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Engineering Bafilomycin High-Producers by Manipulating Regulatory and Biosynthetic Genes in the Marine Bacterium Streptomyces Lo

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Engineering Bafilomycin High-Producers by Manipulating Regulatory and Biosynthetic Genes in the Marine Bacterium Streptomyces Lo marine drugs Article Engineering Bafilomycin High-Producers by Manipulating Regulatory and Biosynthetic Genes in the Marine Bacterium Streptomyces lohii Zhong Li 1,2,3, Shuai Li 1, Lei Du 1 , Xingwang Zhang 1, Yuanyuan Jiang 1,2,3, Wenhua Liu 1, Wei Zhang 1 and Shengying Li 1,4,* 1 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China; [email protected] (Z.L.); [email protected] (S.L.); [email protected] (L.D.); [email protected] (X.Z.); [email protected] (Y.J.); [email protected] (W.L.); [email protected] (W.Z.) 2 Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China 3 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 4 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China * Correspondence: [email protected] Abstract: Bafilomycin A1 is the representative compound of the plecomacrolide natural product family. This 16-membered ring plecomacrolide has potent antifungal and vacuolar H+-ATPase inhibitory activities. In our previous work, we identified a bafilomycin biosynthetic gene cluster (baf ) from the marine bacterium Streptomyces lohii ATCC BAA-1276, wherein a luxR family regulatory gene orf1 and an afsR family regulatory gene bafG were revealed based on bioinformatics analysis. In this study, the positive regulatory roles of orf1 and bafG for bafilomycin biosynthesis are characterized through gene inactivation and overexpression. Compared to the wild-type S. lohii strain, the knockout Citation: Li, Z.; Li, S.; Du, L.; Zhang, of either orf1 or bafG completely abolished the production of bafilomycins. The overexpression of X.; Jiang, Y.; Liu, W.; Zhang, W.; Li, S. orf1 or bafG led to 1.3- and 0.5-fold increased production of bafilomycins, respectively. A genetically Engineering Bafilomycin engineered S. lohii strain (SLO-08) with orf1 overexpression and inactivation of the biosynthetic genes High-Producers by Manipulating orf2 and orf3, solely produced bafilomycin A1 with the titer of 535.1 ± 25.0 mg/L in an optimized Regulatory and Biosynthetic Genes in fermentation medium in shaking flasks. This recombinant strain holds considerable application the Marine Bacterium Streptomyces potential in large-scale production of bafilomycin A1 for new drug development. lohii. Mar. Drugs 2021, 19, 29. https://doi.org/md19010029 Keywords: Streptomyces; bafilomycin; regulatory gene; biosynthesis; fermentation optimization Received: 2 December 2020 Accepted: 8 January 2021 Published: 11 January 2021 1. Introduction Publisher’s Note: MDPI stays neu- Bafilomycins, which are mainly produced by Streptomyces, belong to the plecomacrolide tral with regard to jurisdictional clai- (i.e., a 16- or 18-membered macrolactone connected to a 6-membered hemiacetal ring via ms in published maps and institutio- a three-carbon linker) subfamily of polyketide natural products. These 16-membered nal affiliations. ring macrolides have shown diverse biological activities including antibacterial [1], an- tifungal [2], antitumor [3], and anti-osteoporotic [4] activities. Since bafilomycin A1,B1, and C1 were first isolated from Streptomyces griseus sp. sulphurus (TÜ 1922) in 1983 [5], nearly thirty bafilomycin derivatives have been discovered [5–7]. Essentially, most of these Copyright: © 2021 by the authors. Li- derivatives (Figure1) are generated from the bafilomycin A core structure through various censee MDPI, Basel, Switzerland. 1 known [8–10] and unknown tailoring steps during their biosynthetic pathways. This article is an open access article distributed under the terms and con- As the first and archetypal compound of bafilomycins, bafilomycin A1 has attracted + ditions of the Creative Commons At- much attention because it is a potent and specific inhibitor of vacuolar H -ATPase (V- tribution (CC BY) license (https:// ATPase), which is an important drug target for osteoporosis [4]. This compound may also be creativecommons.org/licenses/by/ applied in antitumor therapy due to its potent autophagy inhibitory activity, which prevents 4.0/). autophagosome-lysosome fusion in cells by targeting the sarco/endoplasmic reticulum Mar. Drugs 2021, 19, 29. https://doi.org/10.3390/md19010029 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, 29 2 of 14 2+ Ca -ATPase (SERCA) pump [11]. Moreover, bafilomycin A1 has been demonstrated to show promising prospects in the field of combined pharmacotherapy. For example, bafilomycin A1 and FK506 have displayed marked synergistic antifungal activities against the fungal pathogen Cryptococcus neoformans [12]; the combined treatment with bortezomib plus bafilomycin A1 has been proved to be capable of enhancing the cytocidal effect and inducing U266 myeloma cells [13]; and the inhibition of autophagy by bafilomycin A1 can decrease the resistance of gastric cancer cells to 5-fluorouracil in vitro [14]. Intriguingly, bafilomycin A1 was recently reported to be capable of interrupting the function of the viral receptor ACE2 via inhibiting the V-ATPase, thus being considered as a candidate for treating the infections caused by coronaviruses (e.g., COVID-19, SARS-CoV, and MERS- CoV) [15]. Despite these promising results, such a potent drug candidate has not entered clinical application owing to its high toxicity to mammalian cells [16]. Thus, bafilomycin A1 has become an attractive target for medicinal chemists to synthesize unnatural bafilomycin derivatives with lower toxicity [17]. For the purposes of new drug development and diverse bioactivity assays, the demands for bafilomycin A1 are fast growing, which has Mar. Drugs 2021, 19, x FOR PEER REVIEW 2 of 14 naturally led to the demand for bafilomycin A1 high-producing strains since total synthesis of bafilomycin A1 remains highly challenging [18,19]. Figure 1. Structures of representativeFigure 1. Structures bafilomycins. of representative bafilomycins. As the first andTo archetypal engineer acompound bafilomycin of Abafilomycins,1 high-producer, bafilomycin knowledge A1 has on attracted its biosynthetic mech- much attentionanisms because is required.it is a potent To and date, specific at least inhibitor six bafilomycin of vacuolar biosynthetic H+-ATPase gene (V- clusters from ATPase), whichStreptomyces is an importantand drugKitasatospora target forspecies osteoporosis have been[4]. This reported compound by this may and also other laborato- be applied in riesantitumor [8,10,20 therapy–23]. The due modular to its potent and domain autophagy organization inhibitory in five activity, type Iwhich polyketide synthase prevents autophagosome(PKS) genes-lysosome (exemplified fusion by inbafAI cells–bafAV by targeting, Figure 2thea) collinearlysarco/endoplasmic matches re- the structure of ticulum Ca2+-ATPasebafilomycin (SERCA A1.) The pump five open[11]. Moreover, reading frames bafilomycinbafB–F are A responsible1 has been demon- for biosynthesis of the strated to showmethoxymalonate promising prospects extender in the unit field based of combined on bioinformatics pharmacotherapy. analysis. Recently, For ex- we completely ample, bafilomycinelucidated A1 and the FK506 post-PKS have tailoring displayed steps marked (Figure synergistic2b) of the antifungal bafilomycin activities biosynthetic pathway against the fungalin Streptomyces pathogen Cryptococcus lohii ATCC BAA-1276. neoformans Specifically, [12]; the combined the adenylyltransferase treatment with Orf3 activates bortezomib plusfumarate bafilomycin to fumaryl-AMP, A1 has been whose proved fumaryl to be capable moiety of is thenenhancing transferred the cytocidal to C21-hydoxyl group effect and inducingof bafilomycin U266 myeloma A1 by cells the fumaryl [13]; and transferase the inhibition Orf2, of giving autophagy rise to by bafilomycin bafilo- C1. Next, mycin A1 can decreasethe ATP-dependent the resistance amino of gastric synthetase cancer BafY cells catalyzes to 5-fluorouracil the C–N in bond vitro formation [14]. between Intriguingly, bafilomycinbafilomycin A C1 1wasand recently 2-amino-3-hydroxycyclopent-2-enone reported to be capable of interrupting (C5N) tothe form func- bafilomycin B1. tion of the viralThe receptor C5N unit ACE2 is assembledvia inhibiting by thethe acyl-CoAV-ATPase, ligase thus being BafX andconsidered the bifunctional as a enzyme candidate for treatinBafZ [g9, 20the]. infections caused by coronaviruses (e.g., COVID-19, SARS-CoV, and MERS-CoV) [15]. Despite these promising results, such a potent drug candidate has not entered clinical application owing to its high toxicity to mammalian cells [16]. Thus, bafilomycin A1 has become an attractive target for medicinal chemists to synthesize un- natural bafilomycin derivatives with lower toxicity [17]. For the purposes of new drug development and diverse bioactivity assays, the demands for bafilomycin A1 are fast growing, which has naturally led to the demand for bafilomycin A1 high-producing strains since total synthesis of bafilomycin A1 remains highly challenging [18,19]. To engineer a bafilomycin A1 high-producer, knowledge on its biosynthetic mecha- nisms is required. To date, at least six bafilomycin biosynthetic gene clusters from Strep- tomyces and Kitasatospora species have been reported by this and other laboratories [8,10,20–23]. The modular and domain organization in five type I polyketide synthase (PKS) genes (exemplified by bafAI–bafAV, Figure 2a) collinearly
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