Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

Metabolic Engineering 13 (2011) 492–498

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Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose Zhen Kang a, Yang Wang a, Pengfei Gu a, Qian Wang a,b, Qingsheng Qi a,b,n a b

State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China National Glycoengineering Research Center, Shandong University, Jinan 250100, People’s Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 January 2011 Received in revised form 5 April 2011 Accepted 13 May 2011 Available online 23 May 2011

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemAM from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli. & 2011 Elsevier Inc. All rights reserved.

Keywords: 5-aminolevulinic acid Escherichia coli Metabolic engineering C5 pathway

1. Introduction 5-Aminolevulinic acid (ALA), a five-carbon amino acid, is a key intermediate involved in the biosynthesis of tetrapyrrole (Smith and Rogers, 1988). ALA has recently attracted much attention for its potential applications in tumor-localizing and photodynamic therapy for various cancers (Bhowmick and Girotti, 2010; Mikolajewska et al., 2010; Sakamoto et al., 2010). In addition, ALA is also used as selective biodegradable herbicide and insecticide in agriculture due to its nontoxicity to crops, animals and humans (Edwards et al., 1984; Sasaki et al., 2002). In living organisms, there are two major pathways described for ALA biosynthesis (Sasaki et al., 2002). One is the C5 pathway, which occurs in higher plants, algae and many bacteria including Escherichia coli (Schon et al., 1986). In the C5 pathway, glutamate is ligated to RNA to form glutamyl-tRNA through the action of glutamyl-tRNA synthetase (GluRS, encoded by gltX gene) (McNicholas et al., 1997). Subsequently, a NADPH-dependent glutamyl-tRNA reductase (HemA, encoded by hemA) catalyzes the reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA) (Li et al., 1989; Schauer et al., 2002). GSA is an unstable intermediate, which is then quickly converted to ALA through the action of glutamate-1-semialdehyde aminotransferase (HemL, n Corresponding author at: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China. Fax: þ86 531 88565610. E-mail address: [email protected] (Q. Qi).

1096-7176/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymben.2011.05.003

encoded by hemL) (Jahn et al., 1992). The other is the C4 pathway, which is present in mammals, birds, yeast and purple non-sulfur photosynthetic bacteria. In this pathway, ALA is formed through catalyzation of 5-aminolevulinate synthase, which condenses the amino acid glycine and succinyl-CoA, an intermediate of tricarboxylic acid (TCA) cycle (Burnham, 1970). In E. coli, ALA biosynthesis is through the C5 pathway and is tightly regulated by feedback inhibition of heme, the end product of the C5 pathway (Woodard and Dailey, 1995). Although researchers found that overexpression of hemA led to an increased ALA accumulation, it was relatively difficult to manipulate this pathway in terms of ALA production (Chen et al., 1994). Therefore, people focused on ALA production via the C4 pathway. The photosynthetic bacterium Rhodobacter sphaeroides was found to accumulate ALA at certain conditions or after mutagenesis (Neidle and Kaplan, 1993; Nishikawa et al., 1999; Tangprasittipap et al., 2007). Through metabolic engineering, recombinant E. coli was also able to produce ALA from the C4 pathway through biotransformation. In this aspect, the ALA sythase from R. sphaeroides was introduced into E. coli by genetic engineering (van der Werf and Zeikus, 1996). However, since biosynthesis of glycine and succinyl-CoA was also tightly regulated in E. coli, glycine and succinate (the precursor of sccinyl-CoA) have to be added in the medium artificially to provide more substrates for ALA biosynthesis. Production of ALA via the C4 pathway in E. coli has been studied by several groups (Kiatpapan and Murooka, 2001; Lin et al., 2009; Xie et al., 2003). To provide an inhibitory effect on the 5-aminolevulinate dehydratase (HemB) and to increase ALA production in

Z. Kang et al. / Metabolic Engineering 13 (2011) 492–498

recombinant E. coli, both glucose and/or levulinic acid also have to be added in the medium (Choi et al., 2008; Liu et al., 2010). In this study, we developed a new strategy to produce ALA in recombinant E. coli via the C5 pathway (Fig. 1). To improve ALA production, heterologous stabilized HemA from Salmonella arizona was introduced and coexpressed with HemL in E. coli. Furthermore, an ALA exporter in E. coli was identified and overexpressed. Through metabolic engineering, the recombinant E. coli produced 4.13 g/L ALA (a yield of 0.168 g ALA/g glucose) in modified minimal medium using glucose as the sole carbon source.

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2. Materials and methods 2.1. Strains and plasmids construction All strains, plasmids and oligonucleotides used in this study were summarized in Tables 1 and 2. Molecular cloning and manipulation of plasmids were done with E. coli DH5a. hemA gene was amplified from wild type E. coli MG1655 genome with primers hemA-F and hemA-R, digested with enzyme HindIII and PstI, and ligated into pUC19, which was cut with the same restriction enzymes. The new plasmid was named as pDEA.

Fig. 1. Schematic presentation of ALA production from glucose in E. coli via C5 pathway. G6P, glucose-6-phosphate; Pyr, pyruvate; Ket, a-ketoglutarate; Glu, glutamate; GlntRNA, Glutamyl-tRNA; GSA, glutamate 1-semialdehyde aminotransferase; ALA, 5-aminolevulinic acid; PBG, porphobilinogen.  means the effect of overexpression of gltX on hemB transcription.

Table 1 Strains and plasmids used in this study. Strain and plasmid

Relevant properties

Source or reference

Lab stock

E. coli MG1655 S. arizona E. coli DDA E. coli DMA E. coli DDMA E. coli DU19 E. coli DEA E. coli DEX E. coli DEL E. coli DA E. coli DXL E. coli DXA E. coli DAL E. coli DAL20 E. coli DALA E. coli DALS E. coli DALU E. coli DDAL E. coli DMAL E. coli DDMAL

F-endA1glnV44thi  1recA1relA1gyrA96deoRnupGF80 dlacZDM15D (lacZYA-argF)U169,hsdR17(rK  mK þ ),l  Wild type Wild type E. coli DH5a DDppA::CmR E. coli DH5a DMppA::KmR E. coli DH5a DDppADMppA::KmR E. coli DH5a harboring pUC19 E. coli DH5a harboring pUC-hemA E. coli DH5a harboring pDEX E. coli DH5a harboring pDEL E. coli DH5a harboring pDA E. coli DH5a harboring pDXL E. coli DH5a harboring pDXA E. coli DH5a harboring pDAL E. coli DH5a harboring pDAL and pCL1920 E. coli DH5a harboring pDAL and pCLRA E. coli DH5a harboring pDAL and pCLYS E. coli DH5a harboring pDAL and pCLUA E. coli DH5a DDppA::CmR harboring pDAL E. coli DH5a DMppA::KmR harboring pDAL E. coli DH5a DDppADMppA::KmR harboring pDAL

Plasmids pUC19 pCL1920 pDEX pDEA pDEL pDA pDXL pDXA pDAL pCLRA pCLYS pCLUA

Cloning vector, AmpR lacPOZ0 Cloning vector, spcR, low copy number pUC19 containing gltX gene (E. coli) pUC19 containing hemA gene (E. coli) pUC19 containing hemL gene (E. coli) pUC19 containing the mutated hemA gene (S. arizona ), hemAM pUC19 containing gltX (E. coli) and hemL (E. coli) pUC19 containing gltX (E. coli) and hemAM (S. arizona ) pUC19 containing hemAM (S. arizona ) and hemL (E. coli) pCL1920 containing rhtA (E. coli) pCL1920 containing yeaS (E. coli) pCL1920 containing udhA (E. coli)

Lab stock Lab stock This work This work This work This work This work This work This work This work This work This work

Strains E. coli DH5a

Lab stock Lab stock This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

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Table 2 Primers used in this study. The base underlined means the restriction enzyme sites, the base in bold means the Shine-Dalgarno sequence added artificially. Primers

Sequence

gltX-F gltX-R hemA-F hemA-R hemAM-F hemAM-R hemL-F hemL-R rhtA-F rhtA-R yeaS-F yeaS-R udhA-F udhA-R DppA-pKDF DppA-pKDR DppA testF DppA testR MppA-pKDF MppA-pKDR MppA testF MppA testR

50 -TCCCTGCAGAAAGGAGGATATACATATGAAAATCAAAACTCGCTTCGCGC-30 50 -GGCGTCGACTTACTGCTGATTTTCGCGTTCAGCAATAAAATCC-30 50 -AGCAAGCTTCTAGGAGGATATACATATGACCCTTTTAGCACTCGGTATCAAC-30 50 -AAACTGCAGCTACTCCAGCCCGAGGCTGTCGCGCAGA -30 50 -CCCGTCGACAAAGGAGGATATACATATGACCAAGAAGCTTTTAGCACTCGGTATCAAC-30 50 -AAATCTAGACTACTCCAGCCCGAGGCTGTCGCGCAGA-30 50 -ACAGGATCCAAAGGAGGATATACATATGAGTAAGTCTGAAAATCTTTACAGCG-30 50 -AATGAGCTCTCACAACTTCGCAAACACCCGACGTGCAGCA-30 50 -CCGAAGCTTTTAATAAGGAGGATATACATATGCCTGGTTCATTACGTAAAATGCCGG-30 50 - GCCCTGCAGTTAATTAATGTCTAATTCTTTTATTTTGCTCTC-30 50 -CCGAAGCTTTTAATAAGGAGGATATACATATGTTCGCTGAATACGGGGTTCTGAAT-30 50 -ATTCTGCAGTTAGGATTGCAGCGTCGCCAGTC-30 50 -AGCGTCGACAAGGAGGAATATACATGCCACATTCCTACGATTACGATGCCATA-30 50 -TTTGGATCCGGGGTTGTTTATCTGCCGTGCATTAAGCC-30 50 -AAGGGTTAAAACAACAAACATCACAATTGGAGCAGAATAGTGTAGGCTGGAGCTGCTTC-30 50 -GCATTTTTGCCTTTGCCATCAGTCTTGTATGGCTTTTAAATGGGAATTAGCCATGGTCC-30 50 -CCGCACTGTTACACTGATG-30 50 -CTTATCGGACAGGGAATGAA-30 50 -CCAGAGATTCAGGTCATTCGCGATCTGTTTGAAGGTCTGGTGTAGGCTGGAGCTGCTTC-30 50 -GGTATATTGATAAATTGGTGCAATCGGTGCTTGCTCCATATGGGAATTAGCCATGGTCC-30 50 -GGCGACAGATGCTGATTAT-30 50 -GGATAACCTTTCAGCCACG-30

Primers for RT-PCR gapA-F gapA-R gltX-F gltX-R hemA-F hemA-R hemL-F hemL-R hemB-F hemB-R

50 -AACTGAATGGCAAACTGACTGGTA-30 50 -TTTCATTTCGCCTTCAGCAGC-30 50 -TGAACTATCTGGTGCGTCTG-30 50 -ATGTAGTGATGGTTCAGCCAC-30 50 -TATCGCAGCCAGGCAGAGCA-30 50 -ATCAAGCGGTTAGTCAGTTTCC-30 50 -ACGATGTTGATGGCAAAGCC-30 50 -AGTTCGGTCACCAGTTGCG-30 50 -TAAACCTGCTGGAGCGTA-30 50 -GCACGACTTTCTCTTCATCTAT-30

To enhance stability of HemA, hemA gene from S. arizona genome was amplified and mutated with primers hemAM-F and hemAM-R to insert two codons (AAGAAG) encoding amino acid Lys between Thr-2 and Leu-3 at N terminus as described previously (Wang et al., 1999a). After purification and digestion with SalI and XbaI, the SalI–hemAM–XbaI fragment was ligated into pUC19 cut with the same restriction enzymes to generate plasmid pDA. gltX and hemL were amplified from wild type E. coli 1655 genome with primers gltX-F and gltX-R, and hemL-F and hemL-R, respectively. After purification and digestion, the fragments PstI–gltX–SalI, BamHI–hemL–SacI, and PstI–gltX–SalI and BamHI–hemL–SacI were ligated into pUC19 cut with the same restriction enzymes to obtain plasmids pDEX, pDEL and pDXL, respectively. Subsequently, the digested fragments PstI–gltX–SalI and BamHI–hemL– SacI were inserted into pDA with the same restriction enzymes to generate plasmids pDXA and pDAL, respectively. rhtA, yeaS and udhA were amplified from wild type E. coli 1655 genome with primers rhtA-F and rhtA-R, yeaS-F and yeaS-R and udhA-F and udhA-R, respectively. The digested fragments HindIII–rhtA–PstI, HindIII–yeaS–PstI and SalI–udhA–BamHI were inserted into pCL1920 with the same restriction enzymes to generate plasmids pCLRA, pCLYS and pCLUA, respectively. To enhance the translation efficiency, the Shine-Dalgarno sequence (AGGAGGA) was artificially added in the upstream of ATG start. 2.2. Gene inactivation The mutants E. coli DHDA (DDppA::CmR) and E. coli DHMA (DMppA::KmR) were created using the one-step inactivation method (Datsenko and Wanner, 2000). This method includes following steps: amplification of the resistance gene

with polymerase chain reaction (PCR) using pKD4 (KmR) or pKD3 (CmR) as template. PCR products flanked by FRT (FLP recognition target) sites and homologous sequences to the gene of interest were purified and transformed into the cells by electroporation (Bio-Rad, Gene Pulser). The knocking out of the target gene in the cells, which carried with plasmid pKD46 that expresses l Red recombinase was happened by recombining the PCR product into the chromosome. Transformants were selected with antibioticresistance plate. The kanamycin or chloramphenicol cassette was removed with the helper plasmid pCP20 that expresses FLP before next mutation. E. coli DHDMA (DDppADMppA::KmR) was obtained by P1 phage transduction with E. coli DHMA as donor strain and E. coli DHDA as acceptor strain. All other genetic operations were performed according to the protocols provided by the manufactures. 2.3. Growth conditions LB medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl, pH 7.2) was used in all DNA manipulations. During cultivation and fermentation, the modified minimal medium was used, which contains 16 g/L (NH4)2SO4, 3 g/L KH2PO4, 16 g/L Na2HPO4  12H2O, 1 g/L MgSO4  7H2O, 0.01 g/L MnSO4  7H2O and 2 g/L yeast extract. Glucose as sole carbon source was added as indicated. Ampicillin (100 mg/mL), chloramphenicol (25 mg/mL), kanamycin (25 mg/mL) or spectinomycin (25 mg/mL) was added to provide selective pressure during cultivation when necessary. To induce the expression of plasmids-borne genes, Isopropyl-b-Dthiogalactopyranoside (IPTG) was added into the cultures with a final concentration of 0.1 mM. Flask cultivations were carried out in 300 mL Erlenmeyer flask supplied with 50 mL modified minimal medium at 37 1C at an

Z. Kang et al. / Metabolic Engineering 13 (2011) 492–498

agitation of 225 rpm. 18 g/L glucose was added initially as the sole carbon source. A 1% (v/v) inoculum from an overnight culture for 12 h was used. Samples were taken and measured with an interval of 4 h. Batch fermentation was performed in 5 L fermentor containing 3.5 L modified minimal medium. A 2% (v/v) inoculum from an overnight culture for 12 h was used. Glucose as a sole carbon source was added at initial with a concentration of 35 g/L. Fermentation was operated at 37 1C. Considering cell growth and ALA stability, the pH was measured by a glass electrode and controlled at 6.0 70.2 with 4 M NaOH. During the fermentation, the dissolved oxygen was monitored using a polarographic oxygen electrode. 2.4. Analytical procedures Optical density (OD) was measured at 600 nm with a spectrophotometer. For analyzing glucose, 1 mL of culture was centrifuged (12,000g for 2 min at 4 1C) and the supernatant was then filtered through a 0.22 mm syringe filter for analysis. The HPLC system was equipped with a cation exchange column (HPX-87 H, BioRad Labs), and a differential refractive index (RI) detector (Shimadzu RID-10 A). A 0.5 mL/min mobile phase using 5 mM H2SO4 solution was applied to the column. The column was operated at 65 1C. To analyze ALA distribution, 30 mL culture of 32 h was centrifuged (12,000g for 2 min at 4 1C). The supernatant was used for extracellular ALA analysis; the cell pellet was washed with 0.85% NaCl solution for two times and disrupted with ultrasonic treatment for intracellular ALA analysis. ALA concentration was analyzed using modified Ehrlich’s reagent (Burnham, 1970).

495

Table 3 ALA production in recombinant E. coli expressing various related genes. A 2% (v/v) inoculum from an overnight culture for 12 h was used. Samples were taken and measured until 48 h. 18 g/L glucose was added initially as sole carbon source. The results are the average of three individual experiments. Strains

Expressed genes

Cell biomass (OD600)

ALA accumulation (mg/L)

E. E. E. E. E. E. E. E. E.

– gltX hemA hemL hemAM hemAM, hemL hemAM, hemL, udhA gltX, hemL gltX, hemAM

9.327 0.84 8.427 0.67 10.767 0.68 12.72 7 1.14 10.217 0.68 9.887 0.74 9.577 0.83 13.74 7 0.74 12.11 7 0.97

31.11 7 3.79 16.83 7 1.45 43.42 7 4.24 23.95 7 1.68 176.167 9.68 2052.297 21.25 2086.297 34.16 26.72 7 1.36 12.017 1.10

coli coli coli coli coli coli coli coli coli

DU19 DEX DEA DEL DA DAL DALU DXL DXA

and E. coli DEL, respectively. The resulting strains were cultivated in modified minimal medium (see ‘‘Section Materials and Methods’’) supplied with 18 g/L glucose for ALA accumulation analysis. Interestingly, we found that the strain DEX showed a decreased ALA accumulation, while E. coli DEA showed an increased 5-ALA accumulation, and E. coli DEL showed almost the same ALA accumulation as E. coli DU19 (Table 3). The result indicated that the reduction of glutamyl-tRNA to glutamate-1-semialdehyde, catalyzed by HemA, is a rate-limiting step and GluRS may negatively affect the ALA biosynthesis in E. coli. Previous reports have proved that HemA is required for ALA biosynthesis in E. coli and overexpression of hemA improved ALA accumulation (Chen et al., 1994; Verderber et al., 1997). This suggested that, to enhance ALA production in E. coli, the activity of HemA should be up-regulated.

2.5. Quantitative real-time PCR (qRT-PCR) The genes and their primers studied in this work were listed in Table 2. The message RNA (mRNA) level was measured by qRTPCR. Samples for mRNA preparation were cultivated 6 h after the addition of 0.1 mM IPTG. The equivalent cells of 6 OD600 were harvested and frozen immediately at 80 1C. mRNA of E. coli DEX (gltX) and E. coli DU19 (control strain) was extracted using the RNeasy Mini Kit (Tiangen). The quantity and purity of the RNA were determined by optical density measurements at 260 and 280 nm. The cDNA was obtained from reverse transcription and qRT-PCR was carried out in a 96-well plate with a total reaction volume of 20 mL per well in MyiQ5768R Real-Time PCR detection TM system using an SYBRs Premix Ex Taq II (Perfect Real Time), according to manufacturer’s specifications (TaKaRa). For all RT-PCR reactions, according to its consistent expression and little variability, gapA was used as a control for normalization between samples. qRTWWCt PCR data were treated using the 2  method described previously (Livak and Schmittgen, 2001) with the software of Opticon Monitor 3.

3. Results and discussion 3.1. Overexpression of the genes involved in the C5 pathway in E. coli 5-Aminolevulinic acid (ALA) can be synthesized in wild type E. coli via the C5 pathway from glutamate, a direct derivative of the tricarboxylic acid (TCA) cycle. This process involves 3 key enzymes, glutamyl-tRNA synthetase (GluRS), glutamyl-tRNA reductase (HemA) and glutamate-1-semialdehyde aminotransferase (HemL). To figure out the rate-limiting step of this pathway, we overexpressed gltX, hemA and hemL separately using multicopy plasmid in E. coli DH5a, generating E. coli DEX, E. coli DEA

3.2. Increasing the ALA accumulation by expression of a modified heterologous HemA The reduction of glutamyl-tRNA to glutamate-1-semialdehyde is the rate-limiting step for ALA biosynthesis in E. coli. This limited function of HemA in E. coli may be due to many reasons (Woodard and Dailey, 1995). The posttranscriptional control of HemA is the most important regulation mechanism involved in this process (Wang et al., 1999b). Pulse-labeling and immunoprecipitation studies indicated that HemA in Salmonella typhimurium was not stable and its stability was greatly decreased in unrestricted cells or when heme was accumulated (Wang et al., 1999a). Due to the instability of HemA to proteolysis, many endeavors, such as using tac or T7 RNA polymerase-directed systems to overproduce S. typhimurium HemA to high levels, were made but all failed (Wang et al., 1999b). At the same time, researchers found that the stability of HemA in S. typhimurium can be significantly increased by inserting two Lysine (codons: AAGAAG) between Thr-2 and Leu-3 at N terminus of the protein. Therefore, we made the same mutation with the HemA from S. arizona, generating HemAM. Since the amino acid sequence of hemA from S. arizona is the same as that from S. typhimurium, we used the hemA gene from S. arizona instead. The resulting E. coli DA, which harbors the mutated HemAM, was able to accumulate 176 mg/L ALA in the medium (Table 3). This amount is 5.7 times more than that of wild type E. coli and 4 times more than the strain DEA, which was overexpressed with native hemA gene from E. coli. In S. typhimurium, HemA is negatively regulated by heme or a heme-dependent process and requires the function of the ATPdependent proteases Lon and ClpAP (Jones and Elliott, 2010; Wang et al., 1999a, 1997). Overexpression of hemA gene from S. typhimurium met with many problems (Choi et al., 1996). However, hemA from E. coli was successfully overexpressed and 3-fold increase could be detected (McNicholas et al., 1997),

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50

The complex formation suggested the efficient and quick transfer of GSA and high accumulation of ALA in the strain DAL. In the C5 pathway, two enzyme reactions need NADPH as cofactor (Fig. 1). Therefore, we overexpressed the udhA (encoding a soluble pyridine nucleotide transhydrogenase, UdhA) gene in E. coli. UdhA catalyzes the transfer of NADP þ between NADPH (Sauer et al., 2004). High amount of UdhA was supposed to provide a possibility to regenerate more NADPH from NADP þ and to improve the catalyzing efficiency of HemA. However, E. coli DALU expressing udhA did not improve the ALA accumulation (Table 3), indicating the availability of NADPH is not a key factor in this process.

45 ALA concentration (mg/L)

40

E. coli DU19 E. coli DEA E. coli DA

35 30 25 20 15 10

3.4. GluRS up-regulated the transcription of hemB gene

5 0

0

4

8

12 16 Cultivation time (h)

20

24

Fig. 2. Comparison of ALA accumulation curve of E. coli DU19, E. coli DEA and E. coli DA. Cultivation was performed in 300 mL Erlenmeyer flask supplied with 50 mL modified minimal medium at 37 1C for 20 h. 18 g/L glucose was added initially as sole carbon source. A 2% (v/v) inoculum from an overnight culture for 12 h was used. Samples were taken and measured with an interval of 4 h. The results are the average of three individual experiments.

indicating that HemA from E. coli was more stable than that from S. typhimurium. In our study, hemA gene from E. coli was at first overexpressed. The expression level of hemA of E. coli was much more than that of hemAM from S. arizona (supplemental information, Fig. 1). However, overexpression of hemA in E. coli only slightly improved the ALA production. This suggested that the enzyme activity of HemA in E. coli is not high. Replacing the stable low active E. coli HemA with mutated high active S. typhimurium HemAM greatly improved the ALA production (Table 3). In addition, we found that the wild type E. coli did not accumulate ALA at the initial growth phase. Only after 20 h of cultivation, E. coli began to accumulate ALA (Fig. 2). Expressions of hemA, both native hemA from E. coli and mutated hemA from S. arizona, resulted in ALA accumulation at the initial growth phase. This will be beneficial to the metabolic production of ALA in E. coli.

3.3. Synergetic effect of HemAM and HemL on ALA production Glutamate-1-semialdehyde aminotransferase catalyzes the pyridoxal 50 -phosphate-dependent reaction, which converts GSA to ALA (Ilag et al., 1991). Although single expression of hemL did not affect the ALA accumulation, HemL was found to form a tight complex with its preceding enzyme HemA in E. coli (Luer et al., 2005). The formation of tight complex guaranteed the efficient and quick transfer of GSA, which is highly reactive, to ALA. Therefore, to obtain a high ALA accumulation in E. coli, we coexpressed hemL gene from E. coli with the mutated hemAM from S. arizona, generating strain DAL. As expected, co-expression of hemL with hemAM achieved a significantly increased ALA production of 2052 mg/L, which was 12.5 times more than that of the strain DA (Table 3). High ALA yield obtained by the strain DAL suggested that HemAM and HemL have a synergetic effect, probably due to the formation of a tight complex in E. coli. The tight complex formation between HemL and HemA in E. coli was previously verified by co-immunoprecipitation experiments and gel permeation chromatography (Luer et al., 2005). We analyzed the amino acid sequence of HemA from E. coli and HemAM from S. arizona; a 95% identity and a 98% similarity were found. Therefore, there is a big possibility to form the tight complex between high stable and active HemAM from S. arizona and HemL from E. coli.

In E. coli, GluRS catalyzed the formation of L-glutamyl-tRNA by the esterification of glutamate to the 30 end of tRNA (Hecht and Chinualt, 1976; Levican et al., 2007). Apart from protein synthesis, only a small fraction of charged glutamyl-tRNA was used to make ALA and heme (Beale et al., 1975). To increase L-glutamyl-tRNA amount for ALA synthesis, we overexpressed gltX gene alone or with other genes. However, no ALA increase in strain DXL was detected (Table 3). Contrarily, both recombinant strains DEX and DXA achieved a reduced ALA production, 16.83 and 12.01 mg/L, respectively (Table 3). We suggested that overexpression of gltX either up-regulates the transcription of hemB gene, which encodes ALA dehydratase, or down-regulates the transcription of the genes involved in ALA biosynthesis. To validate our hypothesis, we performed real-time RT-PCR. As shown in Table 4, overexpression of gltX did not affect the transcription of hemL, but decreased the transcription of hemA. The transcription of hemA in DEX was only 80% of the control strain DU19. Meanwhile, transcription of hemB increased 3.95 fold. The results clearly indicated that expression of gltX has a positive effect on hemB transcription but a negative effect on hemA. The GluRS activity is tightly regulated by the status of heme (Levican et al., 2007). When intracellular heme is in excess, the cells respond by a dramatic decrease in GluRS activity. Actually, we found that the culture of gltX overexpressed strains showed a deeper color than those strains in which gltX was not overexpressed. This indicated that many down-stream porphyrin compounds or heme were accumulated, probably due to the increased hemB expression. 3.5. Overexpression of rhtA accelerated the export and increased the accumulation of ALA To avoid ALA accumulation inside the cells and to produce more ALA in the medium, we carried out the following experiments. First, we knocked out the dppA and/or mppA genes in E. coli. The periplasmic binding proteins—MppA, the L-alanyl-g-Dglutamyl-meso-diaminopimelate binding protein, or DppA, the dipeptide binding protein—actively import the ALA through the interaction with the dipeptide inner membrane ATP-binding cassette transporter (DppBCDF) in E. coli (Letoffe et al., 2006). Inactivation of dppA and/or mppA genes was supposed to reduce Table 4 Effect of gltX overexpression on gene transcription in E. coli DEX. Expression levels of gltX, hemA, hemL and hemB in E. coli DEX are relative to that of the control strain E. coli DU19. The error bars indicate the standard deviation from the mean of three replicates. Genes

Expression amount

gltX hemA hemL hemB

212.797 23.24 0.827 0.01 1.097 0.02 3.95 7 0.36

Z. Kang et al. / Metabolic Engineering 13 (2011) 492–498

3.6. Production of ALA in recombinant strain DALA To produce ALA via C5 pathway in recombinant E. coli, we optimized the cultivation condition and culture medium (optimization process was omitted). We found that the ALA production was greatly enhanced in our modified minimal medium (composition see methods and materials section). Cultivation of

ALA concentration (mg/L)

3250

Extracellular ALA ( mg/L)

Intracellular ALA (mg/L)

E. coli DAL E. coli DALA

2124 7107 2963 7132

148 7 21 123 7 16

20.0

OD(600nm) ALA Glucose

35 30

17.5 15.0

25

12.5

20

10.0

15

7.5

10

5.0

5

2.5

0

0

10

20

30 Time (h)

40

50

60

0.0

OD (600nm)

40

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

ALA (g/L)

Strains

Fig. 4. Batch fermentation of ALA in E. coli DALA. A 2% (v/v) inoculum from an overnight culture for 12 h was used. 35 g/L glucose and 0.1 mM IPTG were added initially. During the fermentation, the pH was controlled optimally at 6.07 0.2.

DAL in LB medium accumulated much less ALA than in the modified minimal medium (data not shown). Supplied with 0.01 g/L FeSO4  7H2O in the medium, ALA accumulation in the strain DAL also dropped to 404.62 from 2052.29 mg/L. Addition of ferric caused the color of the cultures much deeper. This indicated that addition of ferric ion may drive ALA for porphyrins and heme production. To metabolically produce ALA in E. coli, we created the recombinant strain DALA by coexpressing hemAM, hemL and rhtA genes and cultivated it in the optimized minimal medium supplied with 35 g/L glucose. After 48 h cultivation, a high titer of ALA (4.13 g/L) with a yield of 0.168 g ALA per g glucose was achieved (Fig. 4). 4. Conclusion Metabolic engineering provides a powerful tool for regulating the metabolic pathway towards the accumulation of desired compound (Stephanopoulos, 1999). Many bio-based products are now produced in E. coli through metabolic engineering or synthetic biology (Ajikumar et al., 2010; Atsumi et al., 2008; T¨annler et al., 2008; Xia et al., 2010). In this study, we developed a metabolic strategy to produce ALA directly from glucose (Fig. 1). Through metabolic engineering, a robust E. coli that can efficiently accumulate ALA via C5 pathway was constructed. The recombinant E. coli DALA was able to produce 4.13 g/L ALA in a modified minimal medium without adding any other co-substrate or inhibitor. Efficient production of ALA directly from glucose in E. coli provides us a perspective application. Further optimization of the fermentation process should improve the ALA production to even higher level.

2600

1950

1300

650

0

Table 5 Distribution of ALA intracellular and extracellular with or without transporter. 20 g/L glucose as carbon source was added. At 32 h, cells were harvested and washed with 0.85% NaCl solution two times. Subsequently, cells were broken up with ultrasonic treatment. ALA was extracted and analyzed by the method reported previously. The results are the average of three individual experiments.

Glucose (g/L)

ALA assimilation and therefore increase ALA accumulation in the medium. However, cultivation results showed that inactivation of dppA or mppA did not improve ALA production, which suggested that there might be other transporters involved in this process (Fig. 2). Double mutant of dppA or mppA even cannot grow in minimal ALA production medium. Then, we attempted to accelerate the ALA export by overexpression of ALA exporter. To date, there is no report on the efflux mechanisms of ALA. However, numerous secondary exporters responsible for the exportation of natural amino acids have been identified in E. coli (Burkovski and Kramer, 2002). ALA has similar chemical structure to glycylglycine and its physical properties are close to native amino acids carrying uncharged side chains. Hence, we supposed that amino acid exporters with wide substrate specificity might also play roles in ALA export. Both RhtA and YeaS are classified as amino acid secondary transporters and capable of translocating a variety of amino acids and related compounds, such as dipeptide and amino acid analogs, were selected as candidates after deep consideration. rhtA encodes an inner membrane transporter RhtA, which is responsible for threonine and homoserine efflux transport (Livshits et al., 2003) while yeaS encodes a leucine exporter (Kutukova et al., 2005). Overexpression of yeaS resulted in resistance of cells to leucine analogs, glycyl-L-leucine dipeptide, and several other amino acids and their analogs. Herein, we overexpressed yeaS and rhtA in E. coli using low copy plasmid pCL1920, separately, which is compatible with pUC ori plasmids. Excitingly, we found that overexpression of rhtA did increase the ALA accumulation. The strain DALA harboring this gene accumulated 45.9% more ALA than the strain DAL did (Fig. 3), which suggested that RhtA not only exports threonine and homoserine but also plays a role in ALA exportation. Analysis of ALA distribution inside and outside the cells showed that the intracellular ALA of E. coli DAL and E. coli DALA were 148 and 123 mg/L, respectively (Table 5). The results confirmed that RhtA has a broad substrate specificity and can accelerate the ALA production.

497

DDAL

DMAL

DAL20

DALA

DALS

Fig. 3. Effect of transporter modification on ALA production in E. coli. Cultivation was performed in 300 mL Erlenmeyer flask supplied with 50 mL modified minimal medium supplied with glucose for 48 h. Results are the average of three individual experiments. A final ALA accumulation result was measured.

Acknowledgments We thank Professor Marcin Los (University of Gdansk, Poland) for providing us with P1 phage. This research was financially supported by a Grant from the National Natural Science

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Z. Kang et al. / Metabolic Engineering 13 (2011) 492–498

Foundation of China (31070092), a Grant of the National Basic Research Program of China (2011CB707405) and a Grant from Graduate Independent Innovation Foundation of Shandong University (GIIFSDU, yzc09048).

Appendix A. Supplementary Materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ymben.2011.05.003.

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