Biosynthesis in Vitro of Bacillamide Intermediate-Heterocyclic Alacysthiazole by Heterologous Expression of Nonribosomal Peptide Synthetase (NRPS) T
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Journal of Biotechnology 292 (2019) 5–11 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Biosynthesis in vitro of bacillamide intermediate-heterocyclic AlaCysthiazole by heterologous expression of nonribosomal peptide synthetase (NRPS) T Fengli Zhang, Nayila Mulati, Yukun Wang, Yingxin Li, Sanqiang Gong, Loganathan Karthik, ⁎ Wei Sun, Zhiyong Li Marine Biotechnology Laboratory, State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, China ARTICLE INFO ABSTRACT Keywords: Bacillamide C, a potential natural antialgae active compound, is produced by Bacillus atrophaeus C89 derived Bacillus atrophaeus from marine sponge Dysidea avara. A nonribosomal peptide synthetase (NRPS) cluster is hypothesized to be Bacillamides involved in the biosynthesis of bacillamide C. The NRPS with a domain string of A1-PCP1-Cy-A2-PCP2-C can be Heterologous expression divided into three functional modules. After heterologous expression and purification of module A1-PCP1 and Nonribosomal peptide synthetase (NRPS) module Cy-A2-PCP2, their catalytic activities were biochemically proven in vitro by the reaction with the apo- Thiazole PCP domain transformed to the holo-PCP domain through a phosphopantetheinyl transferase, ATP, and substrate amino acids. Five– membered heterocyclic AlaCysthiazole with molecular weight of 172.0389 was detected. This proved the formation of the heterocyclic dipeptide AlaCysthiazole, which is considered to be a building block for the biosynthesis of bacillamide. This study provides a basis for further biosynthesis of bacillamides. 1. Introduction et al., 2017). Even though the biosynthesis of bacillamide C was opti- mized, the yield was very low (Jin et al., 2011; Yu et al., 2015). Bacillamides, which are nonribosomal peptides, can be categorized Therefore, investigating the biosynthetic mechanisms of bacillamide C as bacillamides A, B, C, D, and E (Fig. S1) (Bloudoff et al., 2017; is essential for its mass production and for understanding the biosyn- Ivanova et al., 2007; Jeong et al., 2003; Omura et al., 1975; Socha et al., thetic pathways of other bacillamides. 2007). In our previous study, neobacillamide A and bacillamide C were Nonribosomal peptides are produced by large multifunctional en- isolated from marine Bacillus atrophaeus C89 (Yu et al., 2009). The zymes (Schwarzer and Marahiel, 2001). A nonribosomal peptide syn- tryptamide thiazole (bacillamides A–D) or tryptamide thiazoline (ba- thetase (NRPS) gene cluster was detected in the genome sequence of B. cillamide E) motif as a typical characteristic of bacillamides (Fig. S1), is atrophaeus C89 (Genbank No. NZ_AJRJ01000045.1), which was pre- a building block present in many bioactive cyclic peptides, such as the dicted to synthesize bacillamide C (Liu et al., 2012). Nonribosomal antibiotic zelkovamycin produced by Streptomyces sp. (Tabata et al., peptide synthesis requires at least three functional domains: the ade- 1999), the protein synthesis inhibitors A-21459A and B from Actino- nylation (A) domain, which selects and activates the cognate amino planes sp. (Selva et al., 1996), and the immunosuppressive argyrins acid; the peptidyl carrier protein (PCP) domain, which transports the from the myxobacterium Archangium gephyra (Sasse et al., 2002). Si- activated intermediate; and the condensation (C) domain, which cata- milarly, bacillamides have multiple biological activities, for example, lyzes peptide bond formation, or the cyclization (Cy) domain, which bacillamide A and its derivatives possess substantial algicidal activities catalyzes heterocycle formation (Marshall et al., 2002). In our previous against a range of dinoflagellates and raphidophytes, and exhibit anti- study, the biosynthetic mechanism of bacillamide C was predicted biosis against a particular species of cyanobacteria (Jeong et al., 2003; (Zhang et al., 2018). An NRPS with a domain string of A1-PCP1-Cy-A2- Churro et al., 2009) and bacillamide analogs, thus rendering them PCP2-C can be divided into three functional modules. The A1-PCP1 novel cytotoxic and anti-inflammatory agents (Kumar et al., 2016). module consists of the first A (A1) and the first PCP domain and acti- Bacillamides have been generally synthesized chemically (Fache vates and transports L-alanine. The Cy-A2-PCP2 module consists of a Cy, et al., 2012; Kumar et al., 2016; Li et al., 2009; Wang et al., 2010), only the second A (A2), and the second PCP (PCP2) domains and activates one study reported the biosynthetic pathway of bacillamide E (Bloudoff and carries l-cysteine, catalyzes peptide bond formation, and ⁎ Corresponding author. E-mail address: [email protected] (Z. Li). https://doi.org/10.1016/j.jbiotec.2018.11.024 Received 25 June 2018; Received in revised form 16 October 2018; Accepted 29 November 2018 Available online 11 January 2019 0168-1656/ © 2019 Elsevier B.V. All rights reserved. F. Zhang et al. Journal of Biotechnology 292 (2019) 5–11 heterocyclizes the product 4-carboxylic acid thiazoline derivative. modified Marmur method (Marmur, 1961; Yuwen et al., 2013). The Moreover, the flavin mononucleotide-containing oxidase (Ox) domain primers A1-P1F: 5′-ATGCAAATTAAAGATCTGAGTTCG-3′ and A1-P1R: oxidizes the dehydro heterocycle by a two-electron transfer to produce 5′-GATTACACAAGAACTAAGTTGAGCAATC-3′ were employed to am- heteroaromatic thiazole. The 3rd module consists of the C domain and plify the DNA sequence of module A1-PCP1; in addition, the primers catalyzes peptide bond formation between the intermediates synthe- C1-A2-P2F: 5′−CCAACCCGTGTTAAAGATTTG-3′ and C1-A2-P2R: tized by the A1-PCP1 and Cy-A2-PCP2 modules, and L-tryptophan 5′-GATAAGCGGTTTA TCACTAACATTCAG-3′ were used to amplify that decarboxylated by the next aromatic L-amino acid decarboxylase of module Cy-A2-PCP2. The sequences of the cloned fragments were (AADC) enzyme to produce a bacillamide precursor. Finally, bacilla- examined using T7 promoter and terminator primers. The recombinant mide is synthesized after acetyl modifications at the N-terminus ba- plasmid with phosphopantetheinyl transferase gene (sfp) from Bacillus cillamide precursor (Zhang et al., 2018). In our previous study, the subtilis was provided by Prof. Lin (Huang et al., 2011). AADC enzyme nearest to the NRPS gene cluster in B. atrophaeus C89 was characterized as a highly efficient catalyst for the decarboxylation 2.3. Gene sequence analysis of L-tryptophan to tryptamine (Yuwen et al., 2013). Tryptophan dec- arboxylation is an extremely rare activity among bacteria (Williams Expasy translate tool (http://expasy.org/tools/dna.html) was used et al., 2014), and B. atrophaeus (Yuwen et al., 2013) can produce for translating nucleotide sequence into protein sequence. Amino acid tryptamine as a building block for the biosynthesis of larger natural sequence of NRPS was analyzed using the BLASTP program (https:// products. blast.ncbi.nlm.nih.gov/Blast). Amino acid sequences of the NRPS pro- Heterocyclic chemistry represents a broad and crucial research field tein from various species were aligned using MEGA (Molecular of interest for academic, as well as for industrial, pharmaceutical, and Evolutionary Genetics analysis version 6.0.) for drawing phylogenetic phyto-pharmaceutical fields. The tryptamide thiazole or thiazoline relationship using UPGMA algorithm. motif is a typical characteristic of bacillamides (Yu et al., 2009, 2015). A 4-carboxylic acid thiazole derivative as a bacillamide intermediate 2.4. Expression and purification of recombinant A1-PCP1 and Cy-A2-PCP2 possesses a thiazole cycle. Typically, thiazole is a characteristic struc- ture of natural alkaloids including tantazoles, mirabazoles, and thian- The DNA sequencing in both orientations and the resulting plasmids gazole (Kriek et al., 2007). The thiazole ring is notable as a component were used to transform competent E. coli BL21 (DE3) cells. A single of the vitamin thiamine (B1), and can also be considered as a functional positive colony was inoculated in 5 mL LB medium containing kana- group (Kriek et al., 2007). In some NRPS systems, a single hetero- mycin (50 μg/mL) or ampicillin (100 μg/mL) at 37 °C for 12 h. The cyclization domain is responsible for an amino acid (the acceptor) along overnight culture was used to inoculate LB medium containing anti- with the activated upstream acyl group (the donor), cyclization through biotics and incubated at 37 °C with vigorous shaking until OD600nm an attack across the newly formed amide bond, and dehydration to reached 0.6; isopropyl β-D-1-thiogalactopyranoside was added to the form the stabilized dihydro heterocycle present in the final product medium at a final concentration of 0.5 mM and incubated at 16 °C for (Marshall et al., 2002). 15 h. The cells were centrifuged, resuspended in a binding buffer In our previous study, the AADC enzyme involved in the biosynth- (300 mM NaCl, 20 mM Tris−HCl, pH 8.0), and lysed through sonica- esis of bacillamide C was characterized (Yuwen et al., 2013), and the tion. The debris was removed through centrifugation at 12,000 × g for substrate selection of two recombinant A domains was determined 20 min. The supernatant was filtered through a 0.45-μm Millipore filter (Zhang et al., 2018). Herein, we describe the identification and func- and loaded onto an equilibrated Ni-NTA agarose resin column (Qiagen tional expression of genes encoding an NRPS involved in the bio- Co., Hilden, Germany). The target protein was eluted with an elution