto-D-gluconate
dehydrogenase
2,5-Diketo-
D-gluconate
dehydrogenase
Esterification
Lactonisation
HO
HO
OH
O
CH
2
OH
CH
2
OH
L-Sorbose
HO
OH
O
O
CH
2
OH
COOH
2,5-Diketo-
D-gluconic acid
2,5-Diketo-
D-gluconic
acid pathway
D-Sorbitol
pathway
2-Keto-
L-gulonic acid
HO
HO
OH
O
CH
2
OH
CHO
HO
HO
OH
O
CH
2
OH
COOH
HO
HO
HO
O
CH
2
OH
CO
L-Sorbosone
L-Ascorbic acid
Fig. 2. Microbial pathways for L-ascorbic acid (L-AA) synthesis.
Although up to six routes have been explored for the application of
biotransformation for the production of Reichstein intermediates, only
the two most commercially developed pathways are shown. Both
processes produce 2-keto-L-gulonic acid (2-KLG). As no natural
bacterial strain is capable of efficiently catalyzing either pathway in its
entirety, mixed or sequential fermentations were originally developed
and more recently genetically engineered strains have been created
that allow single step fermentations for either pathway.
under fermentati on condi ti ons [18]. More recentl y,
Sai to and col l eagues i sol ated strai n G624 of G. oxydans
from Japanese peach, whi ch converted D-sorbi tol to
L-sorbose al most quanti tati vel y vi a a membrane-bound
sorbi tol dehydrogenase but was unabl e to synthesi ze
2-KLG [19]. Membrane-bound sorbose dehydrogenase
and cytosol i c sorbosone dehydrogenase were cl oned
from G. oxydansT-100, a strai n that produced 2-KLG
wi th 13% yi el d from D-sorbi tol and expressed i n
strai n G624. After opti mi zati on of the expressi on
system and chemi cal mutagenesi s to bl ock further
2-KLG metabol i sm to L-i donate, G. oxydansstrai n
NB6939/pSDH-tufB1 provi ded ~85% yi el d of 2-KLG
from a 15% D-sorbi tol broth [20]. Recentl y, Asakura and
Hoshi no [21] reported the puri fi cati on and cl oni ng of a
dual speci fi ci ty L-sorbose/L-sorbosone dehydrogenase
capabl e of the sequenti al oxi dati on of L-sorbose through
to 2-KLG in vitro. The authors provi ded evi dence
suggesti ng a peri pl asmi c l ocati on of the protei n rai si ng
the possi bi l i ty of i ts use for the engi neeri ng of an
enti rel y peri pl asmi c D-sorbi tol pathway.
Continuous biocatalytic systems
Vari ous conti nuous bi ocatal yti c systems for the
synthesi s of Rei chstei n i ntermedi ates have been
proposed [22,23]. The onl y approach that has shown
potenti al to evol ve i nto a commerci al appl i cati on i s
based on the 2,5-DKG pathway (Fi g. 4). I n thi s system
D-gl c i s converted to D-gl uconate vi a an NADP-
dependent sol ubl e gl ucose dehydrogenase from
Thermoplasma acidophilum. Further conversi on to
2,5-DKG i s achi eved usi ng cel l preparati ons
contai ni ng membrane bound D-gl uconate and 2-keto-
D-gl uconate dehydrogenase and the cytochrome C
cofactor. Fi nal l y, 2,5-DKG i s converted to 2-KLG by a
sol ubl e, NADPH dependent 2,5-DKG reductase wi th
resul tant regenerati on of NADPfor use by D-gl uconate
dehydrogenase. I t appears that thi s technol ogy has
been i ndependentl y devel oped i n the USA and Chi na.
The Chi nese group devel oped the method at the
l aboratory scal e usi ng resti ng cel l s of G. oxydans
as a source of D-gl uconate and 2-keto-D-gl uconate
dehydrogenase. They achi eved a 16.8% yi el d of 2-KLG
from D-gl c after 30 h wi th a cofactor turnover of 388
[24]. A si mi l ar concept was devel oped i n the USA by a
consortium of companies led by Genencor I nternational
(Pal o Al to, CA, USA) and compri si ng Eastman
Chemi cal Company (Ki ngsport, TN, USA),
Argonne Nati onal Laboratory (Argonne, I L, USA),
El ectrosynthesi s Company (Lancaster, NY, USA) and
Mi croGenomi cs (Carl sbad, CA, USA) through joi nt
fundi ng from the Ameri can Nati onal I nsti tute of
Standards and Technol ogy. I n thi s case, Pantoea citrea
cel l s were mutageni zed to i nacti vate membrane bound
gl ucose dehydrogenase and permeabi l i zed usi ng
organic solvents. These cells acted as a source of the two
membrane-anchored dehydrogenases responsi bl e for
conversi on of D-gl uconate to 2,5-DKG. The bi oreactor
was run for 6 h at 60% oxygen saturati on duri ng whi ch
time initial 2-KLG formation rates were 10 g l
1
h
1
with
i ntegrated producti on rates of 5 g l
1
h
1
and a total
cofactor turnover >500. The bi oreactor was then run
down overni ght wi th l ow agi tati on to gi ve a fi nal ti tre
of 2-KLG of 42 g l
1
and yi el ds from D-gl c approachi ng
100%. No l oss of acti vi ty of the two sol ubl e enzymes
(gl ucose dehydrogenase and 2,5-DKG reductase) was
observed throughout the course of the experi ment [25].
Several advantages for thi s process were envi saged
i ncl udi ng reduced cost through recycl i ng of expensi ve
enzyme cofactors, el i mi nati on of by-product formati on,
cl eaner product recovery, quanti tati ve yi el d, hi gh
vol umetri c producti vi ti es and the abi l i ty to reduce
capital investment by reduction in size of the production
reactor. I t i s expected that thi s technol ogy wi l l soon be
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TRENDS in Biotechnology
D-Sorbitol
D-Gluconate D-Gluconate
L-Sorbose L-Sorbosone
D-Sorbitol L-Sorbose L-Sorbosone
Periplasm
Cytosol
L-Idonate
2-KLG
SLDH
SNDH
2-KR
5-SR
SDH
SNR
SLDH SDH
PPP
2-KLG
S/SNDH
Fig. 3. Synthesis and degradation of 2-keto-L-gulonic acid (2-KLG) by
acetic acid bacteria. Gluconobacter and other acetic acid bacteria
synthesize 2-KLG by partial oxidation of D-sorbitol. Conversion of
D-sorbitol to 2-KLG can occur via either membrane-bound or cytosolic
dehydrogenases. A fully periplasmic pathway is desirable to prevent
D-sorbitol or intermediates being channelled into metabolism via the
action of the cytosolic enzymes L-sorbosone reductase (SNR) and
L-sorbose 5-reductase (5-SR). Furthermore, the activity of 2-keto-L-
gulonate reductase (2-KR) in the cytosol results in the channelling of
2-KLG into central metabolism via L-idonic acid. Similarly, D-sorbitol can
be channelled into central metabolism via D-gluconate. Abbreviations:
PPP, pentose phosphate pathway; SDH, L-sorbose dehydrogenase;
SLDH, D-sorbitol dehydrogenase; SNDH, L-sorbosone dehydrogenase;
S/SNDH, bifunctional L-sorbose/ L-sorbosone dehydrogenase
TRENDS in Biotechnology
NADP
2,5-DKG reductase
(Corynebacterium sp.)
Glucose dehydrogenase
(Thermoplasma acidophilium)
NADPH
2-Keto-L-
gulonic acid
D-Glucose D-gluconic acid
2-Keto-D-
gluconic acid
2,5-Diketo-D-
gluconic acid
2-Keto-
D-gluconate
Dehydrogenase
CytC
Gluconate
Dehydrogenase
CytC
Fig. 4. Biocatalytic process for the synthesis of 2-keto-L-gulonic acid
(2-KLG). The bioreactor contains the soluble enzymes glucose
dehydrogenase and 2,5-DKG reductase together with catalytic quantities
of NADP(H). To link the two soluble enzymes and thus form a redox
couple, whole or treated bacterial cells are added which contain gluconate
dehydrogenase and 2-ketogluconate dehydrogenase activities. The cells
also act as a supply of cofactor for the membrane bound enzymes.
adopted at the i ndustri al scal e. One l i mi tati on i n the
effi ci ency of the conti nuous bi ocatal yti c process i s the
hi gh K
m
(213.5mM) of the 2,5-DKG reductases used,
whi ch were obtai ned from Corynebacteriumsp. [26]. I n
addi ti on, the Corynebacteriumenzymes l ack thermal
stabi l i ty. To i mprove the process, attempts were made
to i sol ate 2,5-DKG reductases wi th more favourabl e
catal yti c properti es. Very recentl y, Eschenfel dt and
col l eagues [27] used degenerate pri mers based on
known 2,5-DKG reductase sequences to ampl i fy
2,5-DKG reductase genes from soil and water samples.
The team successful l y i sol ated and expressed two
novel 2,5-DKG reductases wi th much l ower K
m
val ues
(57 and 67 M) [27] resul ti ng i n the enzymes
di spl ayi ng catal yti c effi ci enci es up to 1000 ti mes hi gher
than the Corynebacteriumenzymes. I n addi ti on, both
enzymes used both NADPH and NADH as cofactor.
One of the two enzymes al so showed hi gher thermal
stabi l i ty than the previ ousl y i sol ated reductases.
Al though data regardi ng the rel ati ve effi ci ency of the
process usi ng al ternati ve enzymes i s not avai l abl e i t i s
probabl e that i mprovements wi l l be apparent.
Bioconversion of 2-KLG to L-AA
Currentl y, the emphasi s of research i nto mi crobi al
transformati ons i s on the producti on of 2-KLG, whi ch
then i s processed further for the synthesi s of L-AA.
There are several probl ems associ ated wi th
conventi onal chemi cal methods for the conversi on
of 2-KLG to L-AA, i ncl udi ng the requi rement for
numerous steps, the use of gaseous hydrogen chl ori de
and/or the requi rement for consi derabl e puri fi cati on
of the fi nal product [28]. To overcome these probl ems,
several methods have been devi sed to al l ow conversi on
of 2-KLG to L-AA. Hydrol ase enzymes have been
used to convert esters of 2-KLG to L-AA al though the
effi ci ency of the process i s uncl ear [28]. Si mi l arl y,
l actonases i sol ated from Zymomonas mobilis,
Escherichia coli and Fusarium oxysporumwere
reported to convert 2-KLG to L-AA [29]. Currentl y,
nei ther method provi des a hi gh enough L-AA yi el d to
be economi cal l y vi abl e. However, the possi bi l i ty of
process i mprovement through better reactor desi gn
and enzyme i mprovement through such methods as
di rected evol uti on and poi nt mutagenesi s remai ns.
There have been recent reports of the i denti fi cati on
of yeast strai ns (Candida blankii and Cryptococcus
dimennae) that can convert 2-KLG to L-AA. However,
the yields were disappointing with accumulation of only
~25 g ml
1
L-AA i n the medi um from 5 mg ml
1
2-KLG
after 48 h i ncubati on [30]. Both speci es can grow on
L-AA as the sol e carbon source [31] and mutagenesi s of
the catabol i c pathway and further i nvesti gati ons i nto
opti mum cul ture condi ti ons coul d i mprove yi el ds.
Direct biosynthesis of L-AA in eukaryotes
The expl oi tati on of eukaryoti c systems natural l y
synthesi zi ng L-AA for the producti on of L-AA enhanced
raw materi al or even the manufacture of L-AA i s i n i ts
i nfancy al though several patents have been fi l ed.
Thi s i s pri mari l y owi ng to our l ack of knowl edge about
the pathway and regul ati on of L-AA bi osynthesi s i n
bi ol ogi cal systems. However, recent advances i n thi s
area obtai ned pri mari l y i n pl ants and yeast and the
ever i ncreasi ng avai l abi l i ty of tool s for data mi ni ng
and genome anal yses are l i kel y to demonstrate the
i mportance of thi s approach i n the comi ng years.
Biotechnological enhancement of plants and
microalgae for the production of L-AA
Attempts have been made to expl oi t mi croal gae
for the di rect producti on of L-AA from i nexpensi ve
feedstocks. Low yi el ds of up to 40 mg l
1
L-AA were
obtai ned by cul turi ng wi l d-type cel l s of the mi croal ga
Chlorella pyrenoidosai n fermenters [32]. After a
random mutagenesi s programme and fermentati on
opti mi zati on, i mproved yi el ds of up to 2 g l
1
L-AA
were achi eved starti ng from 80 g l
1
D-gl c. The bul k of
L-AA produced by the al gae remai ned i ntracel l ul ar
and the process was patented as a method for the
producti on of L-AA enhanced bi omass for ani mal feed
or as di etary suppl ement [33,34]. Accumul ati on of
L-AA i n the fermentati on medi um (~50% of the
total L-AA produced) was obtai ned by swi tchi ng to
col ourl ess aci dophyl i c mi croal gae of the genus
Prototheca[35]. At present, thi s method does not
provi de a commerci al l y competi ti ve opportuni ty for
L-AA bi omanufacture. However, the recent resol uti on
of the pri mary L-AA bi osyntheti c pathway i n hi gher
pl ants [36] (Fi g. 5) wi l l offer addi ti onal tool s for
process i mprovement vi a geneti c engi neeri ng. I n
pl ants, L-AA i s synthesi zed from D-gl c i n a ten-step
pathway vi a the rare sugar L-gal actose (L-gal ). Rapi d
progress has been made i n i denti fyi ng and cl oni ng
cruci al genes rel ated to the bi osyntheti c pathway
wi th publ i shed sequences now avai l abl e for GDP-
mannose pyrophosphoryl ase (enzyme cl assi fi cati on
(EC) 2.7.7.22) [37], GDP-mannose-3,5-epi merase [38],
L-gal actose dehydrogenase [39] and L-gal actono-1,4-
l actone dehydrogenase (EC 1.3.2.3) [40]. Wi th the
cl oni ng of GDP-mannose-3,5-epi merase presenti ng
the opportuni ty for a faci l e method for the synthesi s of
GDP-L-gal actose i t i s expected that the remai ni ng
pathway enzymes wi l l be puri fi ed and thei r genes
cl oned i n the near future.
Al an Berry and co-workers at Bi o-Techni cal
Resources (Mani towoc, WI , USA) reported a
correl ati on between GDP-mannose-3,5-epi merase
acti vi ty and L-AA content of randoml y mutageni zed
strai ns of the uni cel l ul ar al ga Prototheca[41] and
suggested that up-regul ati on of thi s step coul d al l ow
the producti on of al gae and pl ants wi th enhanced L-AA
content. However, to date, there are no reports of
mi croal gae or pl ants wi th enhanced L-AA content as a
resul t of overexpressi on of pathway genes. The one
successful attempt at i ncreasi ng pl ant L-AA content
through geneti c engi neeri ng was reported by Jai n and
Nessl er [42] who expressed the rat gene encodi ng
L-gul ono-1,4-l actone oxi dase (the enzyme i nvol ved i n
the fi nal step i n the ani mal bi osyntheti c pathway) i n
TRENDS in Biotechnology Vol.20 No.7 J uly 2002
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302 Review
l ettuce and observed up to sevenfol d enhancement of
L-AA content i n transgeni c l i nes. I t i s uncl ear why the
expressi on of a mammal i an gene resul ts i n enhanced
L-AA producti on i n pl ants but studi es wi th Arabidopsis
cel l cul tures suggest the presence of secondary
pathways for L-AA bi osynthesi s i n pl ants, i ncl udi ng
one i nvol vi ng L-gul ono-1,4-l actone as an i ntermedi ate
[43]. Further studi es are requi red to characteri ze
the best approach for boosti ng L-AA bi osynthesi s
i n uni cel l ul ar or mul ti cel l ul ar pl ants vi a geneti c
engi neeri ng. The potenti al opportuni ti es range from
i ncreased pl ant performance i n the fi el d [44,45] to
producti on of hi gh L-AA contai ni ng ani mal feedstock to
the generati on of products for the functi onal and heal th
food i ndustry. I n the current cl i mate i t i s unl i kel y that
food crops al tered to express a mammal i an gene woul d
be acceptabl e to the regul ators or general publ i c
al though up-regul ati on of the natural pl ant pathway
vi a geneti c engi neeri ng or more tradi ti onal breedi ng
methods ought to be better recei ved.
Potential for direct L-AA synthesis using yeast
Because of the wi despread use of yeast i n the
bi oi ndustry, the devel opment of strai ns capabl e of
di rect fermentati on of si mpl e sugars i nto L-AA i s very
attracti ve. Several earl y studi es suggested that yeast
cel l s contai n L-AA [46,47] but recent re-assessments
of these cl ai ms usi ng i mproved methodol ogy i ndi cate
that yeast cel l s grown on common carbon substrates
l ack L-AA but contai n i nstead i ts 5-carbon anal ogue
D-erythroascorbi c aci d (D-EAA). Thi s compound ful fi l s
si mi l ar anti oxi dant functi ons i n yeast [48,49] but has
no anti scorbuti c acti vi ty [50]. Yeast cel l s are, however,
known to accumul ate L-AA when grown i n the presence
of the nonphysi ol ogi cal substrates L-gul onol actone
(L-GuL), L-gal actonol actone (L-GL) or L-gal actose
[49,51,52], i ntermedi ates of the L-AA bi osyntheti c
pathways of ani mal s and pl ants, respecti vel y [53,36].
Mounti ng evi dence suggests that bi osynthesi s of L-AA
from these substrates i n yeast occurs vi a the acti vi ty of
enzymes of the D-EAA bi osyntheti c pathway (Fi g. 5).
For exampl e, unl i ke the correspondi ng pl ant enzymes
[36,40], the yeast enzymes D-arabi nose (D-ara)
dehydrogenase and D-arabi nonol actone (D-AL) oxi dase
show broad substrate speci fi ci ti es in vitro, bei ng
capabl e of oxi dati on of not onl y D-ara but al so L-gal ,
L-fucose and L-xyl ose and thei r correspondi ng aci d
l actones respecti vel y [5456]. Pre-i ncubati on of
S. cerevisiaecel l s wi th 30 mM D-ara resul ts i n strong
reducti on (85%) of the abi l i ty of the cel l s to convert
L-[1-
14
C]gal actose i nto L-AA, suggesti ng substrate
competi ti on for the same acti ve si te(s) [52]. Moreover,
expressi on of D-AL oxi dase from S. cerevisiaein
Escherichia coli extends the metabol i c capaci ty of the
organi sm to i ncl ude the abi l i ty to synthesi ze D-EAA
when suppl i ed wi th exogenous D-AL, or L-AA when
suppl i ed wi th exogenous L-GL or L -GuL [57]. These
consi derati ons l ead us to suggest that the enzymes
used for D-EAA bi osynthesi s mi ght al l ow future
expl oi tati on of yeast for the producti on of L-AA [58].
The expl oi tati on potenti al depends on the abi l i ty to
secure a cheap source of starti ng substrate (L-gal or
L-GL). L-GL i s a byproduct of the sugar i ndustry [59]
and processes for i ts expedi ent producti on as a
feedstock for the di rect fermentati on of L-AA by
mi croorgani sms coul d be devel oped. L-Gal i s an
extremel y rare sugar and cheap suppl i es are not
avai l abl e. However, the i sol ati on of genes i nvol ved i n
the producti on of free L-gal duri ng L-AA bi osynthesi s
i n pl ants mi ght provi de useful bi ochemi cal tool s
to extend the metabol i c capaci ty of i ndustri al
mi croorgani sms. Thi s route i s currentl y bei ng
acti vel y pursued i n our l aboratory and by several
other groups worl dwi de. The fact that yeast cel l s have
the capaci ty to synthesi ze L-AA from L-gal actose
suggests that expressi on of onl y three enzymes
(GDP-D-mannose-3,5-epi nerase and those i nvol ved
i n the rel ease of free L-gal from GDP-L-gal ) shoul d be
suffi ci ent to extend thi s syntheti c capaci ty to yeast.
Other sources of useful genes mi ght be mari ne
asci di ans, i n whi ch L-gal bi osynthesi s seems to occur
vi a a novel and as yet undi scovered pathway [60].
TRENDS in Biotechnology Vol.20 No.7 J uly 2002
http://tibtech.trends.com
303 Review
TRENDS in Biotechnology
Plants (a)
(b) Yeasts
HO
OH
OH
OH
O
HO
OH
O
L-Gal DH
NAD NADH
HK PGI PMI
PMM
ATP ADP
L-Galactose
D-Glucose D-Glucose-6-P D-Fructose-6-P D-Mannose-6-P
GDPM PPase GDPME
?
PPi GTP
GDP-L-Galactose D-Mannose-1-P GDP-D-Mannose
L-Galactono-1,4-lactone
OH
OH OH
O
O
CH2OH CH2OH
CH
2OH
OH
L-GL DH
Cyt COX Cyt Cred
L-Ascorbic acid
HO
OH
OH
OH
O
HO
OH
O
CH
2
OH
D-Ara DH
NADP NADPH
D-Arabinose D-Arabinono-1,4-lactone
OH
O
O
CH
2
OH
OH
D-Ara Ox
O
2
H
2
O
2
D-Erythroascorbic
acid
O
O
Fig. 5. Biosynthetic similarities in (a) plant and (b) fungal antioxidant
pathways. Plants synthesize L-AA from D-glucose via a complex nine or
ten step pathway involving phosphorylated sugar intermediates and
sugar nucleotides. The rare sugar L-galactose is an intermediate in the
biosynthetic pathway, which is then sequentially oxidised to L-ascorbic
acid (L-AA) via the actions of the enzymes L-galactose dehydrogenase
and L-galactono-1,4-lactone dehydrogenase, both of which
demonstrate strong substrate specificity. Yeasts synthesize the
5-carbon L-AA analogue, D-EAA via a similar pathway making use
of the 5-carbon sugar intermediate D-arabinose. In this case oxidation
to the final product occurs via D-arabinose dehydrogenase and
D-arabinono-1,4-lactone oxidase. These enzymes lack specificity and
are also capable of oxidising L-galactose, L-galactono-1,4-lactone or
L-gulono-1,4-lactone to L-AA. Abbreviations: D-Ara DH, D-arabinose
dehydrogenase (EC 1.1.1.117); D-AL Ox, D-arabinono-1,4-lactone
oxidase; L-Gal DH, L-galactose dehydrogenase; GDPME,
GDP-D-mannose-3,5-epimerase; GDPM PPase, GDP-D-mannose
pyrophosphorylase (EC 2.7.7.22); L-GL DH, L-galactono-1,4-lactone
dehydrogenase (EC 1.3.2.3); HK, hexokinase (EC 2.7.1.1); PGI,
phosphoglucose isomerase (EC 5.3.1.9); PMI, phosphomannose
isomerase (EC 5.3.1.8); PMM, phosphomannose mutase (EC 5.4.2.8).
Conclusions and future prospects
The Rei chstei n process has come to the end of i ts
rei gn. I t requi res extensi ve pl ants wi th associ ated
fi xed and capi tal costs to real i ze economi es of scal e.
The push for devel opi ng sustai nabl e al ternati ves
to the chemi cal process i s al so i ncreasi ng.
Two-stage fermentati on technol ogy for the
producti on of 2-KLG i s avai l abl e and represents a
cl eaner and more effi ci ent method wi th overal l
producti on cost savi ngs of about a thi rd compared
wi th the Rei chstei n process. The key i ndustri al
pl ayers are showi ng an i ncreased i nterest i n
fermentati on technol ogy. Roche i s upgradi ng i ts
Scotti sh Dal ry pl ant wi th a new two-stage
fermentati on pl ant and a joi nt venture between
Cerestar, BASF and Merck i s to produce 2-KLG
by fermentati on at Krefel d (Germany). Moreover,
new pl ayers are enteri ng the market such as the
US company Archer-Dani el s-Mi dl and (Decatur,
I L, USA) who are expected to begi n produci ng
vi tami n C producti on i mmi nentl y usi ng
fermentati on technol ogy. I n addi ti on, a joi nt venture
between Eastman and Genencor I nternati onal i s
pl anned to enter the market i n 2003 wi th a new
bi ocatal yti c process for the producti on of 2-KLG.
Recent advances i n our understandi ng of L-AA
bi osynthesi s i n pl ants and D-EAA bi osynthesi s i n
yeast wi l l pave the way for the devel opment of novel
methods for di rect L-AA producti on. Geneti c tool s for
the enhancement of L-AA content of pl ant products for
human consumpti on and ani mal fodder are al ready
avai l abl e. The metabol i c capaci ty of yeast cel l s to
synthesi ze L-AA from certai n substrates mi ght yet be
expl oi ted through a whol l y novel approach for the
devel opment of si ngl e-step fermentati on process for
L-AA bi omanufacture.
TRENDS in Biotechnology Vol.20 No.7 J uly 2002
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304 Review
Acknowledgements
The authors gratefully
acknowledge the skilful
assistance of Ian Pitkethly
for preparation of the
figures. SCRI receives
grant-aided support from
the Scottish Executive
Environment and Rural
Affairs Department. RDH
is supported by DEFRA.
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Sti mul i -responsi ve pol ymers are pol ymers that
respond wi th dramati c property changes to smal l
changes i n thei r envi ronment. They can be cl assi fi ed
accordi ng to the sti mul i they respond to as:
temperature-, pH-, i oni c strength-, l i ght-, el ectri c- and
magneti c fi el d-sensi ti ve. Some pol ymers respond to a
combi nati on of two or more sti mul i . Another approach
i s to cl assi fy these pol ymers by thei r physi cal form;
that i s, free chai ns i n sol uti ons, chai ns grafted on a
surface, coval entl y cross-l i nked gel s and reversi bl e or
physi cal gel s. Recent advances i n the desi gn of sti mul i -
responsi ve pol ymers have created opportuni ti es for
novel bi omedi cal appl i cati ons. Sti mul i -responsi ve
changes i n shape, surface characteri sti cs, sol ubi l i ty,
formati on of an i ntri cate mol ecul ar sel f-assembl y and
a sol gel transi ti on enabl ed several novel appl i cati ons
i n the del i very of therapeuti cs, ti ssue engi neeri ng,
cel l cul ture, bi oseparati ons and sensor or actuator
systems. Recent revi ews have summari zed research
progress i n bi omi meti c actuators, i mmobi l i zed
bi ocatal ysts, drug del i very, thermoresponsi ve
surfaces, bi oseparati on and bi oconjugates [1,2]. Thi s
arti cl e expl ores progress made i n sti mul i -responsi ve
pol ymers, wi th speci al emphasi s on recent advances
i n understandi ng the structureproperty rel ati onshi p
i n pol y(N-i sopropyl acryl ami de) (PNI PAAm)-based
systems, reversi bl e sol gel systems, pH-sensi ti ve
pol ymers, hybri d hydrogel s and el ectro- or l i ght-
sensi ti ve pol ymers. Ki neti c and thermodynami c
control of the sti mul i -sensi ti ve response i s cruci al
i n al l appl i cati ons; therefore understandi ng the
structureproperty rel ati onshi p i s essenti al for
Response to stimulus is a basic process of living systems.Based on the lessons
from nature,scientists have been designing useful materials that respond to
external stimuli such as temperature,pH,light,electric field,chemicals and
ionic strength.These responses are manifested as dramatic changes in one of
the following:shape,surface characteristics,solubility,formation of an intricate
molecular self-assembly or a sol-to-gel transition.Applications of stimuli-
responsive,or smart,polymers in delivery of therapeutics,tissue engineering,
bioseparations,sensors or actuators have been studied extensively and
numerous papers and patents are evidence of rapid progress in this area.
Understanding the structureproperty relationship is essential for the further
development and rational design of new functional smart materials.For
example,kinetic and thermodynamic control of the coil-to-globule transition
could be achieved through changes in polymer composition and topology.
Published online: 17 May 2002
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biomedical applications
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