Available online at www.sciencedirect.
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ScienceDirect
Novel fermentation processes for manufacturing plant natural
products
Jingwen Zhou1, Guocheng Du1,2 and Jian Chen1
Microbial production of plant natural products (PNPs), such as
terpenoids, flavonoids from renewable carbohydrate
feedstocks offers sustainable and economically attractive
alternatives to their petroleum-based production. Rapid
development of metabolic engineering and synthetic biology of
microorganisms shows many advantages to replace the
current extraction of these useful high price chemicals from
plants. Although few of them were actually applied on a large
scale for PNPs production, continuous research on these high-
price chemicals and the rapid growing global market of them,
show the promising future for the production of these PNPs by
microorganisms with a more economic and environmental
friendly way. Introduction of novel pathways and optimization
of the native cellular processes by metabolic engineering of
microorganisms for PNPs production are rapidly expanding its
range of cell-factory applications. Here we review recent
progress in metabolic engineering of microorganisms for the
production of PNPs. Besides, factors restricting the yield
improvement and application of lab-scale achievements to
industrial applications have also been discussed.
Addresses
1
Key Laboratory of Industrial Biotechnology, Ministry of Education,
School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi,
Jiangsu 214122, China
2
National Engineering Laboratory for Cereal Fermentation Technology
(NELCF), Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122,
China
Corresponding authors: Zhou, Jingwen (zhoujw1982@jiangnan.edu.cn),
Chen, Jian (jchen@jiangnan.edu.cn)
Current Opinion in Biotechnology 2014, 25:17–23
This review comes from a themed issue on Analytical biotechnology
Edited by Frank L Jaksch and Savaş Tay
S0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.copbio.2013.08.009
Introduction
Plant natural products (PNPs) are chemical compounds or
substances produced by plants, found in nature that usually
has a pharmacological or biological activity. On the basis of
both traditional medicine and plant biochemistry studies,
more and more PNPs were identified for diverse appli-
cations, especially in nutritional and pharmaceutical appli-
cations. These small molecules provide the source and
inspiration for the majority of FDA-approved agents and
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continue to be one of the major sources of inspiration for
drug discovery [1]. The metabolites discovered in plants
may largely avoid the side effect because they must
accumulate in living cells [2].
Most of these compounds can only be only harvested from
their natural sources, which can be time consuming,
expensive and wasteful on the natural resources. A majority
of PNPs with promising applications only exist in their
original plants at a level of microgram to milligram per
kilogram of dry biomass, or even less [3]. Large application
of these PNPs would soon destroy these plant commu-
nities, which even become extinct. Existence of structural
analogues could also severely aggravate the purification
process, leads to extra increase in the costs [4].
Development of novel fermentation processes for man-
ufacturing PNPs on the basis of the metabolic engineer-
ing of microorganisms, has emerged recently as an
interesting and commercially attractive approach due to
several advantages including the utilization of environ-
mentally friendly feedstocks, low energy requirements,
and low waste emission [5]. Recent progress of several
important groups of PNPs that could now be produced by
fermentation processes was summarized here. Factors
restricting the yield improvement and application of
lab-scale achievements to industrial applications have
also been discussed.
Terpenes
Terpenes are a large and diverse class of PNPs that could
be biosynthesized from units of isoprene. Terpenes may
be classified by the number of isoprene units in the
molecule, including hemiterpenes, monoterpenes, ses-
quiterpenes, diterpenes, among others (Figure 1). The
most intensively investigated terpenes for the metabolic
engineering production by microorganisms include arte-
misinin (sesquiterpene) [6], taxol (diterpenes) [7], and
lycopene (tetraterpene) [8], among others.
Among all of these terpenoids, artemisinin attracts the
most intensive interests and are now close to the terminal
market. In 2013, on the basis of the previous systematic
attempts to improve the production of artemisinin,
researchers from Amyris Inc. and other collaborative insti-
tutes, introduced a complete biosynthetic pathway, in-
cluding the discovery of a plant dehydrogenase and a
second cytochrome that provide an efficient biosynthetic
route to artemisinic acid, with fermentation titers of 25 g/L
of artemisinic acid. Combining with an efficient and
Current Opinion in Biotechnology 2014, 25:17–23
18 Analytical biotechnology
Figure 1
D-Glucose
Glyceraldehyde-3-P
MEP/DOXP pathway
Pyruvate 1-Deoxy-D-xylulose-5-P
Acetyl-CoA 2-C-Methyl-D-erythritol-4-P
HMG-CoA Mevalonate
Mevalonate pathway
Squalene
Triterpenes
Biosynthesis of terpenes with enzymes from different origins. The profile was abstracted from the metabolic engineering of S. cerevisiae for the
heterologous biosynthesis of terpenes. MEP/DOXP pathway: 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway, which is
a non-mevalonate pathway existed in plants and most of bacteria. Elements in green represent those metabolites or enzymes are mostly originally
existed in plants only.
scalable chemical process for the conversion of artemisinic
acid to artemisinin, both scientists and engineers are
excited to see that the production of the anti-malaria drug
will soon be finally applied on industrial scale [6].
A majority of the strategies to improve the biosynthesis of
terpenes are firstly developed in artemisinin metabolic
engineering. Some of the general means of enhancing the
terpenoids production include: first, combination of MVA
and MEP pathways. By introducing the MVA pathway
from Saccharomyces cerevisiae, the a-farnesene yield was
increased by 317-fold and reached 380 mg/L [9]. Second,
truncated expression of Hmg1p. The HMG-CoA
reductase is the major bottleneck for tepenoids pro-
duction. Overexpression of the truncated reductase
(tHMG1) could significantly improve the production of
sesquiterpenes [10] and monoterpenes [11] in S. cerevisiae.
Third, regulation of Hmg2p. The instability of Hmg2p is
a bottleneck for the terpenoids production in S. cerevisiae
[12]. A Hmg2p with a point mutation of K6R could
improve the stability of the protein and improve the yield
of cineole [13]. Fourth, reduce the consumption of iso-
prenes to biomass. Introduction of an ERG20 with a
Current Opinion in Biotechnology 2014, 25:17–23
Isopentenyl-PP Dimethylallyl-PP
OPP Monoterpenes
Geranyl-PP
ERG9
OPP Sesquiterpenes
Farnesyl-PP
Diterpenes
OPP
Geranylgeranyl-PP
Tetraterpenes
Phytoene
Current Opinion in Biotechnology
mutation of K197E could significantly enhance the
GPP supply and increased the geraniol production by
10-fold [14]. Replacing the original ERG9 promoter with
the one from HXT2, combining with overexpression of a
a-santalene synthase from Clausena lansium, the yield of
a-santalene was improved to 92 mg/L [15]. Fifth, mod-
ular engineering of heterogenous pathways. By a multi-
variate modular engineering of miltiradiene biosynthesis
pathway from Salvia miltiorrhiza and the original MVA
pathway, the diterpene production was improved to
365 mg/L [16].
However, besides the artemisinin and its precursors, the
yield of most of other terpenes are still very low and still far
away from the industrial application. The situation is even
worse for most of the monoterpenes. For example, with a
serious of pathway optimization, the final yield of linalool
in S. cerevisiae could only produce no more than 150 mg/L
[11]. It should be caused by that most of the microorgan-
isms have low tolerance to these monoterpenes [17]. A
recent breakthrough for the monoterpene production is the
a-pinene, which is a kind of monoterpene that has prom-
ising application as biofuel. By overexpressing a native
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 Plant natural products production by fermentation Zhou, Du and Chen 19
geranyl diphosphate synthase (IspA) from Escherichia coli
and a geranyl diphosphate synthase (GPPS2), co-expres-
sing an a-pinene synthase (Pt30) from Pinus taeda in an E.
coli BL21 (DE3) with a heterologous hybrid mevalonate
pathway, the yield of a-pinene achieved to 0.97 g/L by a
fed-batch strategy [18].
Phenylpropanoids
Phenylpropanoids are a diverse family of organic com-
pounds and can be further divided into coumarins, fla-
vonoids, lignans, strylpyrones, tannins, stilbenoids,
among others (Figure 2). Phenylpropanoids comprise a
highly diverse family of secondary plant metabolites in
plants. The diverse bioactivities of phenylpropanoids
have drawn attention to personal health applications [19].
Microbial production of stilbene now attracts more and
more attention. Trans-resveratrol has already found to
play important roles in the prevention of cardiovascular
diseases and may also provide some protection against
certain types of cancer and diabetes [20]. The first report
of the successful microbial production of resveratrol
appeared in 2003. By introduction of a 4-coumarate:CoA
ligase (4CL) from a hybrid poplar (Populus trichocar-
pa  Populus deltoides) and a stilbene synthase (STS) from
grapevine (Vitis vinifera) in S. cerevisiae, a trace amount of
1.45 mg/L of resveratrol was obtained from the precursor
p-coumaric acid [21]. Since the yield of resveratrol in
yeast could only achieve to a very low level, most of the
following studies used E. coli as the host microorganism.
By screening of combinations with different strains, pro-
moters and enzymes, the resveratrol yield was improved
to 2.31 g/L with p-coumaric acid as precursor and supple-
mentary of cerulenin to inhibit the malonyl-CoA com-
petitive pathway for fatty acids biosynthesis [22]. This
is the highest report of resveratrol according to the current
reports. In December 2012, Swiss biotech firm Evolva,
bought Danish biotech firm Fluxome’s yeast derived
resveratrol business for about 550 000 Euros, further
spurred the global researchers focusing on the similar
PNPs.
Most of the attempts use cinnamic acid or coumaric acid
as precursor for phenylpropanoids biosynthesis. Both
cinnamic acid and coumaric acid can now be only econ-
omically produced from petroleum industry. Their low
water solubility also restricted their initial concentration
from industrial applications. Therefore, the production of
phenylpropanoids from trypsin/phenylalanine or even
glucose, is of great interests for both academic and
industrial researchers. It was generally regarded that
the bottleneck is the low enzyme activity of tyrosine/
phenylalanine ammonia-lyase (PAL/TAL) and the re-
construction of the long pathway from central metabolism
to phenylpropanoids [23]. Expression of TAL/PAL with
higher catalysis activity could significantly enhance the
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production of different phenylpropanoids from tyrosin/
phenylalanine or even glucose [24–26].
The malonyl-CoA, which is another precursor of phenyl-
propanoids, is also a limiting factor. Since overexpression
of autologous ACC in E. coli could result in the hindering
of cell growth, researchers find out that introduction of an
ACC from Corynebacterium glutamicum could partially
solve the problem [27]. Fusion expression of N-terminal
of BPL from E. coli, the C-terminal of BPL from Photo-
rhabdus luminescens and the ACC from C. glutamium, the
production of naringenin and pinocembrin were
improved to 119 mg/L and 429 mg/L, respectively [28].
An and Kim developed another route for the efficient
supply of malonyl-CoA in E. coli by introducing matB and
matC from Rhizobium trifolii. The two genes encoding two
proteins that could uptake the malonate and convert it
into malonyl-CoA in E. coli [29]. On the basis of the
analysis results from a genome-scale model of E. coli,
several genes associated with malonyl-CoA were ident-
ified and modified, improving the nargingenin yield from
p-coumaric acid to 474 mg/L without addition of cerule-
nin [30].
According to the high prices of the above-mentioned
phenylpropanoids, it seems that most of them are now
competitive with the extraction method. However, the
main problems restricted the application of the above
achievements include: First, for unknown reasons, most
of the processes with high yield need two independent
culture processes for either cell growth or enzyme cata-
lysis [22]. Second, most of the high yield could only
achieved with the coumaric acid or cinnamic acid as
precursor, which are low water soluble and high-price.
De novo production of naringenin or pinocembrin could
now only achieve to 108.8 mg/L (in S. cerevisiae [31],
84 mg/L with cerulenin in E. coli [32]) and 40 mg/L
(in E. coli [26]), respectively. Therefore, the precondition
for the industrial application of phenylpropanoids pro-
duction by microorganisms, is to establish a platform that
could use glucose or other low price carbon source to
produce these PNPs in a one-pot culture process with a
high yield.
Alkaloids
Alkaloids are a group of natural products with mostly
basic nitrogen atoms. Alkaloids are the bioactive com-
ponents of most of addictive foods and drugs, such as
coffee, tea, tobacco, cocoa, and opium poppy. A lot of
alkaloids are also the active components of traditional
herbal plants, such as the cocaine, vincristine/cathar-
anthine, atropine, and quinine. Most of these alkaloids
were used as drugs for a long history within the corre-
sponding plants.
Unlike phenylpropanoids and terpenes, alkaloids shared
several totally different pathways derived from the
Current Opinion in Biotechnology 2014, 25:17–23
20 Analytical biotechnology

Figure 2

(a)

O SCoA
Acetyl-CoA

Cell membrane accABCD

COOH COOH
COOH
matA matC fabD
Malonyl-ACP
O OH O OH SCoA
O
Malonic acid Malonic acid Malonyl-CoA
fabB

fabF
Fatty acids Acetoacetyl-ACP Acetyl-ACP

(b) Erythrose 4-phosphate Phosphoenolpyruvate

aroF wt

Chorismate

tyrA pheAfbr
L-Phenylalanine L-Tyrosine
PAL TAL
OH

Lignins
Stilbenes
Lignans
C4H Coumarins
HOOC HOOC
Cinnamic acid Coumaric acid
4CL 4CL
OH

CoASOC CoASOC
4-Cinnamoyl-CoA 4-Coumaryol-CoA
Malonyl-CoA Malonyl-CoA

CHS CHS OH

HO OH HO OH

OH O OH O
Pinocembrin chalcone Naringenin chalcone Aurones
CHI CHI OH

HO O HO O

OH O OH O
Pinocembrin Naringenin

Flavonoids/Isoflavonoids Tannins
Current Opinion in Biotechnology

Current Opinion in Biotechnology 2014, 25:17–23 www.sciencedirect.com

 Plant natural products production by fermentation Zhou, Du and Chen 21
primary metabolism precursors. For their intensive bioac-
tivities, enhancement of the production of alkaloids has
attracted more and more interests from both academic
and industrial fields [33]. However, until recently, the
successful productions of alkaloids in microorganisms are
barely successful. A majority of the metabolic engineering
of microorganisms for alkaloids production are benzyli-
soquinoline alkaloids (BIAs) [34]. Most of the works to
improve the production of BIAs could only be achieved in
plant tissue or cells, especially in Coptis japonica [35].
Market capacity is a dominant factor to decide the fate of
the metabolic engineering studies on a specific PNP.
Compare to artemisinin, lycopene and resveratrol, most
of alkaloids has only very limited market capacities. This
may be the main reason why the studies about these
PNPs are limited. Some of them, for example, like
vinblastine (Velbe1, by Eli Lilly), though the extraction
is difficult and the price is high, chemists developed
complicated and efficient stereochemistry route to syn-
thesize the molecule on industrial scale. In 2008, Minami
et al. reconstructed a BIA biosynthesis pathway with a
monoamine oxidase (MAO) from M. luteus and four other
genes from C. japonica. The artificial pathway could
produce 55 mg/L of reticuline from dopamine [36]. Some
of the pathways need a cytochrome P450 monooxygenase
and a corresponding reductase, which could not be
expressed in E. coli. Utilization of S. cerevisiae could solve
this problem since it could express some active cyto-
chrome P450 monooxygenases. By introducing a cyto-
chrome P450 monooxygenase and a corresponding
reductase in S. cerevisiae, it could produce reticculine from
(R,S)-norlaudanosoline [34].
All of these above mentioned methods require expensive
precursor from chemical synthesis as precursor, make
them difficult to be applied on industrial scale. To avoid
the disadvantages, some of researchers introduced a series
of enzymes for alkaloids production, including tyrosinase,
some genes that could convert tyrosine to L-dopa from
Ralstonia solanacearum, L-dopa decarboxylase from Pseu-
domonas putida [37]. The strain could produce 46 mg/L of
reticuline from glycerol. Though the yield was still very
low (0.15%), the final concentration has already surpassed
the original concentration in Lindera aggregata [38].
From the above research works, it can be seen both the
diversity and the final yield of alkaloids production in
microorganisms were much less comparable to other two
groups of PNPs. Besides the limited economic prospects
of this group of PNPs, from the pure academic view, we
can conclude that this should be caused by several
(Figure 2 Legend) Biosynthesis of phenylpropanoids with enzymes from different origins. (a) Heterologous pathways for malonyl-CoA biosynthesis in
E. coli. (b) The profile was abstracted from the metabolic engineering of E. coli for the heterologous biosynthesis of phenylpropanoids. PAL:
phenylalanine ammonia lyase; TAL: tyrosine ammonia lyase; 4CL: 4-coumarate:CoA ligase; CHS: chalcone synthase, CHI: chalcone isomerase.
Elements in green represent those metabolites or enzymes are mostly originally existed in plants only.
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reasons: first, the diverse metabolic pathways for alkaloids
biosynthesis in their original plants; second, potential
toxicity or other unknown bioactivities that may interrupt
the primary metabolism of host microorganisms for meta-
bolic engineering. Due to the highly diverse in chemical
structures, biosynthesis pathways and their physiological
impacts on microorganisms, the high efficient production
of alkaloids remains to be the most challenging task
among those PNPs.
Perspectives
On the basis of the above summary of the development of
fermentation processes form PNPs, some of the excited
progress has been found. Unfortunately, the good news or
substantial advances are really few. Although the prospect
for the production of PNPs by fermentation processes on
industrial scale is promising and attractive, there are
challenges that we should face. According to the current
status of the field, the following issues should be well
identified and solved.
First, identification and characterization of enzymes
involved in the specific PNP biosynthesis. Rapid devel-
opment of high-throughput sequencing technologies
greatly facilitates the discoveries of new genes and path-
ways in plants [39]. Combination of genome sequencing
and RNA-Seq results in different plant tissues within
different growth periods makes the gene mining easier
than before [40]. Second, rational or semi-rational modi-
fication of enzymes in plants to improve their compat-
ibility in host microorganisms. Achievements in the
protein structure analysis and prediction greatly facilitate
the protein engineering [41]. Combining with the high-
throughput robots, researchers could now perform greatly
more screenings of proteins with specific characteristics
on the basis of the rational or semi-rational strategies [42].
Third, design of more stable integrative and non-indu-
cible expression systems. From the current works about
PNPs, it can be summarized that most of the works are on
the basis of plasmids and inducible expression systems,
which need antibiotics and inducers for reliable
expressions [26,43]. Multiple targets genome manipula-
tion of microorganisms is still a bottleneck for reconstruc-
tion of complicated design of metabolic/regulation
pathways. Invention of new genome engineering strat-
egies, such as TALENs [44], CRISPR/Cas9 [45] and
CRISPR/dCas9 [46] systems, would greatly facilitate
researchers to develop more advanced strategies within
shorter periods. Fourth, development of more reliable in
silico model-based pathway design programs to take
advantage of the development of the systems biology.
On the basis of the development of systems biology, more
Current Opinion in Biotechnology 2014, 25:17–23
22 Analytical biotechnology
and more genome-scale models were developed to
simulate/predict the function of metabolic networks
[47]. Some of them began to involve the regulation
networks to make them more reliable [48]. More and
more successful examples partially or completely on the
basis of in silico models appeared [30]. For the com-
plicated heterologous pathways for PNPs, it is believed
that the in silico model aided strategies would play more
and more important roles. Fifth, enhancement of the
tolerance of host microorganisms to those heterologous
PNPs. A majority of PNPs are good antiseptics.
Although most of them are not as toxic as common
antibiotics, existence of them above a threshold would
lead to the decreased growth of host microorganisms
[49]. This would acutely decrease the final production
of these PNPs. Though the best way seems to be that
we can enhance the tolerances by rational design on the
basis of the understanding of tolerance of host micro-
organisms to diverse PNPs [50], the truth is that the
information is very limited. Therefore, we propose two
substitution strategies, one is to develop the metabolic
engineering on the basis of some microorganisms that
could well resistant to these PNPs, and the other is to
enhance the tolerance of model microorganisms by
adaptive evolution.
Acknowledgements
This work was supported by grants from supported by the Major State Basic
Research Development Program of China (973 Program, 2012CB720806),
the National High Technology Research and Development Program of
China (863 Program, 2012AA022103), the National Natural Science
Foundation of China (31000807, 31370130), the Natural Science
Foundation of Jiangsu Province (BK2011004, BK2010150), the Program for
New Century Excellent Talents in University (NCET-12-0876), the
Foundation for the Author of National Excellent Doctoral Dissertation of
PR China (FANEDD, 2011046), and the Fundamental Research Funds for
the Central Universities (JUSRP51307A).
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Current Opinion in Biotechnology 2014, 25:17–23