Biotechnology Advances 32 (2014) 615–622

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Biotechnological production of muconic acid: current status and future prospects Neng-Zhong Xie a, Hong Liang c, Ri-Bo Huang a,⁎, Ping Xu b,⁎⁎ a

State Key Laboratory of Non-Food Biomass Energy and Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning 530007, People’s Republic of China State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China c State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 20 December 2013 Revised 23 March 2014 Accepted 7 April 2014 Available online 18 April 2014 Keywords: Muconic acid Bio-based chemical Rational design Microbial cell factory Systems metabolic engineering

a b s t r a c t Muconic acid (MA), a high value-added bio-product with reactive dicarboxylic groups and conjugated double bonds, has garnered increasing interest owing to its potential applications in the manufacture of new functional resins, bio-plastics, food additives, agrochemicals, and pharmaceuticals. At the very least, MA can be used to produce commercially important bulk chemicals such as adipic acid, terephthalic acid and trimellitic acid. Recently, great progress has been made in the development of biotechnological routes for MA production. This present review provides a comprehensive and systematic overview of recent advances and challenges in biotechnological production of MA. Various biological methods are summarized and compared, and their constraints and possible solutions are also described. Finally, the future prospects are discussed with respect to the current state, challenges, and trends in this field, and the guidelines to develop high-performance microbial cell factories are also proposed for the MA production by systems metabolic engineering. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MA production from aromatic compounds . . . . . . . . . . . . . . . . The ortho-cleavage pathway of catechol . . . . . . . . . . . . . . . Strategies for efficient MA production from aromatic compounds . . . . MA production from renewable resources via de novo pathways . . . . . . MA production by shunting shikimate synthesis via 3-dehydroshikimate MA production by shunting tryptophan synthesis via anthranilate . . . Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Many unsaturated dicarboxylic acids, such as important platform chemicals like fumaric acid (Xu et al., 2012) and itaconic acid (Klement and Büchs, 2013), have industrial significance for their double bond and two carboxylic groups and therefore can be polymerized to produce synthetic resins and biodegradable polymers. Another

⁎ Corresponding author. Tel.: +86 771 2503902; fax: +86 771 2503916. ⁎⁎ Corresponding author. Tel.: +86 21 34206647; fax: +86 21 34206723. E-mail addresses: [email protected] (R.-B. Huang), [email protected] (P. Xu).

http://dx.doi.org/10.1016/j.biotechadv.2014.04.001 0734-9750/© 2014 Elsevier Inc. All rights reserved.

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potentially important unsaturated dicarboxylic acid is muconic acid (MA), also known as 2,4-hexadienedioic acid. There are three isomers of MA designated cis,cis-MA, cis,trans-MA, and trans,trans-MA (Bui et al., 2013; Burk et al., 2011). Traditional chemical processes for MA production rely on non-renewable petroleum-based feedstock and high concentrations of heavy metal catalysts (Pandell, 1976; Tsuji and Takayanagi, 1978), or the processes produce a mixture of two isomers (cis,cis-MA and cis,trans-MA) from expensive catechol (McKague, 1999); consequently, using these processes results in problems of environmental pollution, petroleum depletion, and/or high cost of a separation process. Thus, a sustainable, environmentally friendly and costeffective biotechnological process based on inexpensive carbohydrate

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raw materials is very desirable. Because of its stereospecific configuration along with the reactive dicarboxylic groups and the conjugated double bonds, MA can undergo a wide variety of reactions as a promising building block or intermediate to produce commodity and specialty chemicals. Such products, including commercially important bulk chemicals such as adipic acid, terephthalic acid, and trimellitic acid, have a wide variety of uses in the manufacture of nylon-6,6, polytrimethylene terephthalate, polyethylene terephthalate, dimethyl terephthalate, trimellitic anhydride, industrial plastics, resins, polyester polyols, food ingredients, pharmaceuticals, plasticizers, cosmetics, and engineering polymers (Fig. 1) (Bui et al., 2012; Bui et al., 2013; Burk et al., 2011; Chen et al., 1992; Coudray et al., 2013; Fink, 2005; Frost et al., 2010, 2013a, 2013b, 2013c; Polen et al., 2013; Schweitzer, 2012). In fact, the worldwide consumption of dimethyl terephthalate was 3.97 million metric tons in 2012 (Bui et al., 2013). MA can also be easily hydrogenated to yield adipic acid, which belongs to the top 50 bulk chemicals (Niu et al., 2002; Weber et al., 2012). With an annual global production of approximately 2.8 million metric tons, adipic acid is a versatile building block for an array of processes in the chemical, pharmaceutical and food industries (Burgard et al., 2011; Cavani and Alini, 2009; Van de Vyver and Román-Leshkov, 2013; Wu et al., 2011). Currently, the industrial process accounting for total adipic acid production relies on the catalytic oxidation of cyclohexanol or a cyclohexanol/cyclohexanone mixture with an excess of HNO3. Such a process requires a high energy input and yields large amount of N2O as by-product, corresponding to 5–8% of the worldwide anthropogenic emissions of N2O. N2O is commonly thought to cause global warming, ozone depletion, acid rain, and smog (Bolm et al., 1999; Cavani and Alini, 2009; Van de Vyver and Román-Leshkov, 2013). With regard to the increasing public awareness of environmental protection and sustainable development, conventional industrial processes for such important bulk chemicals production are undesirable, because of their heavy reliance upon environmentally sensitive and non-renewable feedstocks, high-energy input, and propensity to yield undesirable byproducts. In the last two decades, industrial biotechnology has made significant advancements due to its attractive advantages, such as sustainability, high selectivity, mild operation conditions, “green” catalysts, renewable feedstocks, and water-phase systems (Hjeresen et al., 2001; Olguín et al., 2012; Sheldon, 2005; Shin et al., 2013; Xu et al., 2007). Thus, biotechnological routes to the precursor MA could provide a promising alternative to the conventional method for adipic acid production (Polen et al., 2013; van Duuren et al., 2011a). Recently, the development of biotechnological processes for MA production has been pursued with enthusiasm. Many improvements

have been made in developing microbial cell factories by construction of artificial biosynthetic pathways and optimizations of metabolic networks. Here, we provide a comprehensive and systematic overview of the existing and emerging biological methods for producing MA. The future prospects are discussed, and the guidelines to develop superior microbial cell factories by systems metabolic engineering are also proposed. MA production from aromatic compounds The ortho-cleavage pathway of catechol Aromatic compounds comprise approximately one-quarter of the Earth’s biomass and are the second most widely distributed class of organic compounds in nature (Valderrama et al., 2012). Many aromatic compounds, such as benzoate, toluene, benzene, phenol, aniline, anthranilate, mandelate, and salicylate, are oxidized adaptively by some bacteria with the formation of catechol as the central aromatic intermediate through a variety of ring modification reactions (Harwood and Parales, 1996). The aromatic ring of catechol can be cleaved in two ways, the ortho-cleavage pathway and the meta-cleavage pathway, depending on the type of bacteria (Vaillancourt et al., 2006; Wells and Ragauskas, 2012). In the catechol ortho-cleavage pathway, catechol 1,2-dioxygenase (EC 3.1.11.1, CatA) performs intradiol cleavage of the aromatic ring to yield MA (Fig. 2). Benzoate is stable, water soluble and non-volatile, and thus easy to handle in a water-phase system (Wang et al., 2001; Yoshikawa et al., 1990). These advantages, together with its fairly low price, make benzoate one of the most common raw materials for MA production. Some microorganisms belonging to the genus Pseudomonas, Arthrobacter, Corynebacterium, Brevibacterium, Microbacterium, and Sphingobacterium were reported to metabolize benzoate via the catechol branch of the β-ketoadipate pathway to produce MA (Table 1). Benzoate is first converted to benzoate diol catalyzed by benzoate 1,2-dioxygenase (EC 1.14.12.10) encoded by benABC. Then, the oxidative decarboxylation of benzoate diol to catechol is performed by benzoate diol dehydrogenase (EC 1.3.1.25) encoded by benD (Harwood and Parales, 1996). Ring fission of catechol between the hydroxyl groups is catalyzed by CatA encoded by catA to form MA, and the latter metabolite is then converted into muconolactone by muconate cycloisomerase (EC 5.5.1.1) encoded by catB. Muconolactone is finally converted to tricarboxylic acid cycle intermediates after several more metabolic steps (Fig. 2) (Vaillancourt et al., 2006; Wells and Ragauskas, 2012).

Fig. 1. Summary of the industrial products derived from three isomers of MA.

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Strategies for efficient MA production from aromatic compounds In general, because the metabolite MA is further degraded by muconate cycloisomerase to muconolactone, techniques for MA biosynthesis from aromatic compounds require the generation of mutant strains that are capable of catabolizing aromatic compounds to MA, but are defective in MA assimilation (Fig. 2). To produce MA efficiently, mutants with the following properties will be required: (i) metabolizing an aromatic substrate via catechol by means of the ortho-cleavage pathway; (ii) lacking functional muconate cycloisomerase, hence allowing the accumulation of MA without its further assimilation; (iii) resistant to an aromatic substrate that is toxic to microorganisms; (iv) excreting MA into the medium; (v) showing high CatA activity (modified, based on van Duuren et al., 2012; Yoshikawa et al., 1990). Because aromatic substrates are biologically toxic for microorganisms (Bang and Choi, 1995), the mutants with high tolerance and efficiency are especially important for improving bioprocesses. Recently, Xie et al. (2014) used a benzoate-tolerant mutant of Pseudomonas sp. to develop an efficient process, from which 7.2 g/L MA from 12 g/L benzoate was obtained within 11 h at shake-flask level. To further avoid substrate toxicity, fed-batch processes should be developed to control the substrates concentrations below the inhibitory level (Bang and Choi, 1995). The DO-stat fed-batch culture was commonly employed for growth-inhibiting substrates; Bang and Choi (1995) performed a fed-batch process based on computer-controlled DO-stat feeding, and more than 32.4 g/L MA was accumulated over 40 h by using a mutant strain of Pseudomonas putida BM014. Considering cell concentration is another important factor in highly efficient processes, Choi et al. (1997) developed a cell-recycle bioreactor system with a high cell concentration (40 g/L) to enhance MA productivity. Nutrients were fed for cell growth and enzymes synthesis, and benzoate was fed as the substrate for MA production. After a period of fed-batch operation, continuous production with cell recycling was performed, from which 13.5 g/L MA was accumulated, with an enhanced volumetric productivity of

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5.5 g/L h. This cell-recycle bioreactor for continuous production could be maintained for more than 7 days. Mizuno et al. (1988) used a blocked mutant of Arthrobacter sp. to develop a fed-batch process by feeding 5 g/L benzoate with a small amount of nutrients at 24 h intervals, which gave a concentration of 40 g/L MA after 256 h bioconversion in a 30 L fermentor. To further improve the concentration, yield, and productivity, smaller amounts of benzoate (approximately 2.1 g/L) were added into the culture at 2 h intervals, and 44.1 g/L MA was accumulated over 48 h. The strain P. putida KT2440-JD1, derived from P. putida KT2440 by random mutagenesis, is the first MA-accumulating mutant characterized at the genetic level (van Duuren et al., 2011b, 2012). Transcriptome and proteome analysis indicated that the cat operon in this mutant was no longer induced by 5 mM benzoate for a single point mutation in the conserved DNA-binding domain of CatR, the transcriptional regulator of the cat operon. Since the ben gene cluster contains the catA2 gene encoding a second catechol 1,2-dioxygenase besides catA, the mutant was still able to produce MA from benzoate with a specific production rate at least eight times higher than those reported for other MAproducing strains (van Duuren et al., 2011b, 2012). Another common aromatic substrate is toluene. Utilizing double-blocked, antibioticresistant and muconate-permeable mutation strategies, a mutant of Pseudomonas sp. showed the capacity to produce 45 g/L MA over 4 d in a 14 L fermentor, by successively feeding small amounts of toluene (Chua and Hsieh, 1990). Compared to benzoate, toluene is difficult to control in microbial processes because of its flammability and volatility (Yoshikawa et al., 1990). CatA is a key enzyme in the ortho-cleavage pathway for catalyzing intradiol ring fission of catechol to yield MA (Guzik et al., 2013; Vaillancourt et al., 2006). Most CatAs studied to date are dimmers (αFe3+)2 with identical or similar subunits connected by helical zipper motif. Some metal ions can lead to conformational changes such as a reduction in α-helices and β-sheets in the CatA or replacement of the original Fe3 + at the catalytic domains, resulting in loss of enzymatic

Fig. 2. MA biosynthesis via the ortho-cleavage pathway of catechol from aromatic compounds (modified, based on Harwood and Parales (1996)).

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Table 1 Summary of research studies on the biotechnological production of MA. Microorganism

Substrate

Method

Engineered S. cerevisiae strain Sphingobacterium sp. GCG Engineered S. cerevisiae strain MuA12 Engineered E. coli strain MA-4 Sphingobacterium sp. mutant strain M4115 Corynebacterium lilium ATCC 21793 Engineered E. coli strain AB2834

Glucose Benzoate Glucose Glucose and glycerol Benzoate Benzoate Glucose

Batch Batch Batch Batch Batch Batch Batch

Corynebacterium acetoacidophilum ATCC 13870 Microbacterium ammoniaphilum ATCC 21645 Brevibacterium flavum ATCC 13826 Arthrobacter sp. mutant strain T-8626-11 Pseudomonas sp. mutant strain 1167 Corynebacterium pseudodiphtheriticum mutant strain M2128 P. putida ATCC 31916 P. putida ATCC 31916 Pseudomonas sp. strain B13 P. putida ATCC 31916 P. putida mutant strain BM014 P. putida ATCC 31916 Engineered E. coli strain MYR428 P. putida mutant strain KT2440-JD1 P. putida ATCC 31916 P. putida mutant strain BM014 Engineered E. coli strain WN1/pWN2.248 Arthrobacter sp. mutant strain T8626 Pseudomonas sp. mutant strain DCB-71. Engineered E. coli BL21 (DE3)/pEcatA Engineered E. coli strain WN1/pWN2.248

Benzoate Benzoate Benzoate Benzoate Benzoate Benzoate Catechol Toluene Benzoate Toluene Benzoate Toluene Glucose Benzoate Toluene Benzoate Glucose Benzoate Toluene Catechol Glucose

Batch Batch Batch Batch Batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch Fed-batch

activity. Moreover, transition metals may bind the thiol groups in the protein to deactivate CatA (Guzik et al., 2013; Harwood and Parales, 1996; Vetting and Ohlendorf, 2000). Several attempts to convert catechol to MA by use of immobilized CatA have been reported (Li and Li, 1994; Smith and Retledge, 1989). The use of immobilized CatA rather than free enzymes in biotransformation is advantageous to enhance the stability of the biocatalyst, facilitate enzyme recovery and reuse, eliminate byproducts, and reduce the cost of product recovery and purification (Kalogeris et al., 2006; Smith and Retledge, 1989; Vlakh and Tennikova, 2013). However, the preparation of crude enzyme, immobilization process, and expensive feedstock (catechol) would significantly increase the production cost and hamper its use for industrial production. Recombinant Escherichia coli expressing the catA gene offers a simple method of MA production from catechol. Recently, Kaneko et al. (2011) described a whole-cell biocatalytic process with a high yield and productivity by recombinant E. coli cells expressing the catA gene from P. putida mt-2. The recombinant strain produced 59.0 g/L MA in 12 h with successive additions of catechol. The molar conversion yield almost reached 100% of theoretical value. Since no by-product was accumulated in the reaction mixture, MA was easily separated through an ion-exchange column (Kaneko et al., 2011). The product of CatA is cis,cis-MA, which can be isomerized to cis,trans- or trans,trans-isomers catalyzed by isomerization catalysts. trans,trans-MA can have unique utility over the cis,cis-isomer as a direct synthetic intermediate in Diels-Alder reaction for producing commercially important bulk chemical terephthalic acid (Frost et al., 2013c). Although the biotechnological routes above have made significant progress with high concentrations, yields and productivities, they are established on the basis of the petrochemical industry. An alternative might be to use benzoate, catechol, or related monomeric aromatic compounds as feedstocks derived from lignocellulosic materials (Joffres et al., 2013; Zakzeski and Weckhuysen, 2011); however, at the present state, it is impossible to develop an economically feasible technical route based on the existing conversion and separation technologies of lignocelluloses.

Concentration (g/L) 0.002 0.1 0.14 0.39 0.56 1.4 2.4 2.5 2.5 2.6 4.5 7.2 3.1 5.3 5.7 7.4 12.6 13.5 15.0 16.0 18.5 27.0 32.4 36.8 44.1 45.0 59.0 59.2

Yield (mol/mol)

Reference

0.01% 4% 0.89% 2.2% 28% 4.7% 30%

Weber et al. (2012) Wu et al.(2004) Curran et al. (2013) Sun et al. (2013) Wu et al. (2006) Imada et al. (1989) Draths and Frost (1994), Frost and Draths (1996, 1997) Imada et al. (1989) Imada et al. (1989) Imada et al. (1989) Imada et al. (1989) Xie et al. (2014) Liu et al. (2003) Maxwell (1986, 1988, 1991) Maxwell (1982) Schmidt and Knackmuss (1984) Hsieh (1984) Choi et al. (1997) Hsieh et al. (1985) Yocum et al. (2013) van Duuren et al. (2012) Hsieh (1990) Bang and Choi (1995), Bang et al. (1996) Niu et al. (2002) Mizuno et al. (1988) Chua and Hsieh (1990) Kaneko et al. (2011) Bui et al. (2011, 2013)

8.5% 8.5% 8.8% 91% 61% 47% 100% – 91% – – – – 100% – 100% 22% 96% – 100% 30%

MA production from renewable resources via de novo pathways At the present stage, none of the aforementioned methods eliminate the problem of toxic and petroleum-based raw materials. Draths and Frost (1994) first reported an alternative route for MA biosynthesis by an engineered E. coli strain from D-glucose, which can be derived from starch and lignocellulose found in corn, sugar cane, sugar beets, wood pulp, and other biomass resources. This promising approach based on a de novo biosynthetic pathway allows for more environmentally friendly and sustainable production of MA than methods reported previously. For their outstanding contribution to microbial synthesis of chemicals, the U.S. Environmental Protection Agency (EPA) presented one of its 1998 Presidential Green Chemistry Challenge Awards to Frost and Draths (Bolm et al., 1999).

MA production by shunting shikimate synthesis via 3-dehydroshikimate Biocatalytic conversion of D-glucose into MA requires constructing an artificial biosynthetic pathway that is not known to exist naturally (Fig. 3, indicated in box). This novel pathway constructed by metabolic engineering is based on the shikimic acid pathway, which is present in microorganisms and plants as the biosynthetic route of aromatic amino acids. The shikimic acid pathway begins with the condensation of central intermediates phosphoenolpyruvate (PEP) and erythrose-4phosphate (E4P) to form 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) catalyzed by DAHP synthase (EC 2.5.1.54) in E. coli. DAHP synthase is a critical factor for regulating carbon flux from central metabolism into the shikimic acid pathway. In E. coli, DAHP synthase, occurring as three different isozymes encoded by the aroF, aroG and aroH, is regulated via feedback inhibition by the individual aromatic amino acids L-tyrosine, L-phenylalanine, and L-tryptophan, respectively (Chen et al., 2013; Ikeda, 2006). In the second step, DAHP is converted into 3-dehydroquinate (DHQ) catalyzed by DHQ synthase (EC 4.2.3.4, encoded by aroB). Thereafter, DHQ is converted by DHQ dehydratase (EC 4.2.1.10, encoded by aroD) to form 3-dehydroshikimate (DHS), which is then reduced to shikimic

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acid by shikimate dehydrogenase (EC 1.1.1.25, encoded by aroE), whereby NADPH is consumed (Ghosh et al., 2012). A shikimate dehydrogenase-deficient E. coli mutant was preferred as the host organism to constitutively express heterologous genes aroZ (encoding 3-dehydroshikimate dehydratase, AroZ), aroY (encoding protocatechuate decarboxylase, AroY) and catA (Bui et al., 2013; Niu et al., 2002). In the absence of shikimate dehydrogenase, the shikimic acid pathway is blocked at the level of DHS, and the transformant is unable to convert DHS into shikimic acid and thus accumulating elevated intracellular levels of DHS. The intermediate DHS serves as a substrate for AroZ to produce protocatechuate, thus redirecting carbon flux from the shikimic acid pathway into the artificial biosynthetic pathway. Then, AroY catalyzes the conversion of protocatechuate to catechol, and CatA subsequently converts catechol to MA (Fig. 3). In wild-type E. coli, the shikimic acid pathway is tightly regulated (Yocum et al., 2013). Approaches to increase carbon flow from central metabolism into the shikimic acid pathway involve deregulation of feedback inhibition and transcriptional repression, and overexpression of critical genes by genetic manipulation (Berry, 1996; Bongaerts et al., 2001; Chávez-Béjar et al., 2012; Ghosh et al., 2012; Gosset, 2009; Ikeda, 2006). The supply of intracellular precursors is one of the critical factors affecting MA yield and productivity. Inactivation of pyruvate kinases and the PEP-dependent phosphotransferase system (PTS) and amplified expression of transketolase and transaldolase could effectively increase the supply of the precursors E4P and PEP for DAHP synthesis. For example, a 19.9-fold increase of carbon flux from glucose into the shikimic acid pathway was found when using a PTS-negative mutant, additionally inactivating both pyruvate kinases and overexpressing the transketolase (Gosset et al., 1996). Additionally, disruption of the global regulatory gene csrA could increase gluconeogenesis and decrease glycolysis, thus significantly increasing intracellular PEP concentration (Tatarko and Romeo, 2001). Overcoming the transcriptional repression and feedback inhibition of DAHP synthase, by inactivation of the transcriptional repressor TyrR and amplified expression of feedback inhibition resistant DAHP synthase, could also significantly increase carbon flux into the shikimic acid pathway for synthesis of DAHP at a higher rate. When an increase in carbon flux was redirected to the shikimic acid pathway, DHQ synthase was found to be a rate-

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limiting enzyme of the shikimic acid pathway (Dell and Frost, 1993), and therefore the catalytic activity of DHQ synthase should be increased to take full advantage of elevated carbon flux into the pathway. Niu et al. (2002) developed an engineered E. coli WNl/pWN2.248 by inactivation of shikimate dehydrogenase, overexpression of DHQ synthase and transketolase, plasmid-borne aroFFBR (FBR, feedback inhibition resistant), and the heterologous genes aroZ, aroY and catA. This engineered strain produced 36.8 g/L MA with a yield of 22% (mol/mol) within 48 h of cultivation under fed-batch conditions. Through further optimization of the fermentation process using the same engineered strain, the MA concentration increased to 59.2 g/L (30% yield, mol/mol) after 88 h in the fed-batch culture (Bui et al., 2013), which represents the highest reported concentration of MA under fed-batch conditions. To further improve the performance of the microbial cell factory and lower production cost, continued efforts should be made to solve several problems. First, the aroE gene encoding shikimate dehydrogenase of E. coli WNl/pWN2.248 was completely inactivated leading to a block in the biosynthesis of aromatic amino acids and other aromatic metabolites (Fig. 3). As a result, when culturing this auxotroph in minimal salts medium, it would be necessary to supply L-phenylalanine, L-tyrosine, L-tryptophan, p-aminobenzoic acid, p-hydroxybenzoic acid and 2,3-dihydroxybenzoic acid, or shikimic acid for cell growth and enzymes synthesis (Bui et al., 2013; Niu et al., 2002). To circumvent this problem, homologous recombination mediated approaches may be used to construct engineered strains by replacing wild-type aroE gene with a leaky aroE mutant, which would allow a limited flow of carbon to shikimic acid for synthesis of aromatic amino acids and other aromatic metabolites, thus eliminating the dependence on exogenous addition and reducing the production cost. Other problems include the need for maintaining multi-copy plasmids with antibiotics and inducing expression with an expensive chemical inducer (isopropyl βD-1-thiogalactopyranoside), which is unstable and costly for use in industrial-scale fermentation. Therefore, improved processes are needed to stably integrate expression cassettes into the chromosome by using multi-copy genes as well as efficient promoters for high-level constitutive expression (Yocum et al., 2013). Furthermore, the specific activity of heterologous enzymes AroZ, AroY, and CatA varies widely among organisms, so it is better to introduce those enzymes with higher

Fig. 3. MA biosynthesis by shunting shikimate synthesis via 3-dehydroshikimate. E4P, erythrose 4-phosphate; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-D-arabino-heptulosonate-7phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; AroZ, 3-dehydroshikimate dehydratase; AroY, protocatechuate decarboxylase; CatA, catechol 1,2-dioxygenase (modified, based on Draths and Frost (1994)).

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specific activity or optimized codons. Meanwhile, the specific activity and stability of DAHP synthase, DHQ synthase, transketolase and transaldolase can be improved by directed enzyme evolution. Saccharomyces cerevisiae, with several beneficial properties (e.g., lower pH fermentations, high tolerance, and resistance to microbial contamination), is another common host organism for bio-based chemicals production. More recently, several attempts were also made to produce MA from glucose by engineered S. cerevisiae strains expressing heterologous aroZ, aroY and catA genes (Fig. 3) (Curran et al., 2013; Weber et al., 2012). Metabolic engineering, including knockout of ARO3 and zwf1 genes, overexpression of TKL1 and aro4FBR, and balance of heterologous enzyme levels, was used to reduce feedback inhibition and increase precursor availability. Those genetic modifications led to a final concentration of 141 mg/L MA by nearly 24-fold over the initial strain (Curran et al., 2013). Further substantial improvements of engineered strains based on rational metabolic design may allow for higher concentrations and yields. Nevertheless, because the metabolic system of S. cerevisiae is too complex to accurately predict the consequences of metabolic modifications compared to E. coli, S. cerevisiae is more difficult to genetically manipulate for target production (Kondo et al., 2013; Weber et al., 2012), which suggests that it may be not an ideal host organism for MA production. MA production by shunting tryptophan synthesis via anthranilate In E. coli, the shikimic acid pathway proceeds via a number of intermediates to chorismate, a branch point for the three aromatic amino acids (Fig. 4) (Bongaerts et al., 2001; Gosset, 2009). In the tryptophan biosynthetic pathway, the reaction converting chorismate to anthranilate is catalyzed by anthranilate synthase, which is composed of TrpE and the N-terminal domain of TrpD. The C-terminal domain of TrpD has anthranilate phosphoribosyl transferase activity, which catalyzes the conversion of anthranilate to N-phosphoribosyl anthranilate (Balderas-Hernández et al., 2009). According to Balderas-Hernández et al., high gram per liter levels of anthranilate can be obtained by amplified expression of transketolase, glucokinase, galactose permease, and feedback inhibition resistant DAHP synthase, as well as inactivation of PTS in an anthranilate phosphoribosyl transferase-deficient E. coli mutant. The final engineered strain was able to produce 14 g/L of anthranilate from glucose after 34 h of cultivation under fed-batch condition.

More recently, Sun et al. (2013) established a novel MA-producing pathway by grafting the heterologous anthranilate degradation pathway onto the tryptophan biosynthetic pathway. In this artificial biosynthetic pathway, anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted sequentially to catechol and MA by heterologous anthranilate 1,2-dioxygenase (ADO) from Pseudomonas aeruginosa and CatA from P. putida (Fig. 4). By amplified expression of feedback inhibition resistant anthranilate synthase, and heterologous expression of ADO and CatA, the resultant engineered E. coli MA-3 produced 120.9 mg/L MA coupled with 209.2 mg/L anthranilate after 48 h at a shake-flask level. Higher concentrations of MA were obtained by metabolic network optimization based on an anthranilate phosphoribosyl transferase-deficient E. coli mutant. By overexpressing critical genes, including aroB, aroE, aroL, ppsA, tktA, aroGFBR and trpEFBRG, more carbon flux was directed into the pathway, and the concentration of anthranilate reached 472.2 mg/L. A further increase in anthranilate (689.6 mg/L) was achieved by introducing a glutamine regeneration system by expression of glutamine synthase (Fig. 4). Finally, a concentration of 390.0 mg/L MA without anthranilate accumulation from simple carbon sources was obtained by heterologous expression of ADO and CatA after 32 h in shake-flasks (Sun et al., 2013). In consideration of the high-level production of tryptophan (48.7 g/L) that could be obtained by engineered E. coli (Wang et al., 2013), it may be promising to use this artificial pathway to produce high levels of MA by further optimization of the metabolic network and fermentation process. However, this MA-producing pathway requires input of additional PEP (Bongaerts et al., 2001; Ghosh et al., 2012), which doubles the amount of intracellular precursor PEP consumed to produce MA over the shorter route via 3-dehydroshikimate. In addition to the two heterologous biosynthetic pathways converting intermediates 3-dehydroshikimate or anthranilate into MA (aforementioned), engineered E. coli strains having other artificial synthetic pathways can also produce MA from succinyl-CoA and acetyl-CoA, pyruvate and malonate semialdehyde, pyruvate and succinic semialdehyde, or lysine (Burk et al., 2011). Conclusions and future prospects MA is a promising bulk chemical due to its extensive industrial applications. So far, many efforts have been devoted to develop biotechnological routes to produce MA by transformation of aromatic compounds,

Fig. 4. MA biosynthesis by shunting tryptophan synthesis via anthranilate. E4P, erythrose 4-phosphate; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-D-arabino- heptulosonate-7phosphate; DHQ, 3-dehydroquinate; ADO, anthranilate 1,2-dioxygenase; CatA, catechol 1,2-dioxygenase (modified, based on Sun et al. (2013)).

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making significant progress with respect to both yields and concentrations (Table 1). However, there are growing concerns about sustainability, environmental deterioration, and petroleum depletion. Therefore, microbial MA production by de novo biosynthetic pathways in E. coli from abundant and renewable carbon sources with low costs will be the preferred method in the long run. More recently, fermentative production of MA from glucose was successfully carried out by engineered E. coli with a concentration of 59.2 g/L (30% yield, mol/mol) (Bui et al., 2013). Still, with a view to high concentration, the yield and productivity generally needed for the production of bulk chemicals suitable for commercial application (Kumar et al., 2013), efforts should be made to further improve this biotechnological route to be competitive when compared with petroleum-based technologies. Future researches should focus on systematic and integrative strategies to improve the concentration, yield and productivity by rational metabolic design for industrialscale fermentation. Recent advances in systems metabolic engineering, which integrates metabolic engineering with systems biology, synthetic biology, protein engineering, and evolutionary engineering, provide significant opportunities to further understand metabolic and regulatory mechanisms, identify suitable targets, and offer a set of methodological and strategic tools for rational design and optimization of metabolic networks and downstream processing (Lee et al., 2011, 2012; Sagt, 2013; Yang et al., 2013). Combining with advanced gene manipulation techniques, such as multicistronic expression systems, marker-recycle gene deletion, protein engineering, cell genome editing, and synthesis of very long DNA fragments, systems metabolic engineering is becoming an increasingly powerful tool for designing rational strategies to develop superior microbial cell factories for bio-based chemicals and materials production (Kondo et al., 2013). According to the hitherto published reports (Lee et al., 2012; Park et al., 2007; Sagt, 2013; Shimizu, 2013; Shin et al., 2013; Stephanopoulos, 2012; Yang et al., 2013), we present a scheme for developing high performance E. coli cell factories for MA production by systems metabolic engineering as follows: (i) construction of a genome-wide metabolic model based on multi-omics analyses; (ii) computer simulation for prediction of MA production to screen candidate metabolic engineering target genes; (iii) modification of the metabolic model through manipulation of target genes by using computer simulation and an extraction strategy for rational strain improvement; (iv) optimization of metabolic networks and construction of robust biocatalysts through synthetic biology, protein engineering and pathway engineering to remove negative regulations, delete competing pathways, maintain flux balance, improve tolerance levels, and amplify the rate-controlling biosynthetic pathways; (v) evaluation of various fermentation performance data, including the product yield, concentration, productivity and byproduct formation, and refinement of the strain design by repeating the above procedures; (vi) optimization of medium and process and scaled-up MA fed-batch fermentation using the final engineered strain suitable for industrial-scale fermentation. A convenient way is to use industrial strains for aromatic amino acids production as the parent strains to develop superior microbial cell factories by systems metabolic engineering. Compared to wild-type E. coli, the performance of industrial strains has been improved considerably by a history of classical mutagenesis and selection, which indicates that some beneficial redirection of carbon flux from central metabolism into the shikimate pathway has already occurred. Acknowledgments The work was supported by National Natural Science Foundation of China (31360207), Guangxi Science Foundation (2012GXNSFBA053063) and Guangxi Academy of Sciences (12YJ25SW01).

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