Synthetic and Systems Biotechnology xxx (2017) 1e9

Contents lists available at ScienceDirect

Synthetic and Systems Biotechnology

journal homepage: http://www.keaipublishing.com/en/journals/synthetic-
and-systems-biotechnology/

Metabolic engineering for the microbial production of isoprenoids:

Carotenoids and isoprenoid-based biofuels
Fu-Xing Niu a, b, 1, Qian Lu a, b, 1, Yi-Fan Bu a, b, Jian-Zhong Liu a, b, *
a
Biotechnology Research Center and Biomedical Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
b
South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou
510275, China

a r t i c l e i n f o a b s t r a c t

Article history: Isoprenoids are the most abundant and highly diverse group of natural products. Many isoprenoids have
Received 28 April 2017 been used for pharmaceuticals, nutraceuticals, flavors, cosmetics, food additives and biofuels. Caroten-
Received in revised form oids and isoprenoid-based biofuels are two classes of important isoprenoids. These isoprenoids have
3 August 2017
been produced microbially through metabolic engineering and synthetic biology efforts. Herein, we
Accepted 9 August 2017
briefly review the engineered biosynthetic pathways in well-characterized microbial systems for the
production of carotenoids and several isoprenoid-based biofuels.
Keywords:
© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co. This
Carotenoids
Isoprenoid-based biofuel
is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
Metabolic engineering 4.0/).
Synthetic biology

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Carotenoid products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. b-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Zeaxanthin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.4. Astaxanthin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Isoprenoid-based biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.1. Hemiterpenoid-based biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.2. Monoterpenoid–based biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.3. Sesquiterpenoid–based biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction abundant and highly diverse (structurally and functionally) group

of natural products synthesized in almost all living organisms.
Isoprenoids, also called terpenoids or terpenes, are the most Many isoprenoids have been used for pharmaceuticals, nutraceut-
icals, flavors, cosmetics, food additives and biofuels. Isoprenoids are
usually classified into groups according the number of carbons:
* Corresponding author. Biotechnology Research Centre, School of Life Science, hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15),
Sun Yat-Sen University, Guangzhou 510275, China. diterpenes (C20), triterpenes (C30) and tetraterpenes (carotenoids,
E-mail address: [email protected] (J.-Z. Liu).
C40).
Peer review under responsibility of KeAi Communications Co., Ltd.
1
Contributed equally to this work.
All isoprenoids derive from isopentenyl diphosphate (IPP) and

http://dx.doi.org/10.1016/j.synbio.2017.08.001
2405-805X/© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001
2 F.-X. Niu et al. / Synthetic and Systems Biotechnology xxx (2017) 1e9

its isomer dimethylallyl diphosphate (DMAPP) (Fig. 1). They can be in archaea, fungi, plant cytoplasm and other eukaryotes. The DXP
produced by two metabolic pathways, the mevalonate pathway pathway is mostly found in bacteria and plant plastids. The MVA
(MVA or MEV) [1] and the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway initiates with the condensation of two acetyl-CoAs by
pathway (also called the 2-C-methyl-D-erythritol 4-phosphate thiolase to produce acetoacetyl-CoA. Subsequently, another acetyl-
pathway, MEP pathway) [2]. The MVA pathway is mainly present CoA is condensed with acetoacetyl-CoA to synthesize 3-hydroxy-3-

Fig. 1. Isoprenoid biosynthetic pathway. G3P: Glyceraldehyde 3-phosphate; DXP: 1-deoxy-D-xylulose-5-phosphate; MEP: 2-C-methyl-D-erythritol-4-phosphate; CDP-ME: 4-
diphosphocytidyl-2-C-methyl-D-erythritol; CDP-MEP: 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate; MEcPP: 2-C-methyl-D-erythritol-2,4-cyclodiphosphate; HMB-
PP: 4-hydroxy-3-mehtyl-butenyl 1-diphosphate; HMG-CoA: 3-hydroxy-3-methyl-glutaryl-CoA; Mev-P: Mevalonate 5-phosphate; Mev-PP: Mevalonate diphosphate; IPP: Iso-
pentenyl diphosphate; DMAPP: Dimethylallyl diphosphate; GPP: Geranyl diphosphate; FPP: Farnesyl diphosphate; GGPP: Geranylgeranyl diphosphate; Dxs: 1-deoxy-D-xylulose-5-
phosphate synthase; IspC: 1-D-deoxy-D-xylulose 5-phosphate reductoisomerase; IspD: 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase; IspE: 4-diphosphocytidyl-2-C-
methyl-D-erythritol kinase; IspF: 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase; IspG: 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase; IspH: 4-hydroxy-3-
methyl-2-(E)-butenyl-4-diphosphate reductase; Idi: Isopentenyl diphosphate isomerase; AtoB: Acetoacetyl-CoA synthase; HMGS: Hydroxymethylglutaryl-CoA synthase; HMGR:
Hydroxymethylglutaryl-CoA reductase; MK: Mevalonate kinase; PMK: Phosphomevalonate kinase; PMD: Mevalonate diphosphate decarboxylase; GPPS: GPP synthase; FPPS: FPP
synthase; GGPPS: GGPP synthase; TS: terpene synthase.

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001
 F.-X. Niu et al. / Synthetic and Systems Biotechnology xxx (2017) 1e9 3

methyl-glutaryl-CoA (HMG-CoA) by HMG-CoA synthase. Then, billion, with an expected increase to $1.81 billion by 2022 [7].
mevalonic acid is formed from HMG-CoA using NADPH as a cofactor
by HMG-CoA reductase. Two kinases, mecalonate kinase (MK) and 2.1. Lycopene
phosphomevalonate kinase (PMK), sequentially catalyze the
phosphorylation of mevalonate to produce mevalonate 5- Lycopene is one of the most widely used carotenoids in the
diphosphate (Mev-PP). The final step of the MVA pathway to healthcare product market owing to its excellent performance as an
form IPP is the ATP-driven decarboxylation catalyzed by mevalo- antioxidant and its great potential in the reduction of prostate
nate diphosphate decarboxylase (PMD). The isomerization of IPP by cancer risk in humans. With the development of metabolic engi-
isopentenyl diphosphate isomerase (Idi) leads to DMAPP formation neering, the heterologous expression of the lycopene biosynthetic
and the initiation of isoprenoid biosynthesis (Fig. 1). The overall pathway in Escherichia coli and Saccharomyces cerevisiae has
stoichiometry of the MVA pathway for synthesizing IPP from become a promising strategy for lycopene production.
glucose is given by equation (1). The following strategies have been used to improve lycopene
production in E. coli 1) overexpression of the rate-limiting enzyme's
1.5 Glucose þ 2 NADPH þ 6 NAD ¼ IPP þ CO2 þ 2 ADP þ 2 NADP þ 6 genes; 2) removal of the competing pathways; 3) introduction of a
NADH (1) heterologous MVA pathway; 4) cofactor engineering; 5) genome
modifications, including promoter replacement and chromosomal
The DXP pathway consists of seven enzymatic steps that convert evolution [8]. A shot-gun approach was used to screen native genes
glyceraldehyde 3-phosphate (G3P) and pyruvate to IPP and DMAPP that should be overexpressed in lycopene production. Over-
in a ratio of 5:1 [3]. The DXP pathway starts with the condensation of expression of the dxs, appY, crl and rpoS genes improved lycopene
G3P and pyruvate to produce DXP by DXP synthase (Dxs). This step is production [9]. Flux scanning based on enforced objective flux
crucial and is known as the rate-limiting step of the DXP pathway. (FSEOF) was successfully employed for the identification of gene
DXP is then converted to 2-C-methyl-D-erythritol-4-phosphate amplification targets for improving lycopene production. Co-
(MEP), 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME), 4- overexpression of the dxs, idi and mdh genes enhanced lycopene
diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate (CDP- production [10]. The deletions of gdhA and gpmAB improved lyco-
MEP), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP), 4- pene production [10]. Flux balance analysis also revealed that the
hydroxy-3-mehtyl-butenyl 1-diphosphate (HMB-PP), and IPP and deletions of aceF, fdhF and gdhA enhanced lycopene production by
DMAPP via the series of enzymatic reactions. The overall stoichi- 40% [11]. Zhou et al. reported that the zwf knockout increased
ometry of the DXP pathway for synthesizing IPP from glucose is lycopene production by 130% [12]. Introduction of a heterologous
given by equation (2). MVA pathway into E. coli increased the IPP supply, leading to an
increase in lycopene production [13e16]. Introduction of a heter-
Glucose þ 2 ATP þ 3 NADPH þ NAD ¼ IPP þ CO2 þ 2 ADP þ 3 ologous MVA pathway increased lycopene content up to 198 mg/g
NADP þ NADH (2) dry cell weight (DCW) from 68 mg/g DCW [15]. Our group also
reported that the chromosomal heterologous expression of the
From equations (1) and (2), it can be found that the theoretical optimized S. cerevisiae MVA pathway can further improve lycopene
maximum IPP yield on glucose via the DXP pathway (5/6 ¼ 0.83 C- production [16]. The promoter engineered E. coli LYCOP 20 pro-
mol/C-mol) is higher than that via the MVA pathway (5/9 ¼ 0.56 C- duced lycopene at 529.45 mg/L and 20.25 mg/g DCW in a fed-batch
mol/C-mol). However, the DXP pathway needs one mol more culture [16]. Combined modulating expression of sucAB, sdhABCD
NADPH and 2 mol more ATP than the MVA pathway, indicating that and talB with the regulatory part for increasing ATP and NADPH
the DXP pathway requires more energy and reducing equivalents. availability, and dxs, idi and crtB with the RBS libraries resulted in a
As most isoprenoids were originally discovered in plants, their significant increase in lycopene production to 3.52 g/L with a
extraction from plant materials remains a major production route. content of 50.6 mg/g DCW [17]. Zhu et al. applied the targeted
However, it becomes increasingly difficult to meet their growing engineering strategy to construct an engineered E. coli harboring
demand from plant extraction because of the slow growth rate and the MVA and DXP pathway that produced lycopene at 1.23 g/L
low isoprenoid content of plants. Microbes are an excellent alter- (34.3 mg/g DCW) in a 100-L fed-batch fermentation [18]. Kim et al.
native for overcoming this limitation, as they grow fast, require constructed an engineered E. coli co-expressing the DXP and MVA
little land/water resources, and naturally produce the building pathway that produced lycopene at 1.35 g/L (32 mg/g DCW) in a 2-L
blocks of all isoprenoids: IPP and DMAPP (Fig. 1). In recent years, fed-batch fermentation [19]. Plasmid-based overexpression of
quite a few isoprenoid compounds were overproduced in engi- genes has been the principal strategy for metabolic engineering.
neered microorganisms, including carotenoids, isopentanol, However, plasmid-based expression systems are not suitable
pinene, farnesene, limonene and bisabolene, etc. (Table 1). because of genetic instability and the requirement for constant
Herein, we briefly reviewed recent advances in the metabolic selective pressure to ensure plasmid maintenance. Thus, a chemi-
engineering of microorganisms for isoprenoid production, focusing cally induced chromosomal evolution (CIChE), which is a plasmid-
on carotenoids and isoprenoid-based biofuels. free and high gene copy expression system for engineering E. coli,
was first developed to overcome these drawbacks by Tyo [20]. We
2. Carotenoid products applied the CIChE strategy to construct a lycopene hyper-producer
E. coli that does not carry a plasmid or an antibiotic marker. The
Carotenoids are an important group of natural and liposoluble CIChE E. coli produced lycopene at 33.43 mg/g DCW [21]. De Mey's
pigments with multiple physiological and nutritional functions. group developed a new combinatorial multigene pathway assem-
They were found in plants, fungi, algae and bacteria, displaying bly approach based on Single Strand Assembly methods and Golden
yellow, orange or red color. Carotenoids are widely used as food Gate Assembly, and they applied this approach to optimize the
colorants, food and cosmetics additives, health supplements, ani- lycopene biosynthetic genes in E. coli overexpressing the MEP
mal feeds and nutraceuticals. At least 700 carotenoids have been pathway to obtain a lycopene hyper-producer E. coli that produced
characterized [4]. Carotenoids can be classified into C30, C40 and 448 mg/g DCW of lycopene in a shake flask fermentation [22]. This
C50 carotenoids [5]. More than 95% of known carotenoids are C40 yield is the highest value reported so far.
carotenoids [6]. The global carotenoids market in 2015 was $1.23 S. cerevisiae is another host strain of metabolic engineering for

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001
4 F.-X. Niu et al. / Synthetic and Systems Biotechnology xxx (2017) 1e9

Table 1
Production of isoprenoids by engineered microorganisms.

Isoprenoids Host Approach Culture conditions Yield/Titer References

produced

Lycopene E. coli Systematic (model-based) methods; Shake-flask fermentation 18 mg/g DCW [11]
Combinatorial (transposition-based) methods;
Gene knockout.
Lycopene E. coli Central metabolic genes knockout; Shake-flask fermentation 7.55 mg/g DCW [12]
Amplification of MEP pathway genes
Lycopene E. coli Overexpression of native dxs; Other Shake-flask fermentation 16.8 mg/L [13]
optimization methods (promoters, vectors,
strains)
Lycopene E. coli Introduction of a heterologous MVA pathway; Shake-flask fermentation 198 mg/g DCW [15]
Overexpressing Bacillus licheniformis idi
Lycopene E. coli Optimization of MVA pathway; Promoter Fed-batch fermentation 20.25 mg/g DCW [16]
engineering
Lycopene E. coli Increase ATP and NADPH; Engineering TCA Fed-batch fermentation 50.602 mg/g DCW [17]
modules; Overexpression of dxs\idi\crtE
Lycopene E. coli Application of the targeted engineering strategy Fed-batch fermentation 34.3 mg/g DCW [18]
Lycopene E. coli Co-expression of the DXP and MVA pathway Fed-batch fermentation 32 mg/g DCW [19]
Lycopene E. coli Application of CIChE Shake-flask fermentation 33.43 mg/g DCW [21]
Lycopene E. coli Optimization of the lycopene biosynthetic Shake-flask fermentation 448 mg/g DCW [22]
genes; Overexpressing the MEP pathway (dxs-
idi-ispDF)
Lycopene S. cerevisiae Combination of directed evolution and Fed-batch fermentation 24.41 mg/g DCW [23]
metabolic engineering strategy
Lycopene S. cerevisiae Combination of host engineering and pathway Fed-batch fermentation 55.56 mg/g DCW [25]
engineering
Lycopene Y. lipolytica Deletion of POX1 and GUT2 Shake-flask fermentation 16 mg/g DCW [26]
Lycopene S. avermitilis Activation of the silent lycopene synthetic gene Shake-flask fermentation 82 mg/g DCW [27]
cluster
b-cartoene E. coli Plasmid-expressing the lower MVA pathway Fed-batch fermentation 60 mg/g DCW [28]
and idi from S. cerevisiae, Plasmid-expressing
the upper MVA pathway from Enterococcus
faecalis, Bacillus subtilis dxs and fni, and GPPS2
from Abies grandis; Plasmid-expressing the b-
cartoene synthetic pathway.
b-cartoene E. coli Combined engineering of the MEP, the b- Fed-batch fermentation 3.2 g/L [29]
carotene synthetic, the TCA and the pentose
phosphate (PP) modules by artificial
modulation parts
b-cartoene E. coli Optimizing the biosynthetic pathway Fed-batch fermentation 2.0 g/L [30]
b-cartoene S. cerevisiae Decentralized assemble strategy Shake-flask fermentation 7.41 mg/g DCW [31]
b-cartoene S. cerevisiae Using the inducer/inhibiter-free sequential Fed-batch fermentation 20.79 mg/g DCW, 1156 mg/L [32]
control strategy to sequentially control the
expression of the carotenoid pathway, the MVA
pathway and the competitive squalene
pathway by glucose in the culture broth,
Zeaxanthin E. coli Optimization of the zeaxanthin biosynthetic Shake-flask fermentation 11.95 mg/g DCW [38]
pathway
Zeaxanthin E. coli Introduction of a dynamically controlled TIGR- Fed-batch fermentation 23.16 mg/g DCW [39]
mediated MVA pathway
Astaxanthin E. coli Chromosomal expressing the optimized Shake-flask fermentation 7.50 mg/g DCW [41]
synthetic pathway
Astaxanthin E. coli RBS-modulated expression of the astaxanthin Shake-flask fermentation 5.8 mg/g DCW [42]
biosynthetic genes
Astaxanthin E. coli Plasmid-overexpression of Pantoea ananatis 8.64 mg/g DCW [43]
crtEIB, Pantoea agglomerans crtYZ,
Brevundimonas sp. SD212 crtW and E. coli idi
Astaxanthin S. cerevisiae Introduction of codon-optimized Shake-flask fermentation 4.7 mg/g DCW [44]
Haematococcuspluvialis crtZ and bkt
Astaxanthin S. cerevisiae combinatorial metabolic engineering and Shake-flask fermentation 8.10 mg/g DCW [45]
protein engineering
Astaxanthin C. glutamicum Balanced expression of crtW and crtZ Shake-flask fermentation 0.4 mg/L/h [46]
Isoprene E. coli Introduction of MVA pathway, codon and RBS Shake-flask fermentation 1832 mg/L [48]
optimization, deleted nine relevant genes to
express ispS
Isoprene E. coli Chromosomal expressing the MVA lower 14 L fed-batch fermentation 60 g/L [49]
pathway; Plasmid-expression of the MVA upper
pathway; Plasmid-expression of mvk from
Methanosarcina mazei and isoprene synthase
gene from Populus alba
Isoprene E. coli Overexpression of MEP and MVA pathway; Fed-batch fermentation 24 g/L [50]
Plasmid-expressing mvk from Methanosarcina
mazei and isoprene synthase gene from Populus
alba
Isoprene S. cerevisiae Fed-batch fermentation 2527 mg/L [51]

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001
 F.-X. Niu et al. / Synthetic and Systems Biotechnology xxx (2017) 1e9 5

Table 1 (continued )

Isoprenoids Host Approach Culture conditions Yield/Titer References

produced

Dual metabolic engineering of cytoplasmic and

mitochondrial acetyl-CoA utilization
Isoprene S. cerevisiae Combining the two-level expression system Fed-batch fermentation 3.7 g/L [52]
and directed evolution of ISPS
Isopentenol E. coli Introduction of MVA pathway; Expressing Shake-flask fermentation 1.3 g/L [54]
BsNudF gene
Isopentenol E. coli Constructing the MVA IPP-bypass pathway Shake-flask fermentation 705 mg/L [56]
Isopentenol E. coli RBS engineering of nudB; Expressing the Idi- Shake-flask fermentation 2.23 g/L [57]
NudB fusion protein
Myrcene E. coli Co-overexpression of MVA pathway, AgGPPS Shake-flask fermentation 58.19 mg/L [58]
and ms from Quercus ilex L.
Myrcene E. coli Introducing the MVA lower pathway; Fed-batch fermentation 2.65 g/L [59]
Expressing the MVA upper pathway in
combination with AgGPPS and SabS1
Pinene E. coli Introduction of MVA pathway; Expressing Fed-batch fermentation 0.97 g/L [60]
AgGPPS-Pt30 fusion protein
Pinene E. coli Introduction of MVA pathway; Expressing Shake-flask fermentation 32.4 mg/L [61]
AgPS-AgGPPS fusion protein
Pinene E. coli Introduction of MVA pathway; Expressing Shake-flask fermentation 150 mg/L [62]
PSmut-AgGPPS fusion protein
Limonene E. coli Introduction of MVA pathway, Expressing the Shake-flask fermentation 435 mg/L [63]
AgGPPSeLS fusion protein
Farnesene E. coli Introduction of MVA pathway; Expressing the Shake-flask fermentation 380 mg/L [67]
codon-optimized FS-IspA fusion protein
Farnesene E. coli Application of In vitro reconstitution and Shake-flask fermentation 1.1 g/L [68]
targeted proteomics; Overexpression of Idi with
IspA and AFS in E. coli expressing synthetic MVA
pathway
Farnesene S. cerevisiae Introduction of the artificial acetyl coenzyme 200,000 L bioreactor 130 g/L [69]
biosynthetic pathway (contained Dickeya zeae fed-batch fermentation
aldehyde dehydrogenase (acylating),
Leuconostoc mesenteroides xylulose-5-
phosphate specific phosphoketolase and
Clostridium kluyveri phosphotransacetylase)
with the NADH-consuming HMG-CoA
reductase from Silicibacter pomeroyi
Bisabolene E. coli Co-expressing the codon-optimized AgBIS and Shake-flask fermentation 912 mg/L [70]
the optimized MVA pathway
Bisabolene S. cerevisiae Co-expressing the codon-optimized AgBIS and Shake-flask fermentation 994 mg/L [70]
the optimized MVA pathway
Bisabolene S. cerevisiae Screening the yeast knockout libraries; Co- Fed-batch fermentation 5.2 g/L [72]
expressing the MVA pathway and BIS gene
Farnesol E. coli Co-expressing ispA and the MVA pathway Shake-flask fermentation 135.5 mg/L [73]
Farnesol E. coli Overexpressing ispA, pgpB and the MVA Shake-flask fermentation 526.1 mg/L [74]
pathway
Farnesol S. cerevisiae Overexpressing the truncated HMG-CoA 5L fed-batch fermentation 145 mg/L [75]
reductase

carotenoid production. Xie et al. applied a combined directed 2.2. b-Carotene

evolution and metabolic engineering strategy to construct an
engineered S. cerevisiae that produced 1.61 g/L (24.41 mg/g DCW) of b-Carotene is a carotenoid compound that has been widely used
lycopene in a fed-batch fermentation [23]. Some distantly located in the industrial production of not only pharmaceuticals but also
genetic loci may have potential interactions with the target nutraceuticals, animal feed additives, functional cosmetics, and
pathway. The deletions of these distant genes (YPL062W, YJL064W, food colorants. b-Carotene functions as provitamin A, and it is
ROX1 and DOS2) improved carotenoid production in S. cerevisiae responsible for the synthesis of retinoids. b-Carotene is the cycli-
[24,25]. Chen et al. constructed an engineered S. cerevisiae by zation product of lycopene by lycopene b-cyclase (CrtY) (Fig. 1). The
combining host engineering (distant genetic loci and cell mating heterologous expression of the b-carotene biosynthetic genes in
types) with pathway engineering (enzyme screening and gene fine non-carotenogenic microbiology, e.g., E. coli and S. cerevisiae, has
tuning) for lycopene production, which produced 1.65 g/L become a main alternative means of b-carotene production. E. coli
(55.56 mg/g DCW) of lycopene in a 5-L bioreactor fed-batch co-overexpressing the optimized MEP pathway (Bacillus subtilis dxs
fermentation [25]. Yarrowia lipolytica is another yeast that has and fni, and GPPS2 from Abies grandis) and the MVA pathway pro-
been successfully used for lycopene production. The deletion of duced 3.2 g/L of b-carotene in a fed-batch fermentation [28]. ATP
POX1 and GUT2, which led to an increase in the size of lipid bodies, and NADPH are two important cofactors for terpenoid compounds.
significantly enhanced lycopene production (16 mg/g DCW) in Combined engineering of the MEP, the b-carotene synthetic, the
Y. lipolytica [26]. TCA and the pentose phosphate (PP) modules by artificial modu-
Streptomyces avermitilis has also been successfully used for lation parts resulted in a significant increase in the b-carotene yield.
lycopene production. After activating the silent lycopene synthetic The final strain, E. coli CAR005, produced 2.1 g/L b-carotene with a
gene cluster in S. avermitilis, 82 mg/g DCW of lycopene was pro- yield of 60 mg/g DCW [29]. After integrating the b-carotene
duced in a shake flask fermentation [27]. biosynthetic pathway into the E. coli genome and optimizing the

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001
6 F.-X. Niu et al. / Synthetic and Systems Biotechnology xxx (2017) 1e9

MEP, central metabolic pathway and b-carotene biosynthetic that is produced relative to the total carotenoid content. The CrtW
pathway, the engineered E. coli produced 2.0 g/L of b-carotene in and CrtZ enzymes from different sources show different activities
fed-batch fermentation [30]. Yu's group developed a decentralized and substrate specificities. Thus, optimal astaxanthin biosynthesis
assembly strategy to construct a controllable multigene pathway, requires careful control of the carbon flux along a cooperative
and then they applied this strategy to construct a controllable b- function of these two proteins. It has been suggested that astax-
carotene biosynthetic pathway in S. cerevisiae. The resulting strain anthin biosynthesis proceeds from b-carotene through hydroxyl-
produced 7.41 mg/g DCW of b-carotene [31]. They then established ation first, and then onto ketolation [40]. To increase the
an inducer/inhibiter-free sequential control strategy in S. cerevisiae astaxanthin percentage relative to the total carotenoid content, we
by combining a modified GAL regulation system and a HXT1 compared the conversion efficiency to astaxanthin in four CrtWs,
promoter-controlled squalene synthetic pathway [32]. They which had higher efficiency for astaxanthin production reported in
applied this strategy to sequentially control the expression of the literature, with recombinant E. coli cells that synthesizes zeax-
carotenoid pathway, the MVA pathway and the competitive squa- anthin due to the presence of the P. ananatis crtEBIYZ and found that
lene pathway by glucose in the culture broth, resulting in marked the Brevundimonas sp. SD212 crtW and P. ananatis crtZ genes are the
increase in b-carotene production, which reached 20.79 mg/g DCW best combination for astaxanthin production [41]. After tune-fining
[32]. the crt genes, an astaxanthin producer E. coli ASTA-1 that does not
carry a plasmid or antibiotic marker was constructed. The engi-
2.3. Zeaxanthin neered strain E. coli ASTA-1 produced 7.50 mg/g DCW of astax-
anthin with an astaxanthin ratio of 96.6% relative to the total
Zeaxanthin (3,30 -dihydroxyl-b-carotene) is a yellow oxygenated carotenoid content in a shake flask fermentation [41]. The ratio of
carotenoid composed of 40 carbon atoms that is used as a food astaxanthin to the total carotenoids (96.6%) is the highest value
additive and as a feed additive for fish (color enhancement for the reported to date. Balanced expression of the astaxanthin biosyn-
flesh) and poultry (yolk and skin pigmentation) [33]. Zeaxanthin thetic genes with a compact set of ribosome binding sites led to an
plays a critical role in preventing age-related macular degeneration astaxanthin accumulation of 5.8 mg/g DCW in E. coli [42]. Ma et al.
and cancer and may protect against age-related cataract formation identified and characterized the astaxanthin-producing ability of
[34,35]. The hydroxylation of each ring of b-carotene by b-carotene Sphingomonas sp. ATCC 55669 by complete genome sequencing,
hydroxylase (CrtZ) produces zeaxanthin (Fig. 1). Co-overexpression and then compared the astaxanthin biosynthetic efficiency of the
of the dsx and idi genes in engineered E. coli harboring the zeax- crt genes from different microorganisms in E. coli. The resulting
anthin biosynthetic pathway had an additive effect on zeaxanthin E. coli plasmid-expressing P. ananatis crtEIB, P. agglomerans crtYZ, B.
production, which reached 1.6 mg/g DCW [36]. It has been reported sp. SD212 crtW and E. coli idi produced 8.64 mg/g DCW [43].
that CrtZ is the rate-limiting step in zeaxanthin biosynthesis and a An astaxanthin producing S. cerevisiae was constructed by
higher expression level of crtZ should be required for zeaxanthin integrating two copies of the codon-optimized H. pluvialis crtZ and
production [37,38]. We compared Pantoea ananatis, Pantoea bkt in b-carotene producing S. cerevisiae. The engineered
agglomerans and Haematococcus pluvialis crtZ and reported that S. cerevisiae produced 4.7 mg/g DCW of astaxanthin in a shake-flask
P. ananatis crtZ is superior to those from P. agglomerans or culture [44]. The group recently applied combinatorial metabolic
H. pluvialis for zeaxanthin production [38]. E. coli BETA-1 containing engineering and protein engineering to markedly enhance astax-
pZSBA-2(P37-crtZPAN) produced 11.95 mg/g DCW of zeaxanthin anthin production S. cerevisiae, which reached 8.10 mg/g DCW in
[38]. To balance the expression of the multigene, the tunable shake-flask cultures [45].
intergenic region (TIGR)-mediated MVA pathway was introduced Recently, Corynebacterium glutamicum has been engineered for
into the zeaxanthin-producing strain, E. coli ZEAX, leading to an astaxanthin production, and it reached 1.6 mg/g DCW [46].
increase in zeaxanthin production [39]. However, IPP and FPP are In addition, the titer of astaxanthin is much lower than that of
toxic when they accumulate in E. coli. To avoid the accumulation of other carotenoids (lycopene, b-carotene and zeaxanthin). Because
IPP or FPP, a dynamically controlled TIGR-mediated MVA pathway very few carotenoids were detected in our engineered strain E. coli
was introduced into the zeaxanthin producing strain, E. coli ZEAX, ASTA-1, we guess that the lower astaxanthin yield may be because
markedly enhancing its zeaxanthin production, which achieved the recombinant enzyme (b-carotene hydroxylase and ketolase) or
722.46 mg/L (23.16 mg/g DCW) in a 5.0-L fed-batch fermentation product of their enzymatic reaction affects the formation of the
[39]. carotenoid precursors upstream of phytoene. Therefore, further
efforts focused on astaxanthin production should be carried out.
2.4. Astaxanthin
3. Isoprenoid-based biofuels
Astaxanthin is a highly valued keto-carotenoid with strong
antioxidant activity and singlet oxygen quenching ability. The Methyl branching and cyclic structures are commonly observed
pathway from b-carotene to astaxanthin is a crucial step in the in isoprenoids. The methyl branching structure lowers the freezing
synthesis of astaxanthin. This pathway requires two bifunctional point significantly. The cyclic structures increase the energy density
enzymes: b-carotene hydroxylase CrtZ to add hydroxyl functional and are generally considered valuable features for jet fuels. In
groups to carbons 3 and 30 of b-carotene and b-carotene ketolase recent years, some isoprenoids have been tested and produced as
CrtW to add keto functional groups to carbons 4 and 40 of b-caro- potential diesel and gasoline fuel alternatives because of their
tene (Fig. 1). The two enzymes are bifunctional proteins with lower hygroscopy, higher energy content and good fluidity at low
respect to their substrate specificity. CrtZ can convert not only b- temperatures.
carotene to zeaxanthin but also canthaxanthin to astaxanthin. CrtW
is capable of converting not only b-carotene but also zeaxanthin. 3.1. Hemiterpenoid-based biofuels
Consequently, the heterologous expression of crtZ and crtW in a b-
carotene-producing strain results in the accumulation of eight in- Isoprene (C5H8) is the simplest isoprenoid. It is used to produce
termediates (echinenone, canthaxanthin, adonirubin, b-cryptox- millions of tons of rubber annually and has been suggested as a
anthin, zeaxanthin, adonixanthin, 3-hydroxyechinenone and 30 - liquid fuel [47]. Co-overexpression of Populus trichocarpa codon-
hydroxyechinenone), which affects the percentage of astaxanthin optimized isoprene synthase gene ISPS and the MVA pathway

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001
 F.-X. Niu et al. / Synthetic and Systems Biotechnology xxx (2017) 1e9 7

genes in the 9-gene knockout E. coli AceCo improved isoprene mutant PSD380A. They expressed the PS mutant and GPPS from
production, reaching 1832 mg/L in a shake-flask culture [48]. A. grandis in the engineered E. coli harboring the MVA pathway and
Plasmid-expression of the upper pathway of MVA in concert with achieved 150 mg/L pinene in a shake-flask fermentation [62]. An
the P. alba isoprene synthase gene ISPS plus the mevalonate kinase engineered E. coli expressing the MVA pathway, codon-optimized
and phosphogluconolactonase gene in E. coli integrated the lower GGPS from A. grandi and codon-optimized limonene synthase
pathway of MVA from S. cerevisiae and resulted in production of from Mentha spicata on one plasmid produced 400 mg/L limonene
60 g/L isoprene with a mass yield of isoprene from glucose in a 14-L in a shake-flask fermentation [63].
fed-batch fermentation [49]. Fed-culture of the engineered E. coli Monoterpenes have been reported to be highly toxic, resulting
overexpressing the synergistic dual pathway of MVA and MEP in low microbial production of monoterpene. Overexpression of
resulted in the production of 24.0 g/L isoprene with a yield of efflux pump or tolerance-enhancing genes has become a common
0.267 g/g [50]. The isoprene synthase gene has also been intro- strategy for improving monoterpene production [64,65]. Over-
duced in S. cerevisiae for isoprene production. In recent years, expression of the efflux pump gene (YP-692684) from Alcanivorax
organelle engineering of yeast has attracted increasing attention in borkumensis significantly improved tolerance and enhanced limo-
the biosynthesis of chemicals. Dual metabolic engineering of the nene production [64]. This tolerance engineering strategy has also
cytoplasmic and mitochondrial acetyl-CoA increased isoprene successfully been applied for improving the production of iso-
production in S. cerevisiae, reaching 2527 mg/L in a fed-batch pentenol, olefin and other biofuels [55,64e66].
fermentation [51]. A two-level expression system was developed
for the PGAL1-controlled ISPS by overexpression of GAL4 [52]. 3.3. Sesquiterpenoid–based biofuels
Combining the two-level expression system and directed evolution
of ISPS in S. cerevisiae led to the production of 3.7 g/L in a fed-batch Sesquiterpenoids are one of the largest groups of isoprenoid
fermentation [52]. natural products and have a wide range of activities from antimi-
Ester of isoprenoid alcohols (C5, C10 and C15) have the potential crobial agents (such as phytoalexins capsidiol) to alarm phero-
to be used as replacements for petroleum-based diesels. B. subtilis mones (such as farnesene). Structurally, sesquiterpenoids can be
nudF and E. coli nudB have been introduced into E. coli for iso- acyclic, monocyclic, bi- or even tricyclic with different TPS that
prentol/isoprenol production [53,54]. Overexpression of some iso- catalyze FPP into a large variety of sesquiterpenes. Sesquiterpe-
pentenol tolerance-enhancing genes, such as metR and mdlB, noids are 15 carbons, close to the average length of diesel (C16), but
improved the production of isopentenol in E. coli [55]. A novel IPP- with a branched, rather than a straight-chain structure. Among
bypass MVA pathway was reported for isopentenol production in sesquiterpenoids, farnesol, farnesene and bisabolane have been
E. coli. The IPP-bypass MVA pathway contains the decarboxylation proposed as diesel fuels and produced from IPP.
of mevalonate phosphate by PMD and the hydroxylation of iso- Combinational expression of the heterologous MVA pathway
pentenyl monophosphate (IP) by E. coli phosphatase AphA [56]. and the fused proteins of IspA/AFS led to an approximate 317-fold
George et al. constructed an E. coli with a high yield in 3-methyl-3- increase over the initial production of farnesene in E. coli. The
buten-1-ol production [57]. A titer of 2.23 g/L isoprenol was ob- final engineered E. coli produced approximately 380 mg/L farne-
tained by using an oleyl alcohol overlay in the engineered E. coli. sene in a shake-flask fermentation [67]. In vitro studies on the pu-
This is the highest yield achieved from an engineered stain. rified protein components of MVA and the downstream FPP
pathway have revealed that Idi played a key role in a-farnesene
3.2. Monoterpenoid–based biofuels synthesis in vitro [68]. Based on the in vitro studies, farnesene
production was optimized through overexpression of Idi with IspA
Monoterpenoids are C10 compounds built from two isoprenoid and AFS in E. coli expressing the synthetic MVA pathway. After 96 h
units (one IPP and one DMAP). Monoterpenoids can be divided into of induction, farnesene production reached a concentration of
three major subgroups based on their structural features: 1) acyclic approximately 1.1 g/L in a shake-flask fermentation [68]. Meadows
monoterpenes, such as myrcene and ocimene; 2) monocyclic et al. constructed an artificial cytosolic acetyl coenzyme biosyn-
monoterpenes, such as limonene, menthol, and carvone; 3) bicyclic thetic pathway with a reduced ATP requirement, which contained
monoterpenes, such as pinene, sabinene, and camphor. Dickeya zeae aldehyde dehydrogenase (acylating) (ADA), Leuco-
Co-overexpression of the MVA pathway, A. grandis GPPS2 and nostoc mesenteroides xylulose-5-phosphate specific phosphoketo-
the Quercus ilex L. myrcene synthase gene in E. coli resulted in the lase (PK) and Clostridium kluyveri phosphotransacetylase (PTA)
production of 58.19 mg/L myrcene [58]. E. coli harboring the MVA [69]. Combining the artificial acetyl coenzyme biosynthetic
pathway, A. grandis GPPS2 and the Salvia pomifera sabinene syn- pathway with the NADH-consuming HMG-CoA reductase from
thase gene sabs1 produced 2.65 g/L sabinene in a fed-batch Silicibacter pomeroyi in S. cerevisiae enhanced farnesene produc-
fermentation [59]. tion, which reached 130 g/L in 200, 000-L bioreactor fed-batch
A novel biosynthetic pathway of a-pinene was assembled in fermentation [69].
E. coli BL21(DE3) with the heterologous MVA pathway, codon- Bisabolene, a monocyclic sequiterpene, has been identified as a
optimized GGPS from A. grandis and codon-optimized a-pinene precursor to a potential D2 diesel fuel. To obtain higher titers of
synthase Pt30 from Pinus taeda [60]. The final producing strain bisabolene, bisabolene synthase (BIS) genes from Arabidopsis
YJM28 produced 5.44 mg/L in a shake-flask fermentation and thaliana, Pseudotsuga menziesii, A. grandis and Picea abies have been
0.97 g/L a-pinene in a fed-batch fermentation. Sarria et al. combi- screened in E. coli (harboring the entire MVA pathway in a single
natorically expressed three pinene synthase (PS) and three GPPS plasmid) and S. cerevisiae [70]. Overexpression of the codon-
from conifers in engineered E. coli harboring the MVA pathway. optimized AgBIS in an engineered E. coli expressing the optimized
They achieved approximately 28 mg/L pinene using the best com- MVA pathway resulted in production of 912 mg/L of bisabolene. The
bination (PS and GGPS from A. grandis). Furthermore, they designed same level of bisabolene was also obtained in the engineered
GPPS-PS protein fusions to reduce GPP product inhibition and S. cerevisiae with an overproduction of FPP [70]. Kirby et al. reported
toxicity by substrate channeling, producing 32.4 mg/L pinene in a a novel route from ribulose 5-phosphate (Ru5P) to DXP (nDXP) and
shake-flask fermentation [61]. PS is the rate-limiting enzyme for uncovered two nDXP genes: ribBG108S and yajO. Expression of a Dxr-
pinene biosynthesis. To significantly improve the activity of PS, a- RibB(G108S) fusion improved bisabolene titers more than 4-fold
pinene synthase Pt1 from P. taeda was evolved to obtain a PS [71]. Using a carotenoid-based phenotypic screen of the yeast

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001
8 F.-X. Niu et al. / Synthetic and Systems Biotechnology xxx (2017) 1e9

deletion collection, the genes that affected isoprenoid synthesis in Carotenoids-Global-Outlook-10601158; 2017.
[8] Ma T, Deng ZX, Liu TG. Microbial production strategies and applications of
yeast were identified. Combinations of these deletions and other
lycopene and other terpenoids. World J Microb Biot 2016;32(1). http://
MVA pathway modifications improved the titers of bisabolene dx.doi.org/10.1007/s11274-015-1975-2.
more than 20-fold to 800 mg/L in a flask and 5.2 g/L in a fermen- [9] Kang MJ, Lee YM, Yoon SH, Kim JH, Ock SW, Jung KH, et al. Identification of
tation process [72]. genes affecting lycopene accumulation in Escherichia coli using a shot-gun
method. Biotechnol Bioeng 2005;91(5):636e42. http://dx.doi.org/10.1002/
Farnesol is an important C15 isoprenyl alcohol. Co- bit.20539.
overexpression of the heterologous MVA pathway and ispA in [10] Choi HS, Lee SY, Kim TY, Woo HM. In Silico Identification of gene amplification
E. coli led to the production of 135 mg/L farnesol [73]. Over- targets for improvement of lycopene production. Appl Environ Microb
2010;76(10):3097e105. http://dx.doi.org/10.1128/Aem.00115-10.
expression of IspA and the membrane phosphatase PgpB, along [11] Alper H, Jin YS, Moxley JF, Stephanopoulos G. Identifying gene targets for the
with a heterologous MVA pathway in E. coli, increased farnesol metabolic engineering of lycopene biosynthesis in Escherichia coli. Metab Eng
production to 526.1 mg/L in a shake flask fermentation [74]. A 2005;7(3):155e64. http://dx.doi.org/10.1016/j.ymben.2004.12.003.
[12] Zhou Y, Nambou K, Wei LJ, Cao JJ, Imanaka T, Hua Q. Lycopene production in
farnesol production of 145 mg/L was attained by S. cerevisiae ATCC recombinant strains of Escherichia coli is improved by knockout of the central
200589 with overexpression of HMG-CoA reductase (Hmg1) in a carbon metabolism gene coding for glucose-6-phosphate dehydrogenase.
fermentation culture for 7 days [75]. Biotechnol Lett 2013;35(12):2137e45. http://dx.doi.org/10.1007/s10529-
013-1317-0.
[13] Kim SW, Keasling JD. Metabolic engineering of the nonmevalonate iso-
4. Conclusions pentenyl diphosphate synthesis pathway in Escherichia coli enhances lyco-
pene production. Biotechnol Bioeng 2001;72(4):408e15. http://dx.doi.org/
10.1002/1097-0290(20000220)72:4<408::Aid-Bit1003>3.0.Co;2-H.
Carotenoids and isoprenoid-based biofuels are two classes of [14] Kim SW, Kim JB, Ryu JM, Jung JK, Kim JH. High-level production of lycopene in
important isoprenoids. With advances in metabolic engineering metabolically engineered E. coli. Process Biochem 2009;44(8):899e905.
and synthetic biology, engineered microorganisms have become a http://dx.doi.org/10.1016/j.procbio.2009.04.018.
[15] Rad SA, Zahiri HS, Noghabi KA, Rajaei S, Heidari R, Mojallali L. Type 2 IDI
primary alternative for their production. Most studies on carot- performs better than type 1 for improving lycopene production in metabol-
enoid production are focused on the regulation of carbon flux. Our ically engineered E. coli strains. World J Microb Biot 2012;28(1):313e21.
results from the comparative proteomes demonstrate that zeax- http://dx.doi.org/10.1007/s11274-011-0821-4.
[16] Shen HJ, Hu JJ, Li XR, Liu JZ. Engineering of Escherichia coli for lycopene pro-
anthin overproduction may be associated with not only precursor
duction through promoter engineering. Curr Pharm Biotechnol 2015;16(12):
availability but also cofactor availability, oxidative stress response, 1094e103. http://dx.doi.org/10.2174/1389201016666150731110536.
and membrane storage capacity [39]. Morphology engineering for [17] Sun T, Miao LT, Li QY, Dai GP, Lu FP, Liu T, et al. Production of lycopene by
increasing member storage capacity may be another strategy for metabolically-engineered Escherichia coli. Biotechnol Lett 2014;36(7):
1515e22. http://dx.doi.org/10.1007/s10529-014-1543-0.
improving carotenoid production. [18] Zhu FY, Lu L, Fu S, Zhong X, Hu M, Deng Z, et al. Targeted engineering and
Some isoprenoids have been proposed as biofuels. However, the scale up of lycopene overproduction in Escherichia coli. Process Biochem
levels of isoprenoid-based biofuels are lower than the order of 2015;50(3):341e6. http://dx.doi.org/10.1016/j.procbio.2014.12.008.
[19] Kim YS, Lee JH, Kim NH, Yeom SJ, Kim SW, Oh DK. Increase of lycopene
magnitude of those of carotenoids. Moreover, the titers of mono- production by supplementing auxiliary carbon sources in metabolically
terpenes are lower than those of hemiterpenes and sesquiterpenes. engineered Escherichia coli. Appl Microbiol Biot 2011;90(2):489e97. http://
Toxicity and enzyme activity may be major factors. Tolerance en- dx.doi.org/10.1007/s00253-011-3091-z.
[20] Tyo KEJ, Ajikumar PK, Stephanopoulos G. Stabilized gene duplication enables
gineering and the evolution of enzymes may be effective strategies long-term selection-free heterologous pathway expression. Nat Biotechnol
for improving yields. 2009;27(8). http://dx.doi.org/10.1038/nbt.1555. 760eU115.
[21] Chen YY, Shen HJ, Cui YY, Chen SG, Weng ZM, Zhao M, et al. Chromosomal
evolution of Escherichia coli for the efficient production of lycopene. Bmc
Acknowledgments Biotechnol 2013;13:6. http://dx.doi.org/10.1186/1472-6750-13-6.
[22] Coussement P, Bauwens D, Maertens J, De Mey M. Direct combinatorial
We are grateful to the National Natural Science Foundation of pathway optimization. ACS Synth Biol 2017;6(2):224e32. http://dx.doi.org/
10.1021/acssynbio.6b00122.
China (Grant NO. 21276289), the Natural Science Foundation of [23] Xie WP, Lv XM, Ye LD, Zhou PP, Yu HW. Construction of lycopene-
Guangdong Province (NO. 2015A030311036), the Project of the overproducing Saccharomyces cerevisiae by combining directed evolution
Scientific and Technical Program of Guangdong Province (NO. and metabolic engineering. Metab Eng 2015;30:69e78. http://dx.doi.org/
10.1016/j.ymben.2015.04.009.
2015A010107004) and the Project of the Scientific and Technical [24] Ozaydin B, Burd H, Lee TS, Keasling JD. Carotenoid-based phenotypic screen of
Program of Guangzhou (NO. 201607010028) for their financial the yeast deletion collection reveals new genes with roles in isoprenoid
support. production. Metab Eng 2013;15:174e83. http://dx.doi.org/10.1016/
j.ymben.2012.07.010.
[25] Chen Y, Xiao WH, Wang Y, Liu H, Li X, Yuan YJ. Lycopene overproduction in
References Saccharomyces cerevisiae through combining pathway engineering with host
engineering. Microb Cell Fact 2016;15:113. http://dx.doi.org/10.1186/s12934-
[1] Katsuki H, Bloch K. Studies on the biosynthesis of ergosterol in yeast. For- 016-0509-4.
mation of methylated intermediates. J Biol Chem 1967;242(2):222e7. [26] Matthaus F, Ketelhot M, Gatter M, Barth G. Production of lycopene in the non-
[2] Rohmer M, Knani M, Simonin P, Sutter B, Sahm H. Isoprenoid biosynthesis in carotenoid-producing yeast Yarrowia lipolytica. Appl Environ Microb
bacteria: a novel pathway for the early steps leading to isopentenyl diphos- 2014;80(5):1660e9. http://dx.doi.org/10.1128/Aem.03167-13.
phate. Biochem J 1993;295(Pt 2):517e24. [27] Bai CX, Zhang Y, Zhao XJ, Hu YL, Xiang SH, Miao J, et al. Exploiting a precise
[3] Rohdich F, Zepeck F, Adam P, Hecht S, Kaiser J, Laupitz R, et al. The deoxy- design of universal synthetic modular regulatory elements to unlock the
xylulose phosphate pathway of isoprenoid biosynthesis: studies on the microbial natural products in Streptomyces. P Natl Acad Sci U. S. A
mechanisms of the reactions catalyzed by IspG and IspH protein. P Natl Acad 2015;112(39):12181e6. http://dx.doi.org/10.1073/pnas.1511027112.
Sci U. S. A 2003;100(4):1586e91. http://dx.doi.org/10.1073/pnas.0337742100. [28] Yang JM, Guo LZ. Biosynthesis of beta-carotene in engineered E. coli using the
[4] Hornero-Mendez D, Britton G. Involvement of NADPH in the cyclization re- MEP and MVA pathways. Microb Cell Fact 2014;13:160. http://dx.doi.org/
action of carotenoid biosynthesis. FEBS Lett 2002;515(1e3):133e6. http:// 10.1186/s12934-014-0160-x.
dx.doi.org/10.1016/S0014-5793(02)02453-5. [29] Zhao J, Li QY, Sun T, Zhu XN, Xu HT, Tang JL, et al. Engineering central
[5] Wang F, Jiang JG, Chen Q. Progress on molecular breeding and metabolic metabolic modules of Escherichia coli for improving beta-carotene production.
engineering of biosynthesis pathways of C30, C355, C40, C45, C50 carotenoids. Metab Eng 2013;17:42e50. http://dx.doi.org/10.1016/j.ymben.
Biotechnol Adv 2007;25(3):211e22. http://dx.doi.org/10.1016/ [30] Li YF, Lin ZQ, Huang C, Zhang Y, Wang ZW, Tang YJ, et al. Metabolic engi-
j.biotechadv.2006.12.001. neering of Escherichia coli using CRISPR-Cas9 meditated genome editing.
[6] Heider SAE, Peters-Wendisch P, Wendisch VF, Beekwilder J, Brautaset T. Metab Eng 2015;31:13e21. http://dx.doi.org/10.1016/j.ymben.
Metabolic engineering for the microbial production of carotenoids and related [31] Xie WP, Liu M, Lv XM, Lu WQ, Gu JL, Yu HW. Construction of a controllable
products with a focus on the rare C50 carotenoids. Appl Microbiol Biot beta-Carotene biosynthetic pathway by decentralized assembly strategy in
2014;98(10):4355e68. http://dx.doi.org/10.1007/s00253-014-5693-8. Saccharomyces cerevisiae. Biotechnol Bioeng 2014;111(1):125e33. http://
[7] BCC-Research. The global market outlook (2016-2022). https://www. dx.doi.org/10.1002/bit.25002.
marketresearch.com/Stratistics-Market-Research-Consulting-v4058/ [32] Xie WP, Ye LD, Lv XM, Xu HM, Yu HW. Sequential control of biosynthetic

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001
 F.-X. Niu et al. / Synthetic and Systems Biotechnology xxx (2017) 1e9 9

pathways for balanced utilization of metabolic intermediates in Saccharo- jam.12871.

myces cerevisiae. Metab Eng 2015;28:8e18. http://dx.doi.org/10.1016/ [54] Zheng YN, Liu Q, Li LL, Qin W, Yang JM, Zhang HB, et al. Metabolic engineering
j.ymben.2014.11.007. of Escherichia coli for high-specificity production of isoprenol and prenol as
[33] Sajilata MG, Singhal RS, Kamat MY. The carotenoid pigment zeaxanthin - a next generation of biofuels. Biotechnol Biofuels 2013;6:57. http://dx.doi.org/
review. Compr Rev Food Sci F 2008;7(1):29e49. http://dx.doi.org/10.1111/ 10.1186/1754-6834-6-57.
j.1541-4337.2007.00028.x. [55] Foo JL, Jensen HM, Dahl RH, George K, Keasling JD, Lee TS, et al. Improving
[34] Moeller SM, Jacques PF, Blumberg JB. The potential role of dietary xantho- microbial biogasoline production in Escherichia coli using tolerance engi-
phylls in cataract and age-related macular degeneration. J Am Coll Nutr neering. Mbio 2014;5(6). http://dx.doi.org/10.1128/mBio.01932-14.
2000;19(5):522se7s. e01932e14.
[35] Nishino H, Murakoshi M, Tokuda H, Satomi Y. Cancer prevention by carot- [56] Kang A, George KW, Wang G, Baidoo E, Keasling JD, Lee TS. Isopentenyl
enoids. Arch Biochem Biophys 2009;483(2):165e8. http://dx.doi.org/10.1016/ diphosphate (IPP)-bypass mevalonate pathways for isopentenol production.
j.abb.2008.09.011. Metab Eng 2016;34:25e35. http://dx.doi.org/10.1016/j.ymben.2015.12.002.
[36] Albrecht M, Misawa N, Sandmann G. Metabolic engineering of the terpenoid [57] George KW, Thompson MG, Kang A, Baidoo E, Wang G, Chan LJG, et al.
biosynthetic pathway of Escherichia coli for production of the carotenoids Metabolic engineering for the high-yield production of isoprenoid-based C5
beta-carotene and zeaxanthin. Biotechnol Lett 1999;21(9):791e5. http:// alcohols in E. coli. Sci Rep-Uk 2015;5:11128. http://dx.doi.org/10.1038/
dx.doi.org/10.1023/A:1005547827380. srep11128.
[37] Nishizaki T, Tsuge K, Itaya M, Doi N, Yanagawa H. Metabolic engineering of [58] Kim EM, Eom JH, Um Y, Kim Y, Woo HM. Microbial synthesis of myrcene by
carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Ba- metabolically engineered Escherichia coil. J Agric Food Chem 2015;63(18):
cillus subtilis. Appl Environ Microb 2007;73(4):1355e61. http://dx.doi.org/ 4606e12. http://dx.doi.org/10.1021/acs.jafc.5b01334.
10.1128/Aem.02268-06. [59] Zhang HB, Liu Q, Cao YJ, Feng XJ, Zheng YN, Zou HB, et al. Microbial production
[38] Li XR, Tian GQ, Shen HJ, Liu JZ. Metabolic engineering of Escherichia coli to of sabinene-a new terpene-based precursor of advanced biofuel. Microb Cell
produce zeaxanthin. J Ind Microbiol Biot 2015;42(4):627e36. http:// Fact 2014;13:20. http://dx.doi.org/10.1186/1475-2859-13-20.
dx.doi.org/10.1007/s10295-014-1565-6. [60] Yang JM, Nie QJ, Ren M, Feng HR, Jiang XL, Zheng YN, et al. Metabolic engi-
[39] Shen HJ, Cheng BY, Zhang YM, Tang L, Li Z, Bu YF, et al. Dynamic control of the neering of Escherichia coli for the biosynthesis of alpha-pinene. Biotechnol
mevalonate pathway expression for improved zeaxanthin production in Biofuels 2013;6:60. http://dx.doi.org/10.1186/1754-6834-6-60.
Escherichia coli and comparative proteome analysis. Metab Eng 2016;38: [61] Sarria S, Wong B, Martin HG, Keasling JD, Peralta-Yahya P. Microbial synthesis
180e90. http://dx.doi.org/10.1016/j.ymben.2016.07.012. of pinene. ACS Synth Biol 2014;3(7):466e75. http://dx.doi.org/10.1021/
[40] Scaife MA, Burja AM, Wright PC. Characterization of cyanobacterial beta- sb4001382.
carotene ketolase and hydroxylase genes in Escherichia coli, and their appli- [62] Tashiro M, Kiyota H, Kawai-Noma S, Saito K, Ikeuchi M, Iijima Y, et al. Bacterial
cation for astaxanthin biosynthesis. Biotechnol Bioeng 2009;103(5):944e55. production of pinene by a laboratory-evolved pinene-synthase. ACS Synth
http://dx.doi.org/10.1002/bit.22330. Biol 2016;5(9):1011e20. http://dx.doi.org/10.1021/acssynbio.6b00140.
[41] Liu JZ, Lu Q, Bu YF. Engineered strain with high zeaxanthin or astaxanthin [63] Alonso-Gutierrez J, Chan R, Batth TS, Adams PD, Keasling JD, Petzold CJ, et al.
ratio and its application. China patient 201710036290.9, 2017. Metabolic engineering of Escherichia coli for limonene and perillyl alcohol
[42] Zelcbuch L, Antonovsky N, Bar-Even A, Levin-Karp A, Barenholz U, Dayagi M, production. Metab Eng 2013;19:33e41. http://dx.doi.org/10.1016/
et al. Spanning high-dimensional expression space using ribosome-binding j.ymben.2013.05.004.
site combinatorics. Nucleic Acids Res 2013;41(9):e98. http://dx.doi.org/ [64] Dunlop MJ. Engineering microbes for tolerance to next-generation biofuels.
10.1093/nar/gkt151. Biotechnol Biofuels 2011;4:32. http://dx.doi.org/10.1186/1754-6834-4-32.
[43] Ma T, Zhou YJ, Li XW, Zhu FY, Cheng YB, Liu Y, et al. Genome mining of [65] Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD, et al. Engi-
astaxanthin biosynthetic genes from Sphingomonas sp ATCC 55669 for het- neering microbial biofuel tolerance and export using efflux pumps. Mol Syst
erologous overproduction in Escherichia coli. Biotechnol J 2016;11(2):228e37. Biol 2011;7:487. http://dx.doi.org/10.1038/msb.2011.21.
http://dx.doi.org/10.1002/biot.201400827. [66] Mingardon F, Clement C, Hirano K, Nhan M, Luning EG, Chanal A, et al.
[44] Zhou PP, Ye LD, Xie WP, Lv XM, Yu HW. Highly efficient biosynthesis of Improving olefin tolerance and production in E. coli using native and evolved
astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ AcrB. Biotechnol Bioeng 2015;112(5):879e88. http://dx.doi.org/10.1002/
and bkt. Appl Microbiol Biot 2015;99(20):8419e28. http://dx.doi.org/10.1007/ bit.25511.
s00253-015-6791-y. [67] Wang C, Yoon SH, Jang HJ, Chung YR, Kim JY, Choi ES, et al. Metabolic engi-
[45] Zhou PP, Xie WP, Li AP, Wang F, Yao Z, Bian Q, et al. Alleviation of metabolic neering of Escherichia coli for alpha-farnesene production. Metab Eng
bottleneck by combinatorial engineering enhanced astaxanthin synthesis in 2011;13(6):648e55. http://dx.doi.org/10.1016/j.ymben.2011.08.001.
Saccharomyces cerevisiae. Enzyme Microb Tech 2017;100:28e36. http:// [68] Zhu FY, Zhong XF, Hu MZ, Lu L, Deng ZX, Liu TG. In vitro reconstitution of
dx.doi.org/10.1016/j.enzmictec.2017.02.006. mevalonate pathway and targeted engineering of farnesene overproduction
[46] Henke NA, Heider SAE, Peters-Wendisch P, Wendisch VF. Production of the in Escherichia coli. Biotechnol Bioeng 2014;111(7):1396e405. http://
marine carotenoid astaxanthin by metabolically engineered Corynebacterium dx.doi.org/10.1002/bit.25198.
glutamicum. Mar Drugs 2016;14(7):124. http://dx.doi.org/10.3390/ [69] Meadows AL, Hawkins KM, Tsegaye Y, Antipov E, Kim Y, Raetz L, et al.
md14070124. Rewriting yeast central carbon metabolism for industrial isoprenoid produc-
[47] Rabinovitch-Deere CA, Oliver JWK, Rodriguez GM, Atsumi S. Synthetic biology tion. Nature 2016;537(7622). http://dx.doi.org/10.1038/nature19769. 694-þ.
and metabolic engineering approaches to produce biofuels. Chem Rev [70] Peralta-Yahya PP, Ouellet M, Chan R, Mukhopadhyay A, Keasling JD, Lee TS.
2013;113(7):4611e32. http://dx.doi.org/10.1021/cr300361t. Identification and microbial production of a terpene-based advanced biofuel.
[48] Kim JH, Wang CL, Jang HJ, Cha MS, Park JE, Jo SY, et al. Isoprene production by Nat Commun 2011;2:483. http://dx.doi.org/10.1038/ncomms1494.
Escherichia coli through the exogenous mevalonate pathway with reduced [71] Kirby J, Nishimoto M, Chow RWN, Baidoo EEK, Wang G, Martin J, et al.
formation of fermentation byproducts. Microb Cell Fact 2016;15:214. http:// Enhancing terpene yield from sugars via novel routes to 1-Deoxy-D-Xylulose
dx.doi.org/10.1186/s12934-016-0612-6. 5-Phosphate. Appl Environ Microb 2015;81(1):130e8. http://dx.doi.org/
[49] Whited GM, Feher FJ, Benko DA, Cervin MA, Chotani GK, McAuliffe JC, et al. 10.1128/Aem.02920-14.
Development of a gas-phase bioprocess for isoprene monomer production [72] Ozaydin B, Burd H, Lee TS, Keasling JD. Carotenoid-based phenotypic screen of
using metabolic pathway engineering. Ind Biotechnol 2010;6(3):152e63. the yeast deletion collection reveals new genes with roles in isoprenoid
http://dx.doi.org/10.1089/ind.2010.6.152. production. Metab Eng 2013;15:174e83. http://dx.doi.org/10.1016/
[50] Yang C, Gao X, Jiang Y, Sun BB, Gao F, Yang S. Synergy between methylery- j.ymben.2012.07.010.
thritol phosphate pathway and mevalonate pathway for isoprene production [73] Wang C, Yoon SH, Shah AA, Chung YR, Kim JY, Choi ES, et al. Farnesol pro-
in Escherichia coli. Metab Eng 2016;37:79e91. http://dx.doi.org/10.1016/ duction from Escherichia coli by harnessing the exogenous mevalonate
j.ymben.2016.05.003. pathway. Biotechnol Bioeng 2010;107(3):421e9. http://dx.doi.org/10.1002/
[51] Lv X, Wang F, Zhou P, Ye L, Xie W, Xu H, et al. Dual regulation of cytoplasmic bit.22831.
and mitochondrial acetyl-CoA utilization for improved isoprene production in [74] Wang CL, Park JE, Choi ES, Kim SW. Farnesol production in Escherichia coli
Saccharomyces cerevisiae. Nat Commun 2016;7:12851. http://dx.doi.org/ through the construction of a farnesol biosynthesis pathway - application of
10.1038/ncomms12851. PgpB and YbjG phosphatases. Biotechnol J 2016;11(10):1291e7. http://
[52] Wang F, Lv XM, Xei WP, Zhou PP, Zhu YQ, Yao Z, et al. Combining Gal4p- dx.doi.org/10.1002/biot.201600250.
mediated expression enhancement and directed evolution of isoprene syn- [75] Ohto C, Muramatsu M, Obata S, Sakuradani E, Shimizu S. Overexpression of
thase to improve isoprene production in Saccharomyces cerevisiae. Metab Eng the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for pro-
2017;39:257e66. http://dx.doi.org/10.1016/j.ymben.2016.12.011. duction of prenyl alcohols. Appl Microbiol Biot 2009;82(5):837e45. http://
[53] Gupta P, Phulara SC. Metabolic engineering for isoprenoid-based biofuel dx.doi.org/10.1007/s00253-008-1807-5.
production. J Appl Microbiol 2015;119(3):605e19. http://dx.doi.org/10.1111/

Please cite this article in press as: Niu F-X, et al., Metabolic engineering for the microbial production of isoprenoids: Carotenoids and isoprenoid-
based biofuels, Synthetic and Systems Biotechnology (2017), http://dx.doi.org/10.1016/j.synbio.2017.08.001