Bioresource Technology 166 (2014) 64–71

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Metabolic engineering of Escherichia coli for improving shikimate synthesis from glucose Xianzhong Chen a,⇑, Mingming Li a, Li Zhou a, Wei Shen a, Govender Algasan c, You Fan a, Zhengxiang Wang b a b c

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education, Tianjin University of Science & Technology, Tianjin 300457, China Department of Biotechnology & Food Technology, Faculty of Applied Sciences, Durban University of Technology, P.O. Box 1334, Durban 4001, South Africa

h i g h l i g h t s  Combined engineering quinate and acetate pathway decreased byproducts production.  Modular expression of aroG, ppsA and tktA genes enhanced shikimate titer.  Fed-batch process increased fermentation performance.

a r t i c l e

i n f o

Article history: Received 2 March 2014 Received in revised form 10 May 2014 Accepted 12 May 2014 Available online 21 May 2014 Keywords: Shikimate Escherichia coli Metabolic engineering Aromatic amino acid biosynthesis

a b s t r a c t Shikimate is a key intermediate for the synthesis of the neuraminidase inhibitors. Microbial production of shikimate and related derivatives has the benefit of cost reduction when compared to traditional methods. In this study, an overproducing shikimate Escherichia coli strain was developed by rationally engineering certain metabolic pathways. To achieve this, the shikimate pathway was blocked by deletion of shikimate kinases and quinic acid/shikimate dehydrogenase. EIICBglc protein involved in the phosphotransferase system, and acetic acid pathway were also removed to increase the amount of available phosphoenolpyruvate and decrease byproduct formation, respectively. Thereafter, three critical enzymes of mutated 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (encoded by aroGfbr), PEP synthase (encoded by ppsA), and transketolase A (encoded by tktA) were modularly overexpressed and the resulting recombinant strain produced 1207 mg/L shikimate in shake flask cultures. Using the fed-batch process, 14.6 g/L shikimate with a yield of 0.29 g/g glucose was generated in a 7-L bioreactor. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Shikimate (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) is a hydroaromatic intermediate produced in the common pathway for aromatic amino acid biosynthesis. Nowadays, it has become a promising candidate as an attractive building block for the synthesis of biologically important compounds due to its unique structure (Kramer et al., 2003). More importantly, it has been widely used as a chiral template for the synthesis of the antiviral drug (Tamiflu) used in the treatment of swine/avian flu (Knop et al., 2001; Ghosh et al., 2012). Shikimate has also been applied in the fields of organic chemistry and cosmetic industries (Ghosh et al., 2012). Currently, shikimate is mainly produced by chemical synthesis or extraction from the fruit of Illicium spp. However, these processes ⇑ Corresponding author. Tel./fax: +86 510 85918122. E-mail address: [email protected] (X. Chen). http://dx.doi.org/10.1016/j.biortech.2014.05.035 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

are complicated with the high cost and/or limitations of raw materials making it difficult to meet the increasing worldwide requirements due to the global pandemic of influenza (Ghosh et al., 2012). Thus, microbial fermentation process for shikimate production has been viewed as an alternative, sustainable approach to achieve large scale production of this expensive chemical (Kramer et al., 2003; Ghosh et al., 2012). The shikimate pathway is responsible for the biosynthesis of aromatic amino acids and a large number of other aromatic compounds in plants and microorganisms. (Herrmann and Weaver, 1999; Abbott, 2005). This pathway in Escherichia coli (displayed in Fig. 1) has been widely investigated and metabolically engineered for the biosynthesis of various interesting products (Koma et al., 2012; Kim et al., 2013). In the shikimate pathway, the first reaction for forming 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) was catalyzed by DAHP synthase isoenzmyes encoded by the genes aroF, aroG, and aroH, which are

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Fig. 1. Metabolic network related to shikimate biosynthesis in E. coli. Tandem dashed arrows indicate multiple enzyme reactions. TCA, tricarboxylic acid; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; E4P, erythrose-4-phosphate; ackA, acetate kinase; pta, phosphoacetyl transferase; G-PTS, glucose phosphoenolpyruvate:carbohydrate phosphotransferase system; genes in red color and blue color indicates deletion and overexpression targets, respectively. Genes (in italic) are aroD, 3-dehydroquinate dehydratase; aroK, shikimate kinase I gene; aroL, shikimate kinase II gene; ptsG, IICBGlc, integral membrane glucose permease gene; tktA, transketolase A gene; pykF, pyruvate kinase I; pykA, pyruvate kinase II; ppsA, phosphoenolpyruvate synthase; aroF, aroG, aroH, DAHP synthase isoenzyme genes; ydiB, shikimate dehydrogenase isozyme gene. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

feedback-inhibited by tyrosine, phenylalanine and tryptophan, respectively (Patnaik and Liao, 1994). The resulting DAHP is further catalyzed through three steps to form shikimate. In turn, shikimate is transformed to shikimate-3-phosphate by the shikimate kinase isoenzymes I and II, encoded by the aroK and aroL genes, respectively (Fig. 1). Shikimate-3-phosphate is then transformed to chorismic acid, which then is converted as a common precursor into phenylalanine, tyrosine, tryptophan and other aromatic products. Several metabolic engineering approaches have been developed to overproduce shikimate and other aromatic compounds from E. coli (Knop et al., 2001; Chen et al., 2012; Ghosh et al., 2012;

Koma et al., 2012; Rodriguez et al., 2013). Reported strain constructs have been successfully applied to produce 71 g/L of shikimate with a yield of 0.27 mol/mol glucose and total aromatic compound yield (including shikimate, 3-dehydro shikimate and quinic acid) of 0.34 mol/mol glucose in 1-L fed-batch cultures (Chandran et al., 2003). Escalante et al. (2010) found that the simultaneous inactivation of phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and pykF as part of a strategy could improve shikimate production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol, however the productivity of shikimate

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seemed to be very low. It is difficult to obtain construct strains with a high yield and productivity of shikimate as channeling the flow of carbon to shikimate synthesis is insufficient (Ghosh et al., 2012). Recently, antisense RNA interference and gene deletion were employed to inactivate the aroK gene in a shikimate producing E. coli strain with deleting aroL, ptsHIcrr and ydiB, and overexpressing tktA, glk, aroE and aroB genes. The resulting strain produced 1.85 g/L shikimate using glycerol as carbon source in a 10-L fermentor (Chen et al., 2012). Based on the inactivation of ptsHIcrr, aroK, aroL, pykF and lacI genes in E. coli, Rodriguez et al. (2013) constructed a high-yield shikimate strain through constitutive expression of selected genes from the pentose phosphate and aromatic pathways. The best producing strain accumulated up to 43 g/L of shikimate using a high-substrate batch process in 30 h and relatively low concentrations of acetate and aromatic byproducts were detected (Rodriguez et al., 2013). In addition, Bacillus subtilis as a host was also genetically engineered to overproducing shikimate and 4.67 g/L shikimate and 6.2 g/L dehydroshikimate were produced after inactivation of pyruvate kinase and shikimate kinase activity (Licona-Cassani et al., 2014). Previously, we isolated a wild type strain E. coli B0013 based on its ability to grow quickly using glucose and/or xylose as a carbon source. In this study, we employed this strain to increase shikimate production by systems metabolic engineering. Rational design combined with the scale-up process optimization made the engineered E. coli overproduce shikimate directly from glucose with titer of 15 g/L and a volumetric productivity of 0.26 g/L/h, respectively. 2. Methods 2.1. Bacterial strains, plasmids and oligonucleotides Strains and plasmids used in this study are listed in Table 1 and oligonucleotides used in this study in Table S1 (Supporting information). E. coli JM109 was used as host for plasmid construction. The wild type strain E. coli B0013, which was previously isolated and preserved in our lab, was engineered as the parental strain to originate derivates for overproducing shikimate. Strains were cultivated in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) during recombinant plasmid construction. SOB medium (20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 2.5 mM KCl and 10 mM MgCl2) was used for gene knockout. Various concentrations of antibiotics (ampicillin 100 lg/mL, gentamicin

30 lg/mL, kanamycin 25 lg/mL) were added to culture media of plasmid-bearing E. coli strains. 2.2. Inactivation of genes In this study, shikimate kinase genes aroL and aroK, ptsG encoding EIICBglc protein, quinic acid/shikimate dehydrogenase gene ydiB, pyruvate kinase I gene pykF and acetate kinase-phosphate acetyltransferase gene ackA-pta were rendered inactive using the Red/dif recombination system in E. coli B0013 (Bloor and Cranenburgh 2006; Zhou et al. 2011). The dif sites were targeted by the naturally existing Xer recombinases in E. coli which were enlisted to excise antibiotic resistance genes such as GmR (Gentamicin). Antibiotic-resistant gene cassettes flanked by dif sites and homologous arms of target genes were amplified by PCR with pSK-difGm as template. After DpnI digestion and DNA purification, PCR products was electroporated into E. coli cells harboring plasmid pKD46 expressing Red recombinase. Positive clones were selected using relevant antibiotics and confirmed by PCR analysis. After elimination of plasmid pKD46, resistance genes were removed from the chromosome with native Xer recombinase. Temperature-sensitive plasmids pKD46 were removed by overnight growth at 37 °C. 2.3. DAHP synthase gene (aroG) mutation Directed-site mutagenesis of aroG (mutant sites:D146 N) was introduced by overlap extension polymerase chain reaction (OEPCR) (Heckman and Pease, 2007). Two segments S1 and S2 were cloned from the E. coli 0013 genome with aoG-up/aoGm-dw and aoGm-up/ aoG-dw as primers, respectively. Changed nucleotides were contained in corresponding primers (Table 2). Segments S1 and S2 were spliced together by OE-PCR with primers aoG-up and aoG-dw, S1 and S2 were used as templates with a 1:1 M ratio. The resulting PCR product was purified and ligated into the pMD18-T vector, and the successfully mutated gene was verified by DNA sequencing. The mutant aroG gene was named as aroGfbr. 2.4. Plasmid construction Standard procedures were carried out for polymerase chain reactions (PCR), DNA purifications, enzyme digestions, ligation reactions and plasmid extractions (Sambrook et al., 1989). All constructed plasmids described below were verified by restriction

Table 1 Strains and plasmids used in this study. Plasmids and strains

Relevant genotype and characteristics

Source or Reference

Plasmids pMD18-T vector pSK-difGm pKD46 pMD-ackA-pta’::difGm pTH18kr pTH-aroGfbr pTH-aroGfbr-ppsA pTH-aroGfbr-ppsA-tktA

TA cloning GmR cassette flanked by dif sequence ApR, helper plasmid Disrutpion cassette of ack-pta genes pSC101ori,POlac, KmR pTH18kr carrying aroGfbr pTH18kr carrying aroGfbr and ppsA pTH18kr carrying aroGfbr, ppsA and tktA

TaKaRa, Dalian, China Our lab CGSC (Zhou et al. 2012) (Hashimoto-Gotoh et al. 2000) This study This study This study

Strains E. coli JM109 E. coli W3110 E. coli B0013 SA1 SA2 SA3 SA4 SA4⁄ SA5

Cloning host Control strain, F k IN(rrnD–rrnE)1 rph-1 Wild type B0013, DaroL::dif B0013
DaroL::dif, DaroK::dif B0013
DaroL::dif, DaroK::dif, DptsG::dif B0013
DaroL::dif, DaroK::dif, DptsG::dif, DydiB::dif B0013
DaroL::dif, DaroK::dif, DptsG::dif, DpykF::dif B0013
DaroL::dif, DaroK::dif, DptsG::dif, DydiB::dif, DackA-pta::dif

Our lab CGSC Our lab This study This study This study This study This study This study

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X. Chen et al. / Bioresource Technology 166 (2014) 64–71 Table 2 Cell mass, glucose consumption, shikimate production, and byproduct production by wild-type and engineered strains in shake-flask experiments.a Strains

Biomass (g/L)

Specific growth rate l (1/h)b

Glucose consumed (g/L)

Shikimate (mg/L)

Acetic acid (g/L)

Quinic acid (mg/L)

W3110 B0013 SA1 SA2 SA3 SA4⁄ SA4 SA5

3.42 ± 0.26 3.84 ± 0.23 4.05 ± 0.17 3.94 ± 0.22 5.24 ± 0.34 3.15 ± 0.43 5.71 ± 0.19 6.08 ± 0.21

0.57 ± 0.22 0.83 ± 0.11 0.50 ± 0.07 0.54 ± 0.09 0.38 ± 0.01 0.42 ± 0.04 0.47 ± 0.03 0.50 ± 0.05

14.59 ± 2.53 15.02 ± 2.01 14.45 ± 1.23 12.45 ± 1.57 13.75 ± 1.19 13.22 ± 1.25 12.50 ± 2.12 12.25 ± 1.84

1.31 ± 0.12 1.63 ± 0.19 21.45 ± 2.14 67.81 ± 4.96 417.20 ± 50.01 385.62 ± 32.28 576.17 ± 45.17 670.04 ± 44.23

5.89 ± 0.68 6.45 ± 1.09 6.18 ± 0.82 5.67 ± 0.50 3.06 ± 0.50 2.67 ± 0.75 2.68 ± 0.39 1.15 ± 0.15

ND ND ND 45.45 ± 5.09 128.17 ± 8.22 89.23 ± 9.43 37.62 ± 6.17 40.53 ± 5.43

ND: not detectable. a Data represents the average of three samples (±standard deviations) taken from 27-h shake flask cultures grown on fermentation medium. b The specific growth rate l (1/h) was calculated based on the biomass sample taken at various time points during fermentation process.

enzyme analysis and sequencing. Wild type gene aroGwt, and mutant gene aroGfbr were amplified with primers aoG-up and aoG-dw, and cloned into plasmid pTH18kr via EcoRI and KpnI sites to construct plasmids of pTH-aroGwt and pTH-aroGfbr, respectively. Phosphoenolpyruvate synthase gene ppsA was cloned from E. coli 0013 with primers ppsA-up and ppsA-dw, digested with KpnI and XbaI and cloned into vector pTH-aroGfbr which was previously digested with the same enzymes to construct pTH-aroGfbr-ppsA. Transketolase gene, tktA was amplified with primers tktA-up and tktA-dw. After digestion and purification, the resulting fragment was successively ligated into plasmid pTH-aroGfbr-ppsA via the XbaI and SphI sites to generate the recombinant plasmid pTHaroGfbr-ppsA-tktA. Strategy and maps of all constructed plasmids were displayed as Fig. 2.

2.5. Fermentation media and growth conditions For shake-flask experiments, strains (stored as glycerol stocks at 70 °C) were firstly grown on LB agar plates overnight at 37 °C and subsequently a single colony was transferred to 50 mL of LB medium in a 250-mL flask. After 12 h growth with shaking, cells were harvested by centrifugation and re-suspended in fermentation medium. This suspension was used to inoculate 50 mL fresh fermentation medium at 37 °C, 200 rpm. The fermentation medium (per liter) was described in a previous study (Knop et al. 2001) and revised, contained glucose (15 g), K2HPO4 (7.5 g), ammonium iron (III) citrate (0.3 g), citric acid monohydrate (2.1 g), L-phenylalanine (0.7 g), L-tyrosine (0.7 g), L-tryptophan (0.35 g), and concentrated H2SO4 (1.2 mL). Fermentation medium was adjusted to pH 7.0 by addition of concentrated NH4OH before autoclaving. The following supplements were added immediately prior to initiation of

the fermentation: glucose, MgSO4 (0.24 g), p-hydroxybenzoic acid (0.010 g), potassium p-aminobenzoate (0.010 g), 2,3-dihydroxybenzoic acid (0.010 g), and trace minerals, including (NH4)6(Mo7O24)
4H2O (0.0037 g), ZnSO4
7H2O (0.0029 g), H3BO3 (0.0247 g), CuSO4
5H2O (0.0025 g), and MnCl2
4H2O (0.0158 g). Biomass concentrations were determined and calculations were performed to adjust inoculum size to an OD600 nm of 0.2. For induction, the final concentration of IPTG to 1 mM was added into the culture after 3 h incubation (the OD600 nm of the cultures reached about 2.0). For each bioreactor experiment, fed-batch process was conducted in a 7-L bioreactor (Bioflow110; New Brunswick Scientific Co., Edison, NJ, USA) containing 4 L fermentation medium with an initial glucose concentration of 15 g/L at 37 °C. Growth was initiated by sparging air into the bioreactor at 3 to 7 L/min and the dissolved oxygen concentration maintained above 30% air saturation by agitation at 200 to 1000 rpm. Glucose feeding was employed to maintain a 10–25 g/L concentration in the medium. The pH was controlled at 7 by automatically feeding concentrated NH4OH. Antifoam (Sigma 204) was added manually as needed.

2.6. Analytical methods Cell growth during the cultivations was monitored by measuring the optical density at 600 nm (OD600). A standard curve relating dry cell weight (DCW) to OD was previously constructed (1 OD600 = 0.38 g/L DCW) (Zhou et al., 2012). Glucose concentration was estimated by a glucose biosensor (SBA-40C; Biology Institute of Shandong Academy of Sciences, Tsinan, China). Metabolite concentrations of shikimate, quinic acid, acetic acid, pyruvate, 3dehydroshikimate and 3-dehydroquinic acid were determined by HPLC using an Agilent 1100 series instrument and an Aminex

Fig. 2. Stepwise improvements of the shikimate pathway by modular expression of three critical genes of aroGfbr, ppsA and tktA. Open blocks indicate the origin of replication; shaded block arrows indicate the genes; Note, for each operon, the genes are placed in the reverse order relative to the reaction pathway.

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HPX-87H column (300 mm  7.8 mm; 9 lm) (Bio-Rad, Hercules, USA) maintained at 50 °C. The mobile phase was 5 mM H2SO4, with a flow rate of 0.5 mL/min, at 50 °C. All metabolites were detected with a photodiode array detector at 210 nm.

(Table 2), which indicated that decreased availability of pyruvate (and acetyl coenzyme A) in the pykF mutant might lead to low flux ratios through lactate and acetate formation (Siddiquee et al., 2004).

3. Results and discussion

3.2. Decreased byproduct formation through deletion of ydiB and ackA-pta genes

3.1. Engineering E. coli for shikimate accumulation Previously we isolated an E. coli strain B0013 based on its fast growth rate using glucose and/or xylose. To achieve an overproducing shikimate strain from B0013, we disrupted the shikimate pathway to eliminate shikimate-3-phosphate formation by deletion of aroL and aroK encoding shikimate kinase isoenzymes (Fig. 1). The resulting strain SA2 synthesized shikimate with a concentration of 67 mg/L (Table 2). However strain SA1 only produced 21 mg/L shikimate (Table 2), which indicated that disruption of one of the isoenzymes had little effect on synthesis of shikimate. It should be noted that two engineered strains showed a similar final biomass as the wild type, whereas the average specific growth rate of the both engineered strains was lower than that of the control (Table 2). Previous study also indicated that deletion of the aroK gene is essential for high shikimic acid accumulation in E. coli (Chen et al., 2012). The PTS depends on the expenditure of PEP during glucose transport, which would compete with DAHP synthase to synthesise DAHP (Fig. 1). Evidently, engineering PTS have proved an efficient strategy to alleviate this bottleneck and enhance the production of aromatic compounds (Escalante et al., 2010; Escalante et al., 2012). In this study the ptsG gene was deleted followed by elimination of AroL and AroK resulting in strain SA3 producing a significantly higher level of shikimate. Strain SA3 produced 417 mg/L shikimate, 6-fold higher compared with strain SA2 (Table 2). The average specific growth rate (Table 2) of the engineered strain SA3 decreased to 0.38, still supporting cell growth but at a modest level as compared to the wild type strain B0013 which had a growth rate of 0.83. Meanwhile, it was found that disruption of glucose-PTS had a negative impact on glucose consumption and overall growth pattern (Table 2), which suggested that other glucose transport systems such as mannose-PTS and the galactose transporters could only partly compensate for the elimination of EIIgluc protein in this host. Previous studies indicated that introducing a heterologous glucose transport pathway into an engineered strain can improve cells performance and shikimate yield (Chandran et al., 2003). Glucose transport is worth further investigation in future studies. Pyruvate kinase isoenzymes encoded by the pykF and pykA genes catalyze the formation of pyruvate + Mg-ATP by transferring a phosphate group from PEP to Mg-ADP in the presence of potassium however, pykA contributes little to pyruvate kinase activity (Siddiquee et al., 2004). Previous studies showed that deletion of pykF gene contributed for the enhanced shikimate production (Rodriguez et al., 2013; Licona-Cassani et al., 2014). Here the SA4⁄ strain deficient of pykF was constructed and we discovered glucose consumption and the final cell concentration of the SA4⁄ strain appeared to be slightly lower than that in the wild type and strain SA3, however its growth rate was somewhat higher than that of SA3 strain (Table 2). More unexpectedly, this strain afforded no significant additional increase in shikimate production (Table 2). Recent research suggested that inactivation of PykF in the PTSbackground E. coli caused an increase in anaplerotic flux from PEP to oxaloacetate and an increased flux to the tricarboxylic acid (TCA) cycle, whereas inactivation of PykA caused a reduction in PEP carboxylase and PEP carboxykinase cycling as well as a reduction in flux to TCA (Meza et al., 2012). Furthermore, we found that deletion of pykF gene resulted in a decrease in acetic acid accumulation

The engineered strain SA3 could overproduce shikimate compared with wild type, however we found that quinic and acetic acids also accumulated at a high concentration. These byproducts not only competitively consumed the carbon source and therefore an economic sink, but also had a negative effect on cell growth. Quinic acid was generated from 3-dehyroquinic acid through shikimate dehydrogenase isozyme encoded by ydiB and aroE, respectively. Moreover, the protein products of ydiB and aroE mediate the conversion reaction not only from dehydroquinate to quinate but also from dehydroshikimate to shikimate. Recent studies have shown that overexpression of ydiB does not increase shikimate, while overexpression of aroE increases conversion of dehydroshikimate to shikimate (Juminaga et al., 2012). On the contrary, overexpression of ydiB in E. coli could increase synthesis of quinic acid from 3-dehyroquinate, to produce chlorogenic acid (Kim et al., 2013). To block this catabolic pathway of converting 3-dehyroquinate to quinic acid and drive more precursor to shikimic aicd biosynthesis, ydiB gene was knocked out in SA3 strain to generate strain SA4. Using the resulting strain, production of shikimate increased to 576 mg/L whereas the accumulation of quinic acid decreased to 37 mg/L from 128 mg/L (Table 2), which indicated that mutation of the ydiB gene contributed to minimizing formation of quinic acid and increase shikimate. It is noted that AroE prefers dehydroshikimate to dehydroquinate (Draths et al., 1999), whereas YdiB has nearly the same catalytic efficiency for dehydroshikimate and dehydroquinate (Lindner et al., 2005). The accumulation of quinic acid may be due to the equilibrium of these enzymatic reactions which is contributed to by aroE. Acetate kinase/phosphotransacetylase (ackA-pta) catalyzes the conversion of pyruvate via acetyl-coenzyme A (CoA) and acetyl-phosphate to acetate. And evidently this might be the main pathway for acetate production in E. coli, which is active both aerobically and anaerobically to convert acetyl-CoA to acetate (Yang et al., 1999; Dittrich et al., 2005). Our previous studies revealed that the mutation of pta gene is more effective in reducing acetate and formate yields and increasing lactate yields than the ackA gene mutation in spite of both genes encoding proteins catalyzing reactions in the same pathway for acetate production (Zhou et al., 2011). Here combined inactivation of ackA and pta genes in strain SA4 generated strain SA5. The resulting strain produced shikimate at a concentration of 670 mg/L, 16.3% more when compared with strain SA4. Only 1.15 g/L of acetic acid was accumulated using strain SA5 however, 6.45 g/L acetic acid was observed when the wild type was used (Table 2). Moreover, we found that deletion of ackA and pta genes resulted in the little effect on the cell growth rate and glucose consumption compared with the SA4 strain (Table 2). In our opinion, 1.15 g/L of acetic acid produced by SA5 strain may be contributed by another acetic acid pathway, in which pyruvate oxidase catalyses the decarboxylation of pyruvate to acetic acid and carbon dioxide. 3.3. Modular expression of critical genes to improve shikimate yield Three DAHP synthase isozymes encoded by aroF, aroG and aroH are sensitive to tyrosine, phenylalanine, and tryptophan, respectively. Indeed, transcriptional repression and feedback inhibition of DAHP synthase by aromatic amino acids have been viewed as regulatory mechanisms that tightly regulate carbon flow through

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the shikimate pathway. Moreover, feedback inhibition is quantitatively the major control factor for in vivo catalytic activity of this isoenzyme (Weaver and Herrmann, 1990). Therefore, to de-repress the feedback inhibition caused by phenylalanine, the wild type aroG was mutated to aroGfbr (see Section 2.3, D146 N) (Kikuchi et al., 1997), which was followed by induced expression to verify its function in improving shikimate production. Also, previous studies indicated that the expression levels of the enzyme transketolase I (encoded by tktA) and phosphoenolpyruvate synthase (encoded by ppsA) had a critical impact on the PEP and E4P availability, which were important determinants for in vivo DAHP synthase activity (Patnaik and Liao, 1994; Kramer et al., 2003). A reasonable explanation for the observed ceiling in synthesized shikimate concentrations in strain SA5 is that intracellular PEP and E4P availability becomes a limiting factor, which not adequately accumulated may lead to low shikimate production. Here, we constructed a set of recombinant plasmids to modularly express the aroGfbr, ppsA and tktA genes and evaluate their impact on the fermentation performance of the engineered strains. Strategies for modular expression of the indicated genes were shown in Fig. 3. A previous study indicated that genes close to the promoter are usually induced at much higher levels than those

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distal from the promoter and the reverse arrangement of genes in the operon would benefit for the desired product formation by increasing the protein concentration of the enzymes occurring in the latter part of the pathway (Juminaga et al., 2012). Therefore, the indicated genes were tandemly oriented in opposite directions to corresponding genes for shikimate synthesis in the constructed polycistron in our study (Fig. 2). Expressing multiple genes using a high-copy-number plasmid might lead to a metabolic burden on cells (Flores et al., 2004), which is often reflected in slower growth rates and lower rates and yields of biosynthesized products. Here we used pTH18kr, a low-copy-number plasmid (HashimotoGotoh et al., 2000), to construct a set of recombinants. As depicted in the Fig. 3a and b, three recombinant strains decreased their growth rate and substrate consumption rate compared with strain SA5, which indicated that our modifications may disturb the carbon flow and generate a metabolic burden in the engineered strains. Wild type B0013 had the highest growth rate and glucose consumption, however its final concentration was significantly lower than the engineered strains (Fig. 3d). This might be due to high acetic acid accumulation in the early phase of cell growth (Table 2 and Fig. 3d), and consequently impact on the culture conditions. Further investigation found that all recombinant strains

Fig. 3. Effect of different gene combinations on the production of shikimate fermentation performance. (a) Growth pattern of indicated engineered strains. (b) Glucose consumption kinetic of engineered strains. (c) Effect of modular expression of indicated genes on shikimate and quinic acid production titer. (d) Effect of modular expression of indicated genes on the biomass, acetic acid accumulation and specific growth rate of engineered strains. Broth samples were analysed following a 27-h fermentation period. Fermentation medium containing 15 g/L glucose was used and all data were obtained from shake-flask fermentation experiments. Error bars indicate mean values ± SD from three independent experiments.

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produced more shikimate. Additional expression of aroGfbr gene improved the shikimate production by 34%, reaching 900 mg/L (Fig. 3c). Modular expression of the aroGfbr and ppsA genes resulted in 1080 mg/L shikimate (Fig. 3c). The highest shikimate production at 1207 mg/L was obtained by modular expression of aroGfbr, ppsA and tktA genes, 1.8-fold increase compared with strain SA5. However a slight increase of quinic acid accumulation was found in recombinant strains overexpressing the indicated genes (Fig. 3c). 3.4. Enhanced shikimate production in 7-L bioreactor The highest yield of shikimate at 85 mg/g glucose was generated by strain SA5 /pTH-aroGfbr-ppsA-tktA in shake flask culture (Table 3). Even though the total aromatic compounds (shikimic aicd, quinic acid, and 3-dehydroquinic acid) were calculated, the total yield also was markedly lower than the theoretical level. Titers and yields of shikimate need to be improved to satisfy the industrial potential. Therefore, a 7-L fermentor was used to optimize the shikimate production. Fed-batch process was employed to maintain a 10–25 g/L glucose concentration and also maintaining the dissolved oxygen level at conditions that are beneficial for cell growth and shikimate production. Previous studies indicated that glucose-limited culture conditions that ensued would normally be considered problematic given the reported equilibration of shikimate with quinic acid under glucose-limited conditions (Draths et al., 1999; Knop et al., 2001). Here, we found cultivation of the SA5/pTH-aroGfbr-ppsA-tktA strain by an optimized fed-batch fermentation process synthesized 14.6 g/L of shikimate with a yield of 29 g/100 g glucose (Fig. 4 and Table 3). The titers and yields of shikimate improved 12.1 and 3.4 times respectively, compared with those under shake flask conditions. In addition to shikimate, quinic acid and 3-dehydroquinic acid was synthesized at concentrations of 0.68 g/L and 0.87 g/L, respectively. Indeed, the concentrations of quinic acid and 3-dehydroquinic acid under bioreactor fermentation were higher than that under shake flasks cultures, however their yields from glucose were significantly difference. Table 3 showed that yield of quinic acid reached to 1.36 g/100 g glucose and yield of 3-dehydroquinic acid decreased to 1.74 g/100 g glucose under bioreactor fermentation compared with 0.59 and 2.88 g/100 g glucose under shake flasks process, respectively. Total aromatic compounds yield also reached to 16.65 g/L. Furthermore, the final biomass remarkably increased to 12.8 g/L from 4.9 g/L under shake flasks culture. Correspondingly, the engineered strain SA5/pTH-aroGfbr-ppsA-tktA produced 14.6 g/L shikimate under 7-L fermentor. It should be noted that this yield is still lower than the maximum theoretical yield of shikimate, which could reach 0.83 g/g glucose under the conditions of pyruvate being effectively recycled to PEP via overexpressed PEP synthase in the presence of glucose (Patnaik and Liao, 1994).

Fig. 4. Shikimate and biomass production, and byproducts accumulation by engineered strain SA5/pTH-aroGfbr-ppsA-tktA in 7-L fermentor using fed-batch process. An initial concentration of 15 g/L of glucose was used and a total of 200 g glucose was consumed after 50 h fermentation process.

Meanwhile, our results indicated that engineered strain SA5/pTHaroGfbr-ppsA-tktA produced other aromatic products including 3-dehydroquinic acid and 3-dehydroshikimic acid with a significant lower concentration than previous reports (Knop et al., 2001; Chandran et al., 2003), which might be benefit for extraction process. In the present study, shikimate production level was still lower than the levels previously reported. Our opinion is that the shikimate titer and productivity could be further enhanced through additional metabolic engineering strategies and process optimization.

4. Conclusion Elimination of shikimate kinases combined with deficiency of the phosphotransferase system increased shikimate accumulation significantly. Deletion of genes responsible for acetic acid and quinic acid synthesis resulted in byproduct formation at low concentrations. Modular expression of critical genes improved production titers. The engineered E. coli produced shikimate directly from glucose with a titer of 14.6 g/L in a 7-L bioreactor. Meanwhile, the volumetric and specific shikimate productivity of the engineered strain reached 0.26 g/L/h and 20.2 mg/g cell mass/h, respectively.

Table 3 Comparison of fermentation parameters of the engineered strain SA5/pTH-aroGfbr-ppsA-tktA in shake flasks and 7-L bioreactor experiments.a Parameters

Specific growth rate l (1/h)

QV (SA g/L
h)d

QS (SA mg/g cell mass
h)e

YBiomass/glucose (g/100 g)

YSA/glucose (g/100 g)f

YQA/glucose (g/100 g)f

YDHQ/glucose (g/100 g)f

Shake flasksb 7-L Bioreactorc Fold

0.46 0.21 0.46

0.047 0.26 5.53

9.12 20.2 2.21

28.61 18.23 0.64

8.51 29.25 3.44

0.59 1.36 2.31

2.88 1.74 0.60

a The fermentation process under shake flasks and bioreactor were described in the materials and methods section. Data represent the average of three samples. Cultivation was done in at least triplicates, and the standard deviations were shown. All values are the maximum during fermentations. b The strain was cultivated in the fermentation medium at 37 °C under the shake flasks and the highest yields of indicated compounds were calculated during the overall fermentation process. c The strain was cultivated in the fermentation medium at 37 °C under 7-L bioreactor and the highest yields of indicated compounds were calculated during the overall fermentation process. d QV indicated volume productivity of shikimiate (g/L
h). e QS indicated specific productivity of shikimiate(mg/g cell mass
h). f YSA/glucose, YQA/glucose, and YDHQ/glucose indicate yield of shikimate, quinic acid and dehyroquinate from glucose, respectively.

X. Chen et al. / Bioresource Technology 166 (2014) 64–71

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