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Genetic Engineering of an Industrial Strain of Streptomyces Clavuligerus for Further Enhancement of Clavulanic Acid Production

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Genetic Engineering of an Industrial Strain of Streptomyces Clavuligerus for Further Enhancement of Clavulanic Acid Production Turkish Journal of Biology Turk J Biol (2017) 41: 342-353 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1608-17 Genetic engineering of an industrial strain of Streptomyces clavuligerus for further enhancement of clavulanic acid production 1,2, 1,3 1,4 1,5 1 Aslıhan KURT KIZILDOĞAN *, Güliz VANLI JACCARD , Alper MUTLU , İbrahim SERTDEMİR , Gülay ÖZCENGİZ 1 Department of Biological Sciences, Faculty of Arts and Science, Middle East Technical University, Ankara, Turkey 2 Department of Agricultural Biotechnology, Faculty of Agriculture, Ondokuz Mayıs University, Samsun, Turkey 3 Department of Physiology, University of Lausanne, Lausanne, Switzerland 4 Center for Quantitative Analysis of Molecular and Cellular Biosystems (BIOQUANT), University of Heidelberg, Heidelberg, Germany 5 Department of Molecular Biology-Genetics and Biotechnology, Graduate School of Science Engineering and Technology, İstanbul Technical University, İstanbul, Turkey Received: 05.08.2016 Accepted/Published Online: 25.11.2016 Final Version: 20.04.2017 Abstract: An industrial clavulanic acid (CA) overproducer Streptomyces clavuligerus strain, namely DEPA, was engineered to further enhance its CA production. Single or multiple copies of ccaR, claR (pathway-specific activators), and cas2 (CA synthase) genes under the control of different promoters were introduced into this strain. CA titers of the resulting recombinants were analyzed by HPLC in a dynamic fashion and compared to the vector-only controls and a wild-type strain of S. clavuligerus while their growth was monitored throughout fermentation. The addition of an extra copy of ccaR, under control of its own promoter or constitutive ermE* promoter (PermE*), led to 7.6- and 2.3-fold increased volumetric levels of CA in respective recombinants, namely the AK9 and ID3 strains. Its highly stable multicopy expression by the glpF promoter (PglpF) provided up to 25.9-fold enhanced volumetric CA titers in the respective recombinant, IDG3. claR expression controlled with its own promoter or ermE* and glpF-mediated amplification in an industrial strain brought about only about 1.2-fold increase in the volumetric CA titers. An extra copy of cas2 integration with PermE* into the S. clavuligerus DEPA genome led to 3.8-fold higher volumetric CA production by GV61. Conclusively, multicopy expression of ccaR under PglpF can result in significantly improved industrial high-titer CA producers. Key words: Clavulanic acid, industrial Streptomyces clavuligerus, strain improvement, promoters, ccaR overexpression 1. Introduction CA and clavam biosynthesis (Medema et al., 2010). In Clavulanic acid (CA) is one of the most important addition, cephamycin C and CA clusters are consecutively secondary metabolites naturally produced by Streptomyces positioned on the chromosome as a 60-kb β-lactam clavuligerus. It is a member of very powerful class of supercluster (Ward and Hodgson, 1993). Characterization β-lactamase inhibitors with very weak antibiotic activity of the CA gene cluster is still an ongoing process. However, (minimal inhibitory concentration around 25–125 µg/ essential genes such as cas2 (clavaminate synthase, CAS, a mL) (Baggaley et al., 1997). CA exerts its effect on rate-limiting enzyme) have already been identified (Song penicillin- and cephalosporin-resistant bacteria in such et al., 2009). CA regulation is accomplished by pleiotropic a way that it irreversibly binds to the serine hydroxyl and pathway-specific regulators at the molecular level. group in the catalytic sites of β-lactamases and leads The best known pathway-specific regulators of CA to the formation of a highly stable and inactive enzyme biosynthesis are CcaR and ClaR, which are transcriptional (Foulstone and Reading, 1982; Baggaley et al., 1997). Its activators encoded by ccaR (located in the cephamycin C industrial production is based on large-scale S. clavuligerus gene cluster), and pathway-specific activators encoded by fermentations, as its chemical synthesis in such volumes is claR, respectively (Liras et al., 2008). not practical (Bentley et al., 1977; Ferguson et al., 2016). Media optimization, genetic and metabolic Three distinct gene clusters, namely CA and 5S clavams engineering, and synthetic biology approaches have located in the S. clavuligerus genome and the paralog been commonly used for obtaining high-titer industrial gene cluster found in the megaplasmid, are involved in metabolites, especially β-lactams (Olano et al., 2008; * Correspondence: [email protected] 342 KURT KIZILDOĞAN et al. / Turk J Biol Nielsen et al., 2009; Baltz, 2011; Özcengiz and Demain, kanamycin 25 µg/mL, chloramphenicol 25 µg/mL; all 2013). The use of random mutagenesis and screening purchased from Sigma). DNA manipulations were carried strategies provided an industrial S. clavuligerus strain that out in E. coli DH5α and then E. coli ET12567/pUZ8002 produces 100-fold higher CA production than its wild- to avoid the restriction barrier of S. clavuligerus before type counterpart (Medema et al., 2011a). However, such conjugation. MS agar (Hobbs et al., 1989) was used to classical techniques are tedious and time-consuming, and grow exconjugants after conjugation. S. clavuligerus DEPA rational technologies are more preferred by researchers. was grown in vegetation medium [per liter: soy flour, 20 Moreover, the combination of classical strain isolation g; dextrin, 10 g; KH2PO4, 0.6 g; glycerol trioleate (GTO), techniques with modern strain engineering strategies 5 mL] as preculture and in fermentation medium [per results in multiple complex phenotypes (Patnaik, 2008). liter: soy flour, 20 g; dextrin, 10 g; KH2PO4, 0.6 g; GTO, In general, the amount of secondary metabolite produced 5 g; MOPS, 10.5 g; oligo elements solution (per liter: by standard strains is much lower than what is required CaCl2, 10 g; MgCl2.6H2O, 10 g; FeCl3, 3 g; ZnCl2, 0.5 g; for industrial-scale production; therefore development MnSO4.H2O; 0.5 g; NaCl, 10 g), 10 mL] for CA production. of a super-producer strain by manipulating high-titer Precultures that reached an OD600 value of around 6 were industrial ones is a very rational option (Nielsen et al., used to inoculate fermentation media in a 1/100 ratio. The 2009; Pickens et al., 2011). In the literature, there is only fermentation cultures were supplemented with GTO (0.8 one relevant report in which a double disruption of the lat mL/40 mL) at 96 h of incubation. Fermentations were and cvm genes (lat::apr-cvm::apr) involved in cephamycin carried out on rotary shakers in baffled flasks at 23.5 °C. C and clavam biosynthesis, respectively, resulted in a For CA bioassay, Klebsiella pneumoniae ATCC 29665 was 10% increment in CA yield in commercial S. clavuligerus grown in TSB (Oxoid) at 30 °C and 200 rpm to get an (Paradkar et al., 2001). OD600 value of 0.9–1.0. Integrative and/or multicopy expressions of cas2, 2.2. Construction of plasmids and strains claR, and ccaR under the control of different promoters Chromosomal DNA of S. clavuligerus DEPA was isolated in wild-type S. clavuligerus have resulted in enhanced CA by using the salting-out procedure described by Pospiech production (Pérez-Llarena et al., 1997; Pérez-Redondo et and Neumann (1995) and used as template DNA for PCR al., 1998; Hung et al., 2007; Baltz, 2011; Guo et al., 2013). cloning of the ccaR, claR, and cas2 genes. Primers designed Indeed, the use of previously reported gene manipulations for PCR amplifications were synthesized by Alpha DNA on industrial strains obtained from random mutation (Montreal, Canada) and are listed in Table 2. PCR reactions strategies might yield more productive strains (Paradkar, were prepared as follows: template DNA (50 ng), 1 µL; 10C 2013). Thus, in the present study, we aimed at enhancing PCR buffer, 2.5 µL; 10 mM dNTP, 1 µL; DMSO, 1 µL; each CA yields of the industrial S. clavuligerus strain DEPA forward and reverse primer (10 µM), 1.25 µL; Taq DNA to obtain a versatile CA super-producer with the use of polymerase (Thermo Fisher Scientific, 5 U/µL), 0.5 µL; different vector systems and promoters by (i) insertion MgCl2 (25 mM), 2 µL; dH2O, 14.5 µL. The PCR conditions of ccaR expressed from its own promoter or ermE* were started with an initial denaturation step (10 min constitutive promoter (PermE*) by the use of the pSET152 at 94 °C), followed by 35 cycles of amplification (45 s at integration vector (Wilkinson et al., 2002) and from a 95 °C for denaturation, 45 s at Tm-5 °C for annealing, 1 strong glycerol inducible glpF promoter (PglpF) in the pSPG min/1 kb at 72 °C for extension), and ended with a final multicopy plasmid (Kurt et al., 2013), (ii) integrative/ extension step of 72 °C for 10 min. Enzymes (all 10 U/µL multicopy expression of claR under the control of its in concentration except for Taq DNA polymerase) used in own promoter and PermE* and PglpF, respectively, and (iii) cloning experiments were from Thermo Fisher Scientific cas2 gene expression with the ermE* promoter. To our (Petaluma, CA, USA). knowledge, this study reports for the first time an absolute 2.2.1. The sources/properties of plasmids and related quantification of high-titer CA production via HPLC promoters in recombinant industrial bacteria constructed using a pSPG (Supplementary Figure 1a) is a pIJ699-derived rational approach. replicable vector with a copy number ranging from 40 to 300 per cell (Kieser and Melton, 1988). PglpF in the 2. Materials and methods pSPG vector is a glycerol-inducible promoter found 2.1. Bacterial strains, plasmids, and culture conditions in a glycerol utilization pathway in S. clavuligerus that The microorganisms and plasmid vectors used in the coordinates GlyR-dependent regulation (Baños et al., study are listed in Table 1. Escherichia coli strains were 2009). In addition, pSET152 (Supplementary Figure grown in either Luria broth (LB) (Merck) or on agar plates 1b) is an integrative vector that is unable to replicate in at 37 °C, supplemented with the appropriate amount Streptomyces, but with the presence of the fC31 (phiC31) of antibiotics when necessary (ampicillin 100 µg/mL, attP-int locus in its sequence, it is inserted into the phiC31 343 KURT KIZILDOĞAN et al.
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