| One hallmark of fungi is their capacity to synthesize diverse biological polyketide natural products with structural variation during synthesis by polyketide synthases (PKSs). With a few exceptions, a majority of fungal PKSs are iterative type 1 PKSs, which are multidomain monomodular megasynthases. The fungal IPKSs can be divided into three major classes according to the function and phylogeny, i.e., the non-reducing, the highly-reducing, and the partial-reducing PKSs. This classification reflects the extent of P-keto reduction, domain architectures and produced metabolites. The non-reducing PKSs (NR-PKSs) cyclize the non-reduced reactive poly-β-keto chains into mono-or polycyclic aromatic polyketides. Recently, the product template (PT) domains, which are unique to the NR-PKSs, have been demonstrated to be embedded in controlling specific aldol cyclization and aromatization of the polyketide precursors. Currently, in order to obtain novel and bioactive natural products, even improve compound yield, the combinatorial biosynthesis methods with genetic manipulation become the focus of chemistry and biology interdisciplinary. In previous study, various polyketides were isolated from the fungus Alternaria alternata M311B derived from soft coal Sarcophyton sp.. Particularly, anthraquinones and benzaquinones showed not only significance in chemical ecology but also various pharmacological activities, which biosynthetic pathway should be studied indepthly. In this study, the PKSs of M311B were obtained by molecular biology experiments. Based on the systematic analyses of sequences, structures and functions of fungal PKSs, the relationship between M311B PKS sequences and metabolites, and structures and functional sites, were predicted by bioinformatics, respectively. It hopes to provide basis information for combinatorial biosynthesis of M311B PKSs.The acquisition of M311B PKS sequencesIn this study, a total of eight PKS sequences were obtained from the marine-derived fungus A. alternata M311B by degenerate PCR. Based on the phylogenetic analysis, the PksA and Pksl of A. alternata M311B are classified as the NR-PKSs, which are the first NR-PKSs found in A. alternata. It is suggested that the M311B PksA and Pksl may be involved in benzoquinone and anthraquinone syntheses. The PksJ may be related with the biosynthesis of AOH and AME.Bioinformatical analysis of the sequences, structures and functions of fungal polyketide synthase product template domainsThe NR-PKSs are involved in the biosynthesis of benzoquinone and anthraquinone. The PT domains, specifically in fungal NR-PKSs, mediate the regioselective cyclization of polyketides dominating the final structures. Analyses of PT domains have key roles in predicting the structural types of the products from NR-PKS protein sequences, and modifying/changing the structures of the compounds by combinatorial biosynthesis. Thus, based on the large-scale data mining, the NR-PKSs especially the sequences, structures and functions of PT domains were researched systematically in this study.All of the known NR-PKS amino acid sequences were collected from the database by searching and selecting. Based on the phylogenetic analysis, the collected NR-PKSs comprised by 661 sequences are classified into prominent eight groups (I-VIII) and the group VIII was observed for the first time. The established PT domain phylogenetic tree was similar to that of NR-PKSs, also classified into eight groups (I-VIII). The PT domain phylogenetic tree from the 55 NR-PKSs, which function have been investigated, was established based on their regioselective cyclization modes (C2-C7, C4-C9, and C6-C11) and compound characteristics. The three-dimensional models of PT domains were constructed using three comparative protein modeling methods and refined in the atomic-level by molecular dynamic calculations. It was found that most of the cavity lining residue (CLR) sites in all groups were common, while the regional CLR site mutations resulted in the appearance of finger-like regions with different orientation. The cavity volumes and shapes, even the catalytic dyad positions of PT domains in different groups were corresponding with characteristic cyclization regioselectivity and compound sizes. The conservative residues in PT sequences were responsible for the cyclization functions and the evolution of the key residues resulted in the differentiations of cyclization functions. The above findings may help to better understand the cyclization mechanisms of PT domains and even predict the structural types of the aromatic polyketide products from NR-PKS protein sequences. It could be prospected to manipulate the fungal PKS genes and further regulate the biosynthetic pathway to obtain the target metabolites.The prediction of sequences, structures and functions from M311B PKSsBased on the above results, the functions of the M311B PKSs were analyzed. The domain architecture of each eight PKSs was identified by the Udwary-Merski algorithm in silico. It is the first time to indentify and analyze the SAT and PT domains in A. alternata. According to the analysis of PT domains, it is suggested that the PksA of A. alternata M311B should be responsible for producing 1,3,6,8-THN with C2-C7 cyclization. The M311B Pksl may be involved in anthraquinone syntheses with C6-C11 cyclization. The conservation analyses of all amino acid residues in each domain of M311B PKSs were performed with the evolutionary conservation scores calculated by ConSurf. The amino acid residues which may have a significant impact on function and structure of proteins were analyzed and displayed.In this study, the sequences, structures and functions of fungal PKSs from a marine derived A. alternata M311B were analyzed by molecular biology techniques combined with bioinformatics methods. The findings may elucidate the biosynthetic pathways of secondary metabolites and provided basic information for metabolic regulations ot A. alternata M311B. |
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