412 ISSN 0003-6838, Applied Biochemistry and Microbiology, 2020, Vol. 56, No. 4, pp. 412–419. © Pleiades Publishing, Inc., 2020. Enhanced Tunicamycin Biosynthesis in BldG Overexpressed Streptomyces clavuligerus L. Baş a , Ç. Otur a , and A. Kurt-Kızıldoğan a, * a Department of Agricultural Biotechnology, Faculty of Agriculture, Ondokuz Mayıs University, Samsun, 55139 Turkey *e-mail: aslihan.kizildogan@omu.edu.tr Received August 19, 2019; revised October 21, 2019; accepted February 25, 2020 Abstract—Tunicamycin is a nucleoside type antibiotic with a potent antibacterial activity. Tunicamycin gene cluster in Streptomyces clavuligerus lacks a cluster-situated regulator (CSR). Therefore, there is no informa- tion about its regulation in the cell. To have an insight about the regulation of tunicamycin biosynthesis, the possible effects of BldG pleiotropic regulator involved in the control of secondary metabolite production in S. clavuligerus were investigated. To overexpress bldG in the cell, strains containing multiple copies of the gene expressed from P glpF promoter of S. clavuligerus pLB1, and an additional bldG integrated in the chromosome of S. clavuligerus pLB2, were constructed. S. clavuligerus pLB1 and S. clavuligerus pLB2 fermentations resulted in 16.4- and 13.8-fold higher specific tunicamycin titers, respectively, in comparison to wild type by confirming quantitative reverse-transcription PCR (qRT-PCR) data. However, qRT-PCR expression anal- ysis of tunicamycin genes in S. clavuligerus ΔbldG constructed by Bignell with coworkers [1] showed that gene expressions at T 36 (except for SCLAV_4274 and SCLAV_4275) were from 3.6- to 57.9-fold reduced compared to wild type. The tunicamycin titers were lower in S. clavuligerus ΔbldG than in wild type, as well. Conse- quently, the data presented here is the first report indicating a positive role of BldG on tunicamycin. Keywords: tunicamycin, Streptomyces clavuligerus, bldG, qRT-PCR, overexpression DOI: 10.1134/S000368382004002X The members of genus Streptomyces are versatile producers of a vast array of bioactive secondary metabolites including antibiotics, immunosuppres- sants and anticancer agents such as cephamycin C, clavulanic acid, tunicamycin and holomycin [2]. Sec- ondary metabolite gene clusters are controlled by clus- ter situated regulators (CSRs) at the lowest level, and by higher-level global/pleiotropic regulators, in a com- plex cascade system. Thus, CSRs might have different functions as mentioned by Makitrynskyy et al. [3] and could be (i) an ultimate regulator, (ii) an ultimate reg- ulator having cross-talk function [4], (iii) a true pleio- tropic regulator [5], or (iv) regulator of a distant gene cluster [6]. In turn, global/pleiotropic regulators exert their effects on the gene clusters by controlling CSRs in response to different stimuli such as environmental signals, physiological conditions and developmental stage [7]. Tunicamycin is a fatty acyl nucleoside-type antibi- otic produced by several Streptomyces species including Streptomyces lysosuperificus, Streptomyces chartreusis [8] and Streptomyces clavuligerus [9]. Also, there have been other studies showing different species having tunicamycin gene cluster resembling to that of Strepto- myces [10, 11]. It has great potency against early stage of bacterial cell wall synthesis by targeting MraY (trans- locase I) that catalyzes the formation of peptidoglycan precursor typically referred to as lipid I [12]. In a recent study, it was shown that a marine-derived strain Streptomyces sp. DUT11 produces tunicamycin I, IV and VII showing superior anticomplement activity [13]. Since tunicamycin inhibits eukaryotic protein N-gly- cosylation its clinical use as antibacterial agent is not feasible yet [14]. Tunicamycin is composed of a uracil, an N-acetylglucosamine (GlcNac), an unusual 11-car- bon 2 aminoaldose sugar called tunicamine and an amid-linked fatty acid [15]. The α,β-1″,11′-glycosidic linkage between tunicamine and GlcNac is also unique for this metabolite. The length of N-linked acyl chains is different among tunicamycin variants [16]. Although its structure and function are well known, biosynthetic gene cluster and metabolic path- way studies are limited [16, 17]. A total of 14 genes (tun1(A)-tun14(N)-SCLAV_4287-4274) were identi- fied by genome mining approach coupled with cloning and heterologous expression studies, in S. chartreusis and S. clavuligerus, lacking a CSR within the cluster [18, 19] (Figs. 1a, 1b). Recently, Widdick et al. [20] have reported mutational analysis and transcriptional char- acterization study of tunicamycin gene cluster in S. chartreusis. Since there have been no regulatory genes found in the tunicamycin cluster, a possible regulation at global level needs to be elucidate to get more information for