Prime STAR HS DNA polymerase, restriction endonucleases, Genomic Extraction Kit, Fragment Purification Kit, Agarose Gel DNA Extraction Kit, and Plasmid Purification Kit were purchased from TaKaRa (Dalian, China). DNA sequencing was performed by Sangon (Shanghai, China). Isopropyl-β-D-l-thiogalactopyranoside (IPTG) was obtained from Sangon (Shanghai, China).
Strains, Plasmids, Primers, And Medium
The bacterial strains, plasmids and primers used in this study are listed in Tabe 1.
Strain ARTP-L04 was an L-leucine-producing strain that originated from strain ATCC 13032 by Atmospheric Room Temperature Plasma Mutagenesis (ARTP). E. coli DH5α was used as the host cells for propagating plasmids, and E. coli BL21 (DE3) was used as the host cells for protein expression. The E. coli strain was cultivated in Luria-Bertani (LB) medium at 37oC with vibration at 200 rpm. The C. glutamicum strain was grown in LBG medium (LB containing 5 g/L glucose) at 30oC. When appropriate, kanamycin (30 µg/mL for C. glutamicum, 50 µg/mL for E. coli) and 0.5 mM IPTG were added to the medium.
LBG medium was used as a seed culture medium. The fermentation medium was composed of 90 g/L glucose, 35 g/L (NH4)2SO4, 20 g/L corn steep liquor, 1 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 10 g/L CaCO3, 50 µg/L D-biotin, and 100 µg/L thiamine-HCl. D-biotin and thiamine-HCl were filter-sterilized and aseptically added to the medium. The medium component of CaCO3 was sterilized alone by dry heat sterilization at 160°C for 90 min before being added to the medium. Both media were adjusted to pH 7.5 using KOH.
Constructions Of Plasmids
Routine methods of molecular cloning (PCR, DNA restriction, and ligation) were carried out as previously described (Sambrook et al., 1989). EcoR І and XhoⅠ restriction sites and protective bases were introduced into primers of leuA-F1 and leuA-R1, respectively. Wild-type leuA was amplified with primers of leuA-F1 and leuA-R1 by PCR using genomic DNA of strain ATCC 13032 as a template. With the same primers, another gene fragment (leuAV) was obtained using genomic DNA of strain ARTP-L04 as a template. These two fragments were cleaved with EcoRⅠ and XhoⅠ and then ligated into vector pET28a, resulting in recombinant plasmids pET28a-leuA and pET28a-leuAV (Table 1), respectively. All resulting plasmids were sequenced for identification.
Table 1 Strains, plasmids and primers used in this study
Origin from strain ARTP-L04 was indicated with superscript V. *Underlined bases were the restriction enzyme sites.
Using genomic DNA of strain ARTP-L04 as a template, the gene of leuAV was obtained with primers of leuA-F2 (containing XbaⅠ site) and leuA-R2 (containing EcoR І site) and subsequently ligated into vector pXMJ19, resulting in plasmid pXMJ19-leuAV. With the same primers, plasmid pXMJ19-leuA was also obtained using genomic DNA of strain ATCC 13032 as a template. Meanwhile, the gene of ilvBNV was amplified with primers of ilvBN-F and ilvBN-R using genomic DNA of strain ARTP-L04 as a template. After digestion with HindⅢ and XbaⅠ, the final PCR product was ligated into vector pXMJ-19 (similarly digested), resulting in recombinant plasmid pXMJ19-ilvBNV (Table 1). With XbaⅠand EcoR І, the fragment leuAV was digested and subsequently ligated into plasmid pXMJ19-ilvBNV (similarly digested), resulting in plasmid pXMJ19-ilvBNV/leuAV (Table 1). All resulting plasmids were sequenced for identification.
Constructions Of Recombinant Strains
For protein (α-IMPS) purification, plasmids of pET28a-leuAV and pET28a-leuA were transformed into E. coli BL21 (DE3), thereby resulting in recombinant E. coli strains (Table 1). To verify effect on L-leucine synthesis, plasmids pXMJ19-leuAV, pXMJ19-leuA, and pXMJ19-ilvBNV/leuAV were transformed into C. glutamicum ATCC 13032 by electrotransformation method (Tan et al., 2012), resulting in recombinant C. glutamicum strains (Table 1).
Protein Expression And Purification
Recombinant E. coli strains were grown in LB medium at 37°C till an OD600 of 0.6 was achieved, followed by induction using 0.5 mM IPTG. After 4 h of induction, cells were harvested by centrifugation at 6,000×g for 10 min at 4°C. Cells were disrupted by sonication in buffer A (0.5 M NaCl, 10 mM imidazole, and 20 mM HEPES, pH 8.0), and then the crude extract was obtained. The pET28a vector endows α-IPMS protein with an N-terminal His-tag. Therefore, the α-IPMS protein was purified by Ni-NTA affinity column under buffer B (0.5 mM NaCl, 0.5 mM imidazole, and 20 mM HEPES, pH 8.0). The recombinant protein was then eluted with 0.5 mL elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) and then dialyzed against 50 mM phosphate buffer (pH 7.0). The purified protein was stored in 50% glycerol at -70°C (Wandee et al., 2009).
Enzyme Assay
The α-IPMS activity was analyzed using 5, 5'-dithio-bis (2-nitrobenzoic acid) (DTNB) to detect the formation of coenzyme A (CoA) at a wavelength of 412 nm (Wandee et al., 2009; Kohlaw et al., 1969). The enzyme assay was performed in 150 µL buffer containing 50 µM Tris-HCl (pH 8.5), 20 µM KCl, 0.2 µM acetyl CoA, and 0.5 µM α-ketoisovaleric acid. The reaction was initiated by adding 100 µL purified proteins to 150 µL reaction buffer, followed by incubation at 37oC for 5 min. The reaction was stopped with the addition of 0.75 mL absolute ethanol and 0.5 mL 1 mM DTNB.
Feedback inhibition was determined at different concentrations of L-leucine. The enzyme kinetics curve was obtained by altering L-leucine concentrations. L-leucine was added to reaction mixtures at various final concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.8, 1.0, 2.0, 3.0, 4.0 5.0, 10, and 20 mM).
Enzyme activity was defined as units (of enzyme) per milligram of protein. One unit of the enzyme was defined as the amount catalyzing the formation of 1 µM CoA per minute. Protein concentrations were determined as previously described (Bradford, 1976).
Fermentation
Strains of ATCC 13032/leuA, ATCC 13032/leuAV, ATCC 13032/ilvBNV, and ATCC 13032/ilvBNV-leuAV were grown in medium, and L-leucine levels of these strains were evaluated. Firstly, strains were inoculated in LBG medium by transferring single colonies from LBG plates, followed by incubation at 30°C and 200 rpm. After growing to the exponential phase (14–16 h), the seed culture was inoculated into a 500-mL flask containing 30 mL fermentation medium containing IPTG (0.5 mM). The resulting culture was incubated at 30oC for 72 h at 200 rpm.
Analytical Methods
Samples were collected and centrifuged at 1, 2000×g for 10 min at 4°C. Proteins were purified from the supernatant by trichloroacetic acid (30 g/L) and filtered through a membrane (pore size = 0.22 µm). Amino acids were pre-column derivatized by o-phthaldialdehyde (OPA) and then analyzed on an UltiMate 3000 UHPLC system (Thermo Technologies) equipped with a reverse-phase column (Zorbax Eclipse-AAA) and a UV detector at 338 nm according to the published procedure (Zhang et al., 2014; Zhang et al., 2013).