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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 highprice 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 ([email protected]), Chen, Jian ([email protected])
Current Opinion in Biotechnology 2014, 25:17–23 This review comes from a themed issue on Analytical biotechnology Edited by Frank L Jaksch and Savas¸ 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 applications, especially in nutritional and pharmaceutical applications. These small molecules provide the source and inspiration for the majority of FDA-approved agents and www.sciencedirect.com
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 communities, 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 manufacturing PNPs on the basis of the metabolic engineering of microorganisms, has emerged recently as an interesting and commercially attractive approach due to several advantages including the utilization of environmentally 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, sesquiterpenes, diterpenes, among others (Figure 1). The most intensively investigated terpenes for the metabolic engineering production by microorganisms include artemisinin (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 institutes, introduced a complete biosynthetic pathway, including 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
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Figure 1 D-Glucose
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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 production. 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 isoprenes to biomass. Introduction of an ERG20 with a Current Opinion in Biotechnology 2014, 25:17–23
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, modular engineering of heterogenous pathways. By a multivariate 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 microorganisms 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 promising application as biofuel. By overexpressing a native www.sciencedirect.com
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geranyl diphosphate synthase (IspA) from Escherichia coli and a geranyl diphosphate synthase (GPPS2), co-expressing 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 compounds and can be further divided into coumarins, flavonoids, 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 trichocarpa 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, promoters and enzymes, the resveratrol yield was improved to 2.31 g/L with p-coumaric acid as precursor and supplementary of cerulenin to inhibit the malonyl-CoA competitive 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 economically 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 reconstruction of the long pathway from central metabolism to phenylpropanoids [23]. Expression of TAL/PAL with higher catalysis activity could significantly enhance the www.sciencedirect.com
production of different phenylpropanoids from tyrosin/ phenylalanine or even glucose [24–26]. The malonyl-CoA, which is another precursor of phenylpropanoids, 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 Photorhabdus 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 identified and modified, improving the nargingenin yield from p-coumaric acid to 474 mg/L without addition of cerulenin [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 catalysis [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 production 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 components 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/catharanthine, atropine, and quinine. Most of these alkaloids were used as drugs for a long history within the corresponding plants. Unlike phenylpropanoids and terpenes, alkaloids shared several totally different pathways derived from the Current Opinion in Biotechnology 2014, 25:17–23
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Figure 2
(a) SCoA
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aroF wt Chorismate pheAfbr
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Plant natural products production by fermentation Zhou, Du and Chen 21
primary metabolism precursors. For their intensive bioactivities, 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 benzylisoquinoline 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].
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 metabolic 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 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 synthesize 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 cytochrome P450 monooxygenases. By introducing a cytochrome 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 Pseudomonas 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
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 development of high-throughput sequencing technologies greatly facilitates the discoveries of new genes and pathways 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 modification of enzymes in plants to improve their compatibility in host microorganisms. Achievements in the protein structure analysis and prediction greatly facilitate the protein engineering [41]. Combining with the highthroughput 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-inducible 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 manipulation of microorganisms is still a bottleneck for reconstruction of complicated design of metabolic/regulation pathways. Invention of new genome engineering strategies, 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
(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. www.sciencedirect.com
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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 complicated 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 microorganisms 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
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D et al.: High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 2013. advance online publication. This paper is a milestone of the PNPs production by fermentation processes. They improved the precursor of artemisinin to an incredible level on the basis of the previous attempts with large amount of systematic metabolic engineerings. Combination of the chemical engineering process also avoid the weakness of the current pure biotechnology.
6.
7.
Muchiri R, Walker KD: Taxol biosynthesis: tyrocidine synthetase A catalyzes the production of phenylisoserinyl CoA and other amino phenylpropanoyl thioesters. Chem Biol 2012, 19:679-685.
8.
Zhao J, Li QY, Sun T, Zhu XN, Xu HT, Tang JL, Zhang XL, Ma YH: Engineering central metabolic modules of Escherichia coli for improving b-carotene production. Metab Eng 2013, 17:42-50.
9.
Wang C, Yoon SH, Jang HJ, Chung YR, Kim JY, Choi ES, Kim SW: Metabolic engineering of Escherichia coli for a-farnesene production. Metab Eng 2011, 13:648-655.
10. Asadollahi MA, Maury J, Schalk M, Clark A, Nielsen J: Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiae. Biotechnol Bioeng 2010, 106:86-96. 11. Rico J, Pardo E, Orejas M: Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3methylglutaryl coenzyme A reductase catalytic domain in Saccharomyces cerevisiae. Appl Environ Microbiol 2010, 76:6449-6454. 12. Ignea C, Trikka F, Kourtzelis I, Argiriou A, Kanellis A, Kampranis S, Makris A: Positive genetic interactors of HMG2 identify a new set of genetic perturbations for improving sesquiterpene production in Saccharomyces cerevisiae. Microbial Cell Fact 2012, 11:162.
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).
13. Ignea C, Cvetkovic I, Loupassaki S, Kefalas P, Johnson CB, Kampranis SC, Makris AM: Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids. Microbial Cell Fact 2011, 10:4-22.
References and recommended reading
15. Scalcinati G, Knuf C, Partow S, Chen Y, Maury J, Schalk M, Daviet L, Nielsen J, Siewers V: Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene a-santalene in a fed-batch mode. Metab Eng 2012, 14:91-103.
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Newman DJ, Cragg GM: Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 2012, 75:311-335.
2.
El-Shemy HA, Aboul-Enein AM, Aboul-Enein KM, Fujita K: Willow leaves’ extracts contain anti-tumor agents effective against three cell types. PLoS ONE 2007, 2:e178.
3.
Engels B, Dahm P, Jennewein S: Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. Metab Eng 2008, 10:201-206.
4.
Gazak R, Fuksova K, Marhol P, Kuzma M, Agarwal R, Kren V: Preparative method for isosilybin isolation based on enzymatic kinetic resolution of silymarin mixture. Process Biochem 2013, 48:184-189.
5.
Mavel S, Dikic B, Palakas S, Emond P, Greguric I, de Gracia AG, Mattner F, Garrigos M, Guilloteau D, Katsifis A: Synthesis and biological evaluation of a series of flavone derivatives as potential radioligands for imaging the multidrug resistanceassociated protein 1 (ABCC1/MRP1). Bioorg Med Chem 2006, 14:1599-1607.
Current Opinion in Biotechnology 2014, 25:17–23
14. Fischer MJC, Meyer S, Claudel P, Bergdoll M, Karst F: Metabolic engineering of monoterpene synthesis in yeast. Biotechnol Bioeng 2011, 108:1883-1892. Point mutations of a protein to improve its performance in metabolic engineering is an attractive topic. How to find out the ‘‘point’’, is still a great challenge. This paper may provide some useful clues.
16. Zhou YJJ, Gao W, Rong QX, Jin GJ, Chu HY, Liu WJ, Yang W, Zhu ZW, Li GH, Zhu GF et al.: Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc 2012, 134:3234-3241. 17. Hu FF, Liu JD, Du GC, Hua ZZ, Zhou JW, Chen J: Key cytomembrane ABC transporters of Saccharomyces cerevisiae fail to improve the tolerance to D-limonene. Biotechnol Lett 2012, 34:1505-1509. Previous researches showed that the multi-drug resistance transporters could protect yeast cells from many kinds of PNPs from different origins. In this paper, they show that how incompetent these transporters are under the stress of some specific PNPs. 18. Yang JM, Nie QJ, Ren M, Feng HR, Jiang XL, Zheng YN, Liu M, Zhang HB, Xian M: Metabolic engineering of Escherichia coli for the biosynthesis of a-pinene. Biotechnol Biofuels 2013, 6:60. 19. Sugiyama A, Linley PJ, Sasaki K, Kumano T, Yamamoto H, Shitan N, Ohara K, Takanashi K, Harada E, Hashegawa H: Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases. Metab Eng 2011, 13:629-637. 20. Kiselev KV: Perspectives for production and application of resveratrol. Appl Microbiol Biotechnol 2011, 90:417-425. www.sciencedirect.com
Plant natural products production by fermentation Zhou, Du and Chen 23
21. Becker JVW, Armstrong GO, Merwe MJ, Lambrechts MG, Vivier MA, Pretorius IS: Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the winerelated antioxidant resveratrol. FEMS Yeast Res 2003, 4:79-85. 22. Lim CG, Fowler ZL, Hueller T, Schaffer S, Koffas MAG: High-yield resveratrol production in engineered Escherichia coli. Appl Environ Microbiol 2011, 77:3451-3460. This is the must-to-read paper for those who would like to be invovled in the metabolic engineering of microoroganisms for phenylpropanoids. It contains most of the brilliant ideas about how to improve the yield of this group of PNPs. 23. Park NI, Xu H, Li X, Kim YS, Lee MY, Park SU: Overexpression of phenylalanine ammonia-lyase improves flavones production in transgenic hairy root cultures of Scutellaria baicalensis. Process Biochem 2012, 47:2575-2580. 24. Vannelli T, Wei Qi W, Sweigard J, Gatenby AA, Sariaslani FS: Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi. Metab Eng 2007, 9:142-151. 25. Wang Y, Halls C, Zhang J, Matsuno M, Zhang Y, Yu O: Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metab Eng 2011, 13:455-463. 26. Wu JJ, Du GC, Zhou JW, Chen J: Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy. Metab Eng 2013, 16:48-55. 27. Miyahisa I, Kaneko M, Funa N, Kawasaki H, Kojima H, Ohnishi Y, Horinouchi S: Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster. Appl Environ Microbiol 2005, 68:498-504. 28. Leonard E, Lim KH, Saw PN, Koffas MAG: Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl Environ Microbiol 2007, 73:3877. 29. An JH, Kim YS: A gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putative dicarboxylate carrier protein (MatC) in Rhizobium trifolii. Eur J Biochem 1998, 257:395-402. 30. Xu P, Ranganathan S, Fowler ZL, Maranas CD, Koffas MAG: Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab Eng 2011, 13:578-587. A perfect case that can show how a genome-scale model could be applied to enhance the design process for an enhanced specific PNP. 31. Koopman F, Beekwilder J, Crimi B, Houwelingen A, Hall R, Bosch D, van Maris A, Pronk J, Daran J-M: De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microbial Cell Fact 2012, 11:155. 32. Santos CNS, Koffas M, Stephanopoulos G: Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab Eng 2011, 13:392-400. Few of the earliest attempts to produce flavonoids from glucose. It shows how to reconstruct a complicated long pathway from different origins in microorganisms and make it to be operational. 33. Glenn WS, Runguphan W, O’Connor SE: Recent progress in the metabolic engineering of alkaloids in plant systems. Curr Opin Biotechnol 2013, 24:354-365. The fundamental information about the biosynthesis of alkaloids in plant systems from this review, provides almost all of the potentials for metabolic engineering of microorganisms to produce more diverse alkaloids. 34. Hawkins KM, Smolke CD: Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat Chem Biol 2008, 4:564-573.
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35. Inui T, Kawano N, Shitan N, Yazaki K, Kiuchi F, Kawahara N, Sato F, Yoshimatsu K: Improvement of benzylisoquinoline alkaloid productivity by overexpression of 30 -hydroxy-Nmethylcoclaurine 40 -O-methyltransferase in transgenic Coptis japonica plants. Biol Pharm Bull 2012, 35:650-659. 36. Minami H, Kim JS, Ikezawa N, Takemura T, Katayama T, Kumagai H, Sato F: Microbial production of plant benzylisoquinoline alkaloids. Proc Natl Acad Sci U S A 2008, 105:7393-7398. 37. Nakagawa A, Minami H, Kim JS, Koyanagi T, Katayama T, Sato F, Kumagai H: A bacterial platform for fermentative production of plant alkaloids. Nat Commun 2011, 2:326. 38. Nakagawa A, Minami H, Kim JS, Koyanagi T, Katayama T, Sato F, Kumagai H: Bench-top fermentative production of plant benzylisoquinoline alkaloids using a bacterial platform. Bioeng Bugs 2012, 3:49-53. 39. Schatz M, Witkowski J, McCombie WR: Current challenges in de novo plant genome sequencing and assembly. Genome Biol 2012, 13:243. 40. McGettigan PA: Transcriptomics in the RNA-seq era. Curr Opin Chem Biol 2013, 17:4-11. 41. Marks DS, Hopf TA, Sander C: Protein structure prediction from sequence variation. Nat Biotechnol 2012, 30:1072-1080. 42. Hillerich BS, Payne J: Advances in protein expression: high throughput tools for improving analysis. Science 2013, 340:382. 43. Xu P, Gu Q, Wang WY, Wong L, Bower AGW, Collins CH, Koffas MAG: Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun 2013, 4:1409. An integrated case to show how researchers could optimize a group of multi-gene pathways by trying the possible combinations on the basis of a semi-rational metabolic network design strategy. 44. Sun N, Zhao HM: Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing. Biotechnol Bioeng 2013, 110:1811-1821. 45. Jiang WY, Bikard D, Cox D, Zhang F, Marraffini LA: RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 2013, 31:233-239. Novel genome engineering strategies on the basis of a virus immune system from bacteria. It may revolutionize the current strategies for the direct genetic engineering on genomes. 46. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013, 152:1173-1183. An easier way to achieve the multi-gene regulation in microorganisms with the CRISPR/dCas9 system. 47. McCloskey D, Palsson BO, Feist AM: Basic and applied uses of genome-scale metabolic network reconstructions of Escherichia coli. Mol Syst Biol 2013, 9:661. 48. Gherardini PF, Helmer-Citterich M: Experimental and computational methods for the analysis and modeling of signaling networks. New Biotechnol 2013, 30:327-332. 49. Brennan TCR, Kro¨mer JO, Nielsen LK: Physiological and transcriptional responses of Saccharomyces cerevisiae to Dlimonene show changes to the cell wall but not to the plasma membrane. Appl Environ Microbiol 2013, 79:3590-3600. 50. Liu JD, Zhu YB, Du GC, Zhou JW, Chen J: Exogenous ergosterol protects Saccharomyces cerevisiae from D-limonene stress. J Appl Microbiol 2013, 114:482-491.
Current Opinion in Biotechnology 2014, 25:17–23
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 highprice 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 ([email protected]), Chen, Jian ([email protected])
Current Opinion in Biotechnology 2014, 25:17–23 This review comes from a themed issue on Analytical biotechnology Edited by Frank L Jaksch and Savas¸ 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 applications, especially in nutritional and pharmaceutical applications. These small molecules provide the source and inspiration for the majority of FDA-approved agents and www.sciencedirect.com
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 communities, 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 manufacturing PNPs on the basis of the metabolic engineering of microorganisms, has emerged recently as an interesting and commercially attractive approach due to several advantages including the utilization of environmentally 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, sesquiterpenes, diterpenes, among others (Figure 1). The most intensively investigated terpenes for the metabolic engineering production by microorganisms include artemisinin (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 institutes, introduced a complete biosynthetic pathway, including 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
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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 production. 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 isoprenes to biomass. Introduction of an ERG20 with a Current Opinion in Biotechnology 2014, 25:17–23
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, modular engineering of heterogenous pathways. By a multivariate 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 microorganisms 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 promising application as biofuel. By overexpressing a native www.sciencedirect.com
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-expressing 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 compounds and can be further divided into coumarins, flavonoids, 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 trichocarpa 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, promoters and enzymes, the resveratrol yield was improved to 2.31 g/L with p-coumaric acid as precursor and supplementary of cerulenin to inhibit the malonyl-CoA competitive 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 economically 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 reconstruction of the long pathway from central metabolism to phenylpropanoids [23]. Expression of TAL/PAL with higher catalysis activity could significantly enhance the www.sciencedirect.com
production of different phenylpropanoids from tyrosin/ phenylalanine or even glucose [24–26]. The malonyl-CoA, which is another precursor of phenylpropanoids, 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 Photorhabdus 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 identified and modified, improving the nargingenin yield from p-coumaric acid to 474 mg/L without addition of cerulenin [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 catalysis [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 production 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 components 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/catharanthine, atropine, and quinine. Most of these alkaloids were used as drugs for a long history within the corresponding plants. Unlike phenylpropanoids and terpenes, alkaloids shared several totally different pathways derived from the Current Opinion in Biotechnology 2014, 25:17–23
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Figure 2
(a) SCoA
O
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matA
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aroF wt Chorismate pheAfbr
tyrA L-Phenylalanine
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Current Opinion in Biotechnology 2014, 25:17–23
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Plant natural products production by fermentation Zhou, Du and Chen 21
primary metabolism precursors. For their intensive bioactivities, 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 benzylisoquinoline 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].
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 metabolic 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 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 synthesize 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 cytochrome P450 monooxygenases. By introducing a cytochrome 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 Pseudomonas 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
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 development of high-throughput sequencing technologies greatly facilitates the discoveries of new genes and pathways 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 modification of enzymes in plants to improve their compatibility in host microorganisms. Achievements in the protein structure analysis and prediction greatly facilitate the protein engineering [41]. Combining with the highthroughput 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-inducible 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 manipulation of microorganisms is still a bottleneck for reconstruction of complicated design of metabolic/regulation pathways. Invention of new genome engineering strategies, 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
(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. www.sciencedirect.com
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 complicated 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 microorganisms 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
Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Main A, Eng D et al.: High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 2013. advance online publication. This paper is a milestone of the PNPs production by fermentation processes. They improved the precursor of artemisinin to an incredible level on the basis of the previous attempts with large amount of systematic metabolic engineerings. Combination of the chemical engineering process also avoid the weakness of the current pure biotechnology.
6.
7.
Muchiri R, Walker KD: Taxol biosynthesis: tyrocidine synthetase A catalyzes the production of phenylisoserinyl CoA and other amino phenylpropanoyl thioesters. Chem Biol 2012, 19:679-685.
8.
Zhao J, Li QY, Sun T, Zhu XN, Xu HT, Tang JL, Zhang XL, Ma YH: Engineering central metabolic modules of Escherichia coli for improving b-carotene production. Metab Eng 2013, 17:42-50.
9.
Wang C, Yoon SH, Jang HJ, Chung YR, Kim JY, Choi ES, Kim SW: Metabolic engineering of Escherichia coli for a-farnesene production. Metab Eng 2011, 13:648-655.
10. Asadollahi MA, Maury J, Schalk M, Clark A, Nielsen J: Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiae. Biotechnol Bioeng 2010, 106:86-96. 11. Rico J, Pardo E, Orejas M: Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3methylglutaryl coenzyme A reductase catalytic domain in Saccharomyces cerevisiae. Appl Environ Microbiol 2010, 76:6449-6454. 12. Ignea C, Trikka F, Kourtzelis I, Argiriou A, Kanellis A, Kampranis S, Makris A: Positive genetic interactors of HMG2 identify a new set of genetic perturbations for improving sesquiterpene production in Saccharomyces cerevisiae. Microbial Cell Fact 2012, 11:162.
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).
13. Ignea C, Cvetkovic I, Loupassaki S, Kefalas P, Johnson CB, Kampranis SC, Makris AM: Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids. Microbial Cell Fact 2011, 10:4-22.
References and recommended reading
15. Scalcinati G, Knuf C, Partow S, Chen Y, Maury J, Schalk M, Daviet L, Nielsen J, Siewers V: Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene a-santalene in a fed-batch mode. Metab Eng 2012, 14:91-103.
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Newman DJ, Cragg GM: Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 2012, 75:311-335.
2.
El-Shemy HA, Aboul-Enein AM, Aboul-Enein KM, Fujita K: Willow leaves’ extracts contain anti-tumor agents effective against three cell types. PLoS ONE 2007, 2:e178.
3.
Engels B, Dahm P, Jennewein S: Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. Metab Eng 2008, 10:201-206.
4.
Gazak R, Fuksova K, Marhol P, Kuzma M, Agarwal R, Kren V: Preparative method for isosilybin isolation based on enzymatic kinetic resolution of silymarin mixture. Process Biochem 2013, 48:184-189.
5.
Mavel S, Dikic B, Palakas S, Emond P, Greguric I, de Gracia AG, Mattner F, Garrigos M, Guilloteau D, Katsifis A: Synthesis and biological evaluation of a series of flavone derivatives as potential radioligands for imaging the multidrug resistanceassociated protein 1 (ABCC1/MRP1). Bioorg Med Chem 2006, 14:1599-1607.
Current Opinion in Biotechnology 2014, 25:17–23
14. Fischer MJC, Meyer S, Claudel P, Bergdoll M, Karst F: Metabolic engineering of monoterpene synthesis in yeast. Biotechnol Bioeng 2011, 108:1883-1892. Point mutations of a protein to improve its performance in metabolic engineering is an attractive topic. How to find out the ‘‘point’’, is still a great challenge. This paper may provide some useful clues.
16. Zhou YJJ, Gao W, Rong QX, Jin GJ, Chu HY, Liu WJ, Yang W, Zhu ZW, Li GH, Zhu GF et al.: Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc 2012, 134:3234-3241. 17. Hu FF, Liu JD, Du GC, Hua ZZ, Zhou JW, Chen J: Key cytomembrane ABC transporters of Saccharomyces cerevisiae fail to improve the tolerance to D-limonene. Biotechnol Lett 2012, 34:1505-1509. Previous researches showed that the multi-drug resistance transporters could protect yeast cells from many kinds of PNPs from different origins. In this paper, they show that how incompetent these transporters are under the stress of some specific PNPs. 18. Yang JM, Nie QJ, Ren M, Feng HR, Jiang XL, Zheng YN, Liu M, Zhang HB, Xian M: Metabolic engineering of Escherichia coli for the biosynthesis of a-pinene. Biotechnol Biofuels 2013, 6:60. 19. Sugiyama A, Linley PJ, Sasaki K, Kumano T, Yamamoto H, Shitan N, Ohara K, Takanashi K, Harada E, Hashegawa H: Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases. Metab Eng 2011, 13:629-637. 20. Kiselev KV: Perspectives for production and application of resveratrol. Appl Microbiol Biotechnol 2011, 90:417-425. www.sciencedirect.com
Plant natural products production by fermentation Zhou, Du and Chen 23
21. Becker JVW, Armstrong GO, Merwe MJ, Lambrechts MG, Vivier MA, Pretorius IS: Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the winerelated antioxidant resveratrol. FEMS Yeast Res 2003, 4:79-85. 22. Lim CG, Fowler ZL, Hueller T, Schaffer S, Koffas MAG: High-yield resveratrol production in engineered Escherichia coli. Appl Environ Microbiol 2011, 77:3451-3460. This is the must-to-read paper for those who would like to be invovled in the metabolic engineering of microoroganisms for phenylpropanoids. It contains most of the brilliant ideas about how to improve the yield of this group of PNPs. 23. Park NI, Xu H, Li X, Kim YS, Lee MY, Park SU: Overexpression of phenylalanine ammonia-lyase improves flavones production in transgenic hairy root cultures of Scutellaria baicalensis. Process Biochem 2012, 47:2575-2580. 24. Vannelli T, Wei Qi W, Sweigard J, Gatenby AA, Sariaslani FS: Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi. Metab Eng 2007, 9:142-151. 25. Wang Y, Halls C, Zhang J, Matsuno M, Zhang Y, Yu O: Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metab Eng 2011, 13:455-463. 26. Wu JJ, Du GC, Zhou JW, Chen J: Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy. Metab Eng 2013, 16:48-55. 27. Miyahisa I, Kaneko M, Funa N, Kawasaki H, Kojima H, Ohnishi Y, Horinouchi S: Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster. Appl Environ Microbiol 2005, 68:498-504. 28. Leonard E, Lim KH, Saw PN, Koffas MAG: Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl Environ Microbiol 2007, 73:3877. 29. An JH, Kim YS: A gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putative dicarboxylate carrier protein (MatC) in Rhizobium trifolii. Eur J Biochem 1998, 257:395-402. 30. Xu P, Ranganathan S, Fowler ZL, Maranas CD, Koffas MAG: Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA. Metab Eng 2011, 13:578-587. A perfect case that can show how a genome-scale model could be applied to enhance the design process for an enhanced specific PNP. 31. Koopman F, Beekwilder J, Crimi B, Houwelingen A, Hall R, Bosch D, van Maris A, Pronk J, Daran J-M: De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microbial Cell Fact 2012, 11:155. 32. Santos CNS, Koffas M, Stephanopoulos G: Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab Eng 2011, 13:392-400. Few of the earliest attempts to produce flavonoids from glucose. It shows how to reconstruct a complicated long pathway from different origins in microorganisms and make it to be operational. 33. Glenn WS, Runguphan W, O’Connor SE: Recent progress in the metabolic engineering of alkaloids in plant systems. Curr Opin Biotechnol 2013, 24:354-365. The fundamental information about the biosynthesis of alkaloids in plant systems from this review, provides almost all of the potentials for metabolic engineering of microorganisms to produce more diverse alkaloids. 34. Hawkins KM, Smolke CD: Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat Chem Biol 2008, 4:564-573.
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35. Inui T, Kawano N, Shitan N, Yazaki K, Kiuchi F, Kawahara N, Sato F, Yoshimatsu K: Improvement of benzylisoquinoline alkaloid productivity by overexpression of 30 -hydroxy-Nmethylcoclaurine 40 -O-methyltransferase in transgenic Coptis japonica plants. Biol Pharm Bull 2012, 35:650-659. 36. Minami H, Kim JS, Ikezawa N, Takemura T, Katayama T, Kumagai H, Sato F: Microbial production of plant benzylisoquinoline alkaloids. Proc Natl Acad Sci U S A 2008, 105:7393-7398. 37. Nakagawa A, Minami H, Kim JS, Koyanagi T, Katayama T, Sato F, Kumagai H: A bacterial platform for fermentative production of plant alkaloids. Nat Commun 2011, 2:326. 38. Nakagawa A, Minami H, Kim JS, Koyanagi T, Katayama T, Sato F, Kumagai H: Bench-top fermentative production of plant benzylisoquinoline alkaloids using a bacterial platform. Bioeng Bugs 2012, 3:49-53. 39. Schatz M, Witkowski J, McCombie WR: Current challenges in de novo plant genome sequencing and assembly. Genome Biol 2012, 13:243. 40. McGettigan PA: Transcriptomics in the RNA-seq era. Curr Opin Chem Biol 2013, 17:4-11. 41. Marks DS, Hopf TA, Sander C: Protein structure prediction from sequence variation. Nat Biotechnol 2012, 30:1072-1080. 42. Hillerich BS, Payne J: Advances in protein expression: high throughput tools for improving analysis. Science 2013, 340:382. 43. Xu P, Gu Q, Wang WY, Wong L, Bower AGW, Collins CH, Koffas MAG: Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun 2013, 4:1409. An integrated case to show how researchers could optimize a group of multi-gene pathways by trying the possible combinations on the basis of a semi-rational metabolic network design strategy. 44. Sun N, Zhao HM: Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing. Biotechnol Bioeng 2013, 110:1811-1821. 45. Jiang WY, Bikard D, Cox D, Zhang F, Marraffini LA: RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 2013, 31:233-239. Novel genome engineering strategies on the basis of a virus immune system from bacteria. It may revolutionize the current strategies for the direct genetic engineering on genomes. 46. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013, 152:1173-1183. An easier way to achieve the multi-gene regulation in microorganisms with the CRISPR/dCas9 system. 47. McCloskey D, Palsson BO, Feist AM: Basic and applied uses of genome-scale metabolic network reconstructions of Escherichia coli. Mol Syst Biol 2013, 9:661. 48. Gherardini PF, Helmer-Citterich M: Experimental and computational methods for the analysis and modeling of signaling networks. New Biotechnol 2013, 30:327-332. 49. Brennan TCR, Kro¨mer JO, Nielsen LK: Physiological and transcriptional responses of Saccharomyces cerevisiae to Dlimonene show changes to the cell wall but not to the plasma membrane. Appl Environ Microbiol 2013, 79:3590-3600. 50. Liu JD, Zhu YB, Du GC, Zhou JW, Chen J: Exogenous ergosterol protects Saccharomyces cerevisiae from D-limonene stress. J Appl Microbiol 2013, 114:482-491.
Current Opinion in Biotechnology 2014, 25:17–23
