Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites
For a long time people are using plants not only as crop cultures but also for obtaining of various chemicals. Currently plants remain one of the most important and essential sources of biologically active compounds in spite of progress in chemical or microbial synthesis. In our review we compare po...
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| Опубліковано в: : | Цитология и генетика |
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| Дата: | 2010 |
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Інститут клітинної біології та генетичної інженерії НАН України
2010
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites / Y.V. Sheludko // Цитология и генетика. — 2010. — Т. 44, № 1. — С. 65-75. — Бібліогр.: 90 назв. — рос. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859716820764721152 |
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| author | Sheludko, Y.V. |
| author_facet | Sheludko, Y.V. |
| citation_txt | Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites / Y.V. Sheludko // Цитология и генетика. — 2010. — Т. 44, № 1. — С. 65-75. — Бібліогр.: 90 назв. — рос. |
| collection | DSpace DC |
| container_title | Цитология и генетика |
| description | For a long time people are using plants not only as crop cultures but also for obtaining of various chemicals. Currently plants remain one of the most important and essential sources of biologically active compounds in spite of progress in chemical or microbial synthesis. In our review we compare potentials and perspectives of modern genetic engineering approaches for pharmaceutical biotechnology and give examples of actual biotechnological systems used for production of several promising natural compounds: artemisinin, paclitaxel and scopolamine.
З давніх часів люди використовували рослини не тільки як харчові культури, але і для отримання різноманітних хімічних сполук. Незважаючи на сучасний розвиток методів хімічного синтезу й мікробіологічних біотехнологій, рослини залишаються найважливішим і незамінним джерелом біологічно активних речовин. В огляді ми зіставили можливості й перспективи використання сучасних методів генетичної інженерії в фармацевтичній біотехнології і навели приклади сучасних біотехнологічних систем, які застосовують для одержання деяких цінних натуральних продуктів – артемізініна, паклітаксела і скополаміна.
С давних времен растения использовались людьми не только как пищевые культуры, но и для получения разнообразных химических соединений. Несмотря на современное развитие химических методов синтеза и микробиологических биотехнологий, растения остаются важнейшим и незаменимым источником биологически активных веществ. В обзоре мы сопоставили возможности и перспективы использования современных методов генетической инженерии в фармацевтической биотехнологии и привели примеры новейших систем, используемых для получения некоторых ценных натуральных продуктов – артемизинина, паклитаксела и скополамина.
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Обзорные статьи
УДК 575.854 + 547.94 + 582.923.5
Y.V. SHELUDKO
Institute of Cell Biology and Genetic Engineering, Kiev
E�mail: ysheludko@ukr.net
RECENT ADVANCES IN PLANT
BIOTECHNOLOGY AND GENETIC
ENGINEERING FOR PRODUCTION OF
SECONDARY METABOLITES
For a long time people are using plants not only as crop
cultures but also for obtaining of various chemicals. Currently
plants remain one of the most important and essential sources
of biologically active compounds in spite of progress in chemi�
cal or microbial synthesis. In our review we compare poten�
tials and perspectives of modern genetic engineering approach�
es for pharmaceutical biotechnology and give examples of
actual biotechnological systems used for production of several
promising natural compounds: artemisinin, paclitaxel and
scopolamine.
Introduction. One of the most important tasks
for modern genetic engineering, biotechnology and
pharmacology is search or creation of systems for
high�scale obtaining of valuable natural products –
complex organic compounds produced by living
organisms. Since the earliest time people used
plants not merely as food crops but additionally as
sources of various chemicals: pharmaceuticals,
insecticides, food supplements, dyes etc. Currently
plants remain an essential provider of biologically�
active compounds in spite of development of chem�
ical or microbial technologies. A number of inher�
ent advantages make plants the central and highly
perspective object in natural product biosynthesis
researches e.g. a) ecological and pharmacological
safety; b) high native biosynthetic capabilities
including multistep stereospecific synthesis of
complex organic molecules, eukaryotic type of
biopolymer synthesis and processing; c) possibili�
ties of scaling up of valuable compound produc�
tion using natural potential of plant systems; and
d) economical values.
For primary classification of pharmacological�
ly valuable plant natural substances one can define
a group of mainly low molecular weight com�
pounds, including first of all plant secondary
metabolites, and a group of proteins and peptides
with high molecular weight which are the products
of heterological expression of foreign genes in
plant cells.
All biochemical processes in plant cell can be
conditionally classified as primary and secondary
metabolism. Compounds and processes which are
necessary for growth, development and breeding
belong to primary metabolism. It includes mainly
the metabolism of proteins, nucleic acids, carbohy�
drates and lipids. On the other hand, biosynthesis
and catabolism of variable pigments, alkaloids,
terpenes, phenolics belong to secondary metabo�
lism. All these substances considered to be not
directly essential for plant cell life, and their func�
tion in plants is not always clear [1, 2]. The major�
ity of secondary compounds are considered to be
participating in plant�environment interactions:
they defense plants from pathogens, pests or herbi�
vores, serve as attractants, have allelopathic,
photo�protective or light�harvesting functions
[1–4]. It is not surprising that most of them have a
strong influence upon animal and human organ�
ism. Numerous examples of pharmaceutical appli�
cations of plant secondary metabolites are given in
recent reviews [5–9].
ІSSN 0564–3783. Цитология и генетика. 2010. № 1 65
© Y.V. SHELUDKO, 2010
People studied pharmacological properties of
secondary compounds since great antiquity: men�
tions of medicinal applications of alkaloid�con�
taining plant were found among Chinese,
Mesopotamia and, later, India ancient sources
dated 3000–1000 B. C. [10]. Organic synthesis
progress at the close of XIX century and develop�
ment of chromatographic separation protocols in
the first half of XX century allowed isolation and
identification of numerous organic substances
responsible for pharmacological activities of plant
extracts. However, in spite of considerable success
in modern organic synthesis, plant’s ability to form
biologically active stereoisomers often makes them
the unique and essential source of pharmacologi�
cally�valuable natural products. Moreover, consid�
erable part of synthetic pharmaceuticals has been
developed as modifications of natural substances
of plant origin. As the experts estimate, in USA
nearly 50 % of drugs for cancer chemotherapy are
derivatives of plant extract components [11]. One
should remark that searching of the optimal bal�
ance between drug efficiency and toxicity in the
recent years brought scientists again to substances
isolated from natural sources, first of all from
plants [12].
In recent years an intensive work has been car�
ried out on screening of biological activity and
structural diversity of secondary metabolites.
Nevertheless the biosynthetic potential of plant
cells is considered to be not even half exhausted –
a total amount of substances produced by plants
was estimated in range about 500 thousands [13,
14]. Actual models suggest correlation between
evolution of secondary metabolism in plants and
reciprocal adaptation of pests or pathogens leading
to divergence and stimulating biodiversity in the
both groups [1].
The majority of secondary biosynthetic path�
ways are multistep enzymatic processes with com�
plex and delicate regulation mechanisms on tran�
scriptional and/or posttranscriptional level.
Segregation of intermediates inside of single plant
cell or their transport between different parts of the
whole plant often occurs. All these factors make
investigation and, especially, controlled biotech�
nological production of secondary compounds an
extremely complicated task.
Classification of secondary metabolites may be
based on the chemical structure or biological charac�
teristics of substances. In general, three big groups
of secondary compounds can be assigned: ter�
penes, phenolics and alkaloids, which include the
main part of currently identified compounds.
Their number is estimated to be from more than
50 000 structures to about 100 000 [2, 14–16].
Many terpenes exhibit strong pharmacological
activities against a number of human diseases.
Among them we can mention cardenolides of
Digitalis sp. [17], glycyrrhizin extracted from the
licorice root and calanolides from Calophyllum
with anti�HIV activities [18], antibacterial shikonin
from Lithospermum erythrorhizon [19], monoter�
penoid alkaloid camptothecin isolated from
Camptotheca acuminata and Nothapodytes foetida
[20, 21], artemisinin from Artemisia annua used for
malaria treatment and having additionally cyto�
toxic features [22, 23], and many others. In recent
publications Morimoto et al. reported about suc�
cessful studies of cannabinoid biosynthesis:
5 enzymes were characterized and the correspon�
ding genes were cloned [24, 25]. Heterologous
expression of tetrahydrocannabinolic acid syn�
thase gene resulted in formation of tetrahydro�
cannabinolic acid (precursor of tetrahydrocannabi�
nol) from cannabigerolic acid [24]. This gene was
later expressed in Pichia pastoris cells. High level of
enzymatic activity (app. 1.3 nkat/L) was detected
in culture medium [26].
In spite of impressive scope and wide range of
researches, only several secondary biosynthetic
pathways have been studied in details on the enzy�
matic and gene levels. In our manuscript we will
focus on these examples. Evidently, the frame of
this publication does not allow performing a thor�
ough review of all plant secondary metabolism
research areas. Therefore we will discuss here
biotechnological systems developed for produc�
tion of certain valuable and perspective natural
products.
Artemisinin production. Artemisinin from A.
annua is currently one of the most effective anti�
malarial drugs recommended by WHO during
short�course artemisinin�based combination ther�
apy [27]. Low content of artemisinin in plants
(0.01–1 % DW) and ever�growing demand for
artemisinin�containing pharmaceuticals stimulat�
ed studies on biosynthetic pathway of this com�
pound formation and attempts to enhance its
accumulation in plant systems [28]. Total organic
ISSN 0564–3783. Цитология и генетика. 2010. № 166
Y.V. Sheludko
synthesis of artemisinin was found to be very diffi�
cult and costly process [29]. More perspective were
approaches on improvement of artemisinin pro�
duction in plant tissue under salinity stress condi�
tions [30].
Numerous studies have been carried out in
order to obtain artemisinin from plant cell culture
systems by selection of a highly productive line,
supplying with precursors or elicitation [28, 31].
Additionally a hairy root culture of A. annua was
established [32].
Cloning of several terpene biosynthesis genes
like cotton farnesyl diphosphate synthase and its
overexpression in A. annua hairy roots resulted in
three� to four�fold higher yield of artemisinin [33].
Redirection of amorpha�4,11�diene synthase and
farnesyl diphosphate synthase to the plastids in
transgenic Nicotiana tabacum allowed to enhance
considerably accumulation of one of the artemisinin
precursors, amorpha�4,11�diene [34].
The most promising way to scale up the produc�
tion of artemisinin was cloning and heterologous
expression of genes coding for several consequent
enzymes of mevalonate pathway (amorpha�4,11�
diene synthase, cytochrome P450 monooxygenase
(CYP71AV1), cytochrome P450 oxidoreductase)
from A. annua in Saccharomyces cerevisiae strain
(Fig. 1). As a result, 100 mg/L of artemisinic acid,
direct precursor of artemisinin, were synthesized
in the course of three�step reaction from native
yeast intermediate metabolite farnesyl pyrophos�
phate. Its further conversion to artemisinin is not
complex [35, 36]. Production of artemisinic acid
from S. cerevisiae in bioreactor increased recently
25�fold and reached up to 2.5 g/L [37]. This
example demonstrates efficiency of the present
strategy of secondary pathway genetic engineering
comprising characterization and cloning of respec�
tive genes, regulator elements and correct choice
of heterologous expression system.
Paclitaxel production. Perhaps one of the most
famous cytotoxic natural compounds discovered
during the last decades was diterpene amid pacli�
taxel also known as taxol. Its antitumour activity as
a component of Taxus brevifolia extract is known
since 1965; in 1972 the chemical structure of taxol
was elucidated [38]. In 1992 Taxol® was registered
and appeared in the world pharmaceutical market.
Numerous clinical trials proved its efficiency
against several types of cancer currently making
taxol one of the most perspective anticancer drugs.
Cytotoxic effect of paclitaxel is based on cell divi�
sion blocking by microtubules stabilization [39, 40].
Ever�growing demand for paclitaxel and its low
content in wood of slowly growing yew�trees
(about 0.03 % d. w. in T. brevifolia– bark of sever�
ІSSN 0564–3783. Цитология и генетика. 2010. № 1 67
Recent advances in plant biotechnology and genetic engineering for production
Fig. 1. Part of artemisinin biosynthesis pathway in S. cerevisiae (strain expressing amorphadiene
synthase gene (ADS), cytochrome P450 monooxygenase (CYP71AV1) and NADPH: cytochrome
P450 oxidoreductase (CPR) [35]: 1 – farnesyl pyrophosphate; 2 – amorpha�4,11�diene; 3 – arte�
misinic acid; 4 – artemisinin
al hundred thousands of yew�trees needs to be
extracted to supply world year demand for pacli�
taxel) stimulated researches on chemical and
biotechnology synthesis of this compound. More
than 300 relative compounds have been isolated
and characterised from different Taxus species up
to now [41].
The total chemical synthesis of paclitaxel was
found to be very complex process too expensive for
commercial production. Partial biosynthesis of
paclitaxel and its more active derivatives like
Taxotere® from precursors (for example, baccatin
III) appeared more perspective. Baccatin III was
isolated from yew needles that did not destroy trees
and extended the source of raw materials [40, 42].
High value of paclitaxel and its extremely low
natural supply became a prerequisite for numerous
projects on selection of highly productive Taxus
cell lines and enhancing of paclitaxel biosynthesis
in cell cultures. Results of these studies were sum�
marized in recent publications [43, 44]. Manipu�
lation with cultural medium composition in com�
bination with efficient selection allowed in a num�
ber of cases accumulation of paclitaxel in cells up
to 0.03–0.05 % d. w. that is comparable or even
surpasses the metabolite level in T. brevifolia bark
[45, 46]. Further investigation proved efficiency of
elicitation for taxoid biosynthesis stimulation
because a number of important enzymes of ter�
pene pathway (for instance geranylgeranyl diphos�
phate synthase and taxadiene synthase) are jas�
monate inducible [47, 48].
Tabata reported that development of Taxus cell
suspension selection, cultivation and elicitation
protocol resulted in stable paclitaxel production up
to 295 mg/L [49]. Multiple jasmonate treatments
in bioreactor increased taxoid yield in cell suspen�
sions up to 612 mg/L [50]. Companies of Phyton
Catalytic Inc. (USA) and Samyang Genex (South
Korea) informed about commercial isolation of
paclitaxel from cell cultures [16, 39].
Two alternative pathways of terpene biosynthe�
sis have been described at present time. Both path�
ways lead to production of common terpene pre�
cursors (dimethylallyl diphosphate (DMAPP) and
isopentenyl diphosphate (IPP)) which can be
transformed in more complex molecules in the
course of further conversions. The classic meval�
onate pathway (MVA) which functions in the
cytosol initially was assumed to be the sole source
of the terpenoid precursors IPP and DMAPP. It
supplies the precursors for production of sesquiter�
penes and triterpenes. Alternative pathway named
after the first committed precursor, 2�C�methyl�
D�erythritol�4�phosphate (MEP) is localized in
plastids and is generally used to supply precursors
ISSN 0564–3783. Цитология и генетика. 2010. № 168
Y.V. Sheludko
Fig. 2. Selected stages of paclitaxel biosynthesis: 1 – geranylgeranyl pyrophosphate; 2 – taxa�4(5),11(12)�diene; 3 – baccatin
III; 4 – paclitaxel; TS – taxadiene synthase. Numerous arrows indicate more than one step
for the production of monoterpenoids, diterpenoids
and tetraterpenoids [1, 51]. Moreover, recent stud�
ies showed possibilities for interchanges between
intermediates of the both pathways [52]. Experi�
ments on inhibition of IPP transport from cyto�
plasm to plastids demonstrated that some IPP from
mevalonate pathway might be transfered from the
cytoplasm to the plastids in the course of taxol and
baccatin III biosynthesis. It was also presumed
that different IPP biosynthesis pathways occur
during different growth phases in Taxus cells [53].
Because of diterpenoid origin of paclitaxel, the
special attention was paid to the investigation of
MEP pathway regulation and cloning the appropri�
ate genes. In general, 15 consequent secondary
enzymatic reactions should be accomplished to
form baccatin III– the key precursor of paclitaxel
[54] (Fig. 2). Recent reviews reported cloning and
characterization of 10 genes of taxane biosynthesis
[43, 51 and references cited therein, 54, 55]. In par�
ticular, 2�C�methyl�D�erythritol 2,4�cyclodiphos�
phate synthase gene, which is the 5�th enzyme of
the MEP pathway, was cloned from T. medium [56].
The efficiency of Agrobacterium transformation
of yew cells is low and successful transformation
protocol of Taxus cell suspensions was developed
not long ago [57]. Because of this the majority of
cloned genes were functionally expressed in E. coli
и Saccharomyces cerevisiae [54, 58]. In the last case,
5 genes coding for 5 consequent reaction enzymes
from primary metabolism to the intermediate taxa�
dien�5�α�acetoxy�10�β�ol were installed in a sin�
gle yeast host. It was shown that enzymes encoded
by introduced heterologous genes utilized yeast
isoprenoid precursors. However biosynthesis was
blocked at the first cytochrome P450 hydroxyla�
tion step [54]. In order to enhance the hydroxyla�
tion activity, coexpression of cytochrome P450
reductase with cytochrome P450 oxygenase was
successfully performed in yeast cells [59].
Among plant species, A. thaliana was trans�
formed with recombinant T. baccata taxadiene syn�
thase gene coding for plastid localized enzyme of
one of early stages of paclitaxel biosynthesis cata�
lyzing conversion of geranylgeranyl diphosphate to
taxadiene. It led to accumulation of taxadiene in
Arabidopsis cells [60]. This experiment demon�
strated the perspective of approaches based on
engaging of natural terpenoid precursors of plant
host in taxane biosynthesis paythway. However,
constitutive production of the full�length His�
tagged enzyme in A. thaliana plants caused growth
retardation and decreased the levels of photosyn�
thetic pigments. Although these effects may be
driven by a toxic taxadiene, the lower accumula�
tion of endogenous plastid isoprenoids such as
carotenoids and chlorophylls in transgenic plants
also suggested the alteration of the balance of ger�
anylgeranyl diphosphate pool. Using of inducible
transgene expression system allowed optimization
of taxadiene production which reached 30�fold
higher levels than those in plants constitutively
expressing the transgene [60]. Even higher taxadi�
ene accumulation was observed after expression of
taxadiene synthase in tomato fruits due to redirec�
tion of carotenoid metabolites: about 160 mg of
taxadiene was extracted from 1 kg of freeze dried
fruits [61].
Except for higher plants, taxadiene synthase was
expressed in a moss Physcomitrella patens [62] and
in the yeast S. cerevisiae [63]. Transgenic moss accu�
mulated taxadiene up to 0.05 % of fresh weight.
Transgene expression did not affect significantly
the amounts of the endogenous diterpenoids. In
contrast to other transgenic plants expressing het�
erologous taxadiene synthase, transgenic P. patens
did not exhibit any growth inhibition due to the
alteration of diterpenoid metabolic pools that sug�
gests the perspective of this object for the biotechno�
logical production of paclitaxel and its precursors.
Introduction of T. chinensis taxadiene synthase
alone in S. cerevisiae did not increase the taxadiene
levels because of insufficient levels of the universal
diterpenoid precursor geranylgeranyl diphosphate.
In order to attain a high level of taxadiene and its
intermediate metabolites, geranylgeranyl diphos�
phate synthase from Sulfolobus acidocaldarius and
codon optimized T. chinensis taxadiene synthase
gene were introduced into yeast genome. It result�
ed in 40�fold increase in taxadiene to app.
8.7 mg/L as well as significant amounts of geranyl�
geraniol (app. 33.1 mg/L), suggesting possibility
for further increase of taxadiene level [63].
Scopolamine production. The anticholinergic
tropane alkaloids hyosciamine, its racemic form
atropine, and scopolamine have been known
among the oldest drugs in the medicine because of
their effect on parasympathetic nervous system.
Currently they are widely used in pharmacology as
muscle relaxants. These substances together with a
ІSSN 0564–3783. Цитология и генетика. 2010. № 1 69
Recent advances in plant biotechnology and genetic engineering for production
number of other tropane alkaloids were isolated
mainly from Solanaceae species, although tropane
alkaloids were additionally detected in plants of
several other families [64]. Hyoscyamine is nor�
mally the more abundant alkaloid in Solanacea
species while scopolamine (which is more physio�
logically active and valuable) is produced in greater
quantities only in Duboisia spp and Datura metel
[64, 65]. As it was also shown for other natural
products mentioned above, the chemical synthesis
of these alkaloids has proved to be difficult and not
economically feasible so that plant material is their
only source. World demand for scopolamine was
estimated to be about 10 times greater than that of
hyoscyamine together with atropine [66]. This
provoked the interest to tropane alkaloid biosyn�
thesis pathway and biotechnological production of
scopolamine. Because it was shown that undiffer�
entiated systems such as calluses or cell cultures
have low productivity [67], hairy roots caused by
the infection of plants with A. rhizogenes have been
chosen as an object for attempts to enhance scopo�
lamine production. Owing to their stable and high
productivity, hairy root cultures have been investi�
gated for several decades for biotechnological pro�
duction of the valuable metabolites (progress in
understanding of secondary biosynthesis mecha�
nisms in hairy root cultures was reflected in the
recent reviews [68–71].
Hairy roots of Hyoscyamus muticus may pro�
duce high contents of hyoscyamine, but in many
cases only trace amounts of scopolamine [72].
Sevon et al. described obtaining and analysis of
hairy roots in more than 15 species. Amounts of
scopolamine in the studied cultures varied from
0.2 to 32 mg/g DW. Laborious selection of the
more productive clones and optimization of the
growth conditions was often necessary to reach
these levels of scopolamine accumulation [71].
Thus it is obvious that metabolic engineering of
this biosynthetic pathway or its single steps could
help to improve scopolamine production. In partic�
ular, the conversion of hyoscyamine to the much
more valuable scopolamine could be regarded as
the major goal of these studies.
Early stages of nicotine and tropane alkaloid
biosynthesis are coinciding and discussed together
with further reactions in the recent reviews [65,
73]. The first committed step of both pyridine and
tropane alkaloid metabolism is S�adenosylmethio�
nine (SAM)�dependent methylation of putrescine
catalysed by putrescine N�methyltransferase, form�
ing N�methylputrescine. The overexpression of N.
tabacum putrescine N�methyltransferase (PMT)
gene in scopolamine�rich Duboisia hybrids, Datura
metel, Atropa belladonna and H. muticus caused
increasing in accumulation of the direct metabo�
lite N�methylputrescine (2–4�fold compared to
wild type roots) [74], but there was no significant
increase in either tropane or pyridine�type alka�
loids [74–76] or the effect on the alkaloid level was
only marginal. However, regulation of the expres�
sion of this gene can be crucial for alkaloid pro�
duction in several species: in some transgenic N.
sylvestris lines overexpression of pmt gene increased
the nicotine content, whereas suppression of
endogenous PMT activity severely decreased the
nicotine content and induced abnormal mor�
phologies [75].
Scopolamine is 6,7β�epoxide derivative of
hyoscyamine, formed from hyoscyamine in a two�
step process via 6β�hydroxyhyoscyamine [78] by
enzyme hyoscyamine 6β�hydroxylase (H6H) which
can be classified as 2�oxoglutarate�dependent
dioxygenase (Fig. 3). The enzyme was purified and
characterized from H. niger [79]. The cDNA encod�
ing H. niger H6H has been isolated by Matsuda et
al. [80]. Additionally, H6H cDNA was cloned from
several other scopolamine�producing Solanaceae
species e.g. A. baetica [81], A. belladonna [82],
Anisodus tanguticus [83] etc. Additionally, tropinone
reductase, which catalyzes an earlier reaction of
scopolamine biosynthesis in H. niger, has been
cloned [84]. H6H gene from H. niger was placed
under the control of 35S promoter and introduced
to A. belladonna using A. rhizogenes. The obtained
hairy roots contained up to five�fold higher con�
centrations of scopolamine than wild�type cul�
tures [85]. Hyoscyamine was almost completely
ISSN 0564–3783. Цитология и генетика. 2010. № 170
Y.V. Sheludko
Fig. 3. Conversion of hyoscyamine (1) to scopolamine (2) by
enzyme hyoscyamine 6β�hydroxylase (H6H)
converted to scopolamine in the leaves of trans�
genic A. belladonna plants expressing h6h gene.
The level of scopolamine in the leaves reached up to
1.2 % DW [86]. Later, 35S�h6h gene was intro�
duced into H. muticus producing high amounts of
tropane alkaloids (up to 6 % of the dry weight in
the leaves of mature plant). The best selected
transgenic line produced 17 mg/L scopolamine,
although conversion of hyoscyamine to scopo�
lamine was still incomplete. In these examples
overexpression of a single gene in the pathway has
often led to an improved accumulation of the more
valuable end product.
Further experiments included simultaneous
overexpression of genes encoding PMT and the
downstream H6H in H. niger hairy root cultures. It
resulted in accumulation of significantly higher
amounts of scopolamine (up to 411 mg/L,) in
hairy root lines expressing both pmt and h6h genes
compared with the control cultures (app. nine
times more than that in the wild type) and trans�
genic lines harboring only one of the mentioned
genes (more than two times higher level of scopo�
lamine as compared with the best single�gene
transgenic lines) [87].
Biotransformation was reported to be an alter�
native way for scopolamine production using non�
hyoscyamine�producing transgenic systems fed
with precursor hyoscyamine. Hairy roots of N.
tabacum transformed with 35S�h6h gene have been
studied for the production of scopolamine and
nicotine alkaloids after feeding the cultures with
hyoscyamine. In the optimal conditions the most
productive clones of N. tabacum hairy roots con�
verted up to 45 % of exogenous hyoscyamine to
scopolamine; up to 85 % of the total scopolamine
was released to the culture medium [88]. Recently,
the protocol for bioconversion of hyoscyamine
into scopolamine in bioreactor with N. tabacum
cell suspension cultures was reported [89].
Functionally active H6H was obtained after het�
erologous expression of h6h gene from Brugmansia
candida in S. cerevisiae [90].
Conclusions and future perspectives. In conclu�
sion, cloning and heterologous overexpression of
genes coding for several key enzymes of secondary
metabolism often allowed considerable increasing
of the level of valuable end product. The next step
on the way to obtaining the commercial amounts
of metabolite included correct choice of expres�
sion system and adaptation of the process to biore�
actor scale. However, the efficient control of desired
product synthesis requires a complete knowledge
of all the steps in biosynthetic pathway, regulation
mechanisms and cloning of the respective genes. It
is difficult to forecast the results of introduction
into plant genome of a single or reduced number of
genes. Their overexpression may cause appearance
of multiple rate�limiting steps and did not enhance
production of desirable metabolite. It is necessary
to consider the processes involved in the regulation
of the whole pathway and interconnecting cellular
pathways. Alternatively, translocation of gene clus�
ter encoding the enzymes responsible for sequence
of biochemical conversation in non�plant expres�
sion system can result in creation of highly effi�
cient productive complex.
Ю.В. Шелудько
СОВРЕМЕННЫЕ ДОСТИЖЕНИЯ
БИОТЕХНОЛОГИИ
И ГЕНЕТИЧЕСКОЙ ИНЖЕНЕРИИ
РАСТЕНИЙ ДЛЯ ПОЛУЧЕНИЯ
ВТОРИЧНЫХ МЕТАБОЛИТОВ
С давних времен растения использовались людь�
ми не только как пищевые культуры, но и для получе�
ния разнообразных химических соединений. Несмот�
ря на современное развитие химических методов син�
теза и микробиологических биотехнологий, растения
остаются важнейшим и незаменимым источником
биологически активных веществ. В обзоре мы сопо�
ставили возможности и перспективы использования
современных методов генетической инженерии в
фармацевтической биотехнологии и привели приме�
ры новейших систем, используемых для получения
некоторых ценных натуральных продуктов – артеми�
зинина, паклитаксела и скополамина.
Ю.В. Шелудько
СУЧАСНІ ДОСЯГНEННЯ
БІОТЕХНОЛОГІЇ
ТА ГЕНЕТИЧНОЇ ІНЖЕНЕРІЇ
РОСЛИН ДЛЯ ОТРИМАННЯ
ВТОРИННИХ МЕТАБОЛІТІВ
З давніх часів люди використовували рослини не
тільки як харчові культури, але і для отримання різно�
манітних хімічних сполук. Незважаючи на сучасний
розвиток методів хімічного синтезу й мікробіологіч�
них біотехнологій, рослини залишаються найважливі�
шим і незамінним джерелом біологічно активних ре�
човин. В огляді ми зіставили можливості й перспекти�
ви використання сучасних методів генетичної інжене�
ІSSN 0564–3783. Цитология и генетика. 2010. № 1 71
Recent advances in plant biotechnology and genetic engineering for production
рії в фармацевтичній біотехнології і навели приклади
сучасних біотехнологічних систем, які застосовують
для одержання деяких цінних натуральних продуктів –
артемізініна, паклітаксела і скополаміна.
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ІSSN 0564–3783. Цитология и генетика. 2010. № 1 75
Recent advances in plant biotechnology and genetic engineering for production
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| id | nasplib_isofts_kiev_ua-123456789-66683 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0564-3783 |
| language | English |
| last_indexed | 2025-12-01T08:12:38Z |
| publishDate | 2010 |
| publisher | Інститут клітинної біології та генетичної інженерії НАН України |
| record_format | dspace |
| spelling | Sheludko, Y.V. 2014-07-20T14:48:02Z 2014-07-20T14:48:02Z 2010 Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites / Y.V. Sheludko // Цитология и генетика. — 2010. — Т. 44, № 1. — С. 65-75. — Бібліогр.: 90 назв. — рос. 0564-3783 https://nasplib.isofts.kiev.ua/handle/123456789/66683 575.854 + 547.94 + 582.923.5 For a long time people are using plants not only as crop cultures but also for obtaining of various chemicals. Currently plants remain one of the most important and essential sources of biologically active compounds in spite of progress in chemical or microbial synthesis. In our review we compare potentials and perspectives of modern genetic engineering approaches for pharmaceutical biotechnology and give examples of actual biotechnological systems used for production of several promising natural compounds: artemisinin, paclitaxel and scopolamine. З давніх часів люди використовували рослини не тільки як харчові культури, але і для отримання різноманітних хімічних сполук. Незважаючи на сучасний розвиток методів хімічного синтезу й мікробіологічних біотехнологій, рослини залишаються найважливішим і незамінним джерелом біологічно активних речовин. В огляді ми зіставили можливості й перспективи використання сучасних методів генетичної інженерії в фармацевтичній біотехнології і навели приклади сучасних біотехнологічних систем, які застосовують для одержання деяких цінних натуральних продуктів – артемізініна, паклітаксела і скополаміна. С давних времен растения использовались людьми не только как пищевые культуры, но и для получения разнообразных химических соединений. Несмотря на современное развитие химических методов синтеза и микробиологических биотехнологий, растения остаются важнейшим и незаменимым источником биологически активных веществ. В обзоре мы сопоставили возможности и перспективы использования современных методов генетической инженерии в фармацевтической биотехнологии и привели примеры новейших систем, используемых для получения некоторых ценных натуральных продуктов – артемизинина, паклитаксела и скополамина. en Інститут клітинної біології та генетичної інженерії НАН України Цитология и генетика Обзорные статьи Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites Сучасні досягнeння біотехнології та генетичної інженерії рослин для отримання вторинних метаболітів Современные достижения биотехнологии и генетической инженерии растений для получения вторичных метаболитов Article published earlier |
| spellingShingle | Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites Sheludko, Y.V. Обзорные статьи |
| title | Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites |
| title_alt | Сучасні досягнeння біотехнології та генетичної інженерії рослин для отримання вторинних метаболітів Современные достижения биотехнологии и генетической инженерии растений для получения вторичных метаболитов |
| title_full | Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites |
| title_fullStr | Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites |
| title_full_unstemmed | Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites |
| title_short | Recent advances in plant biotechnology and genetic engineering for production of secondary metabolites |
| title_sort | recent advances in plant biotechnology and genetic engineering for production of secondary metabolites |
| topic | Обзорные статьи |
| topic_facet | Обзорные статьи |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/66683 |
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