Insights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition.

Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER1/2 inside module 1/2 and the ketosynthase domain KS3 inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS4 of module 4 acts as a gatekeeper facilitating cross-module enoylreduction. Additionally, evidence is provided that module 3 and module 6 in the AZL PKS are evolutionarily homologous, which makes evolution-oriented PKS engineering possible. These results reveal intermodular recognition, furthering understanding of the mechanism of the PKS assembly-line, thus providing different insights into PKS engineering. This also reveals that gene duplication/conversion and subsequent combinations may be a neofunctionalization process in modular PKS assembly-lines, hence providing a different case for supporting the investigation of modular PKS evolution.

Bioactive Monoterpenes and Polyketides from the Ascidian-Derived Fungus Diaporthe sp. SYSU-MS4722.

There has been a tremendous increase in the rate of new terpenoids from marine-derived fungi being discovered, while new monoterpenes were rarely isolated from marine-derived fungi in the past two decades. Three new monoterpenes, diaporterpenes A-C (1-3), and one new α-pyrones, diaporpyrone A (6), along with nine known polyketides 4, 5, and 7-13 were isolated from the ascidian-derived fungus Diaporthe sp. SYSU-MS4722. Their planar structures were elucidated based on extensive spectroscopic analyses (1D and 2D NMR and HR-ESIMS). The absolute configurations of 1 and 3 were identified by an X-ray crystallographic diffraction experiment using Cu-Ka radiation, and those of compound 2 were assigned by calculating NMR chemical shifts and ECD spectra. It afforded an example of natural epimers with different physical properties, especially crystallization, due to the difference in intermolecular hydrogen bonding. Compounds 9, 10, and 13 showed moderate total antioxidant capacity (0.82 of 9; 0.70 of 10; 0.48 of 13) with Trolox (total antioxidant capacity: 1.0) as a positive control, and compounds 5 and 7 showed anti-inflammatory activity with IC50 values of 35.4 and 40.8 µM, respectively (positive control indomethacin: IC50 = 35.8 µM).

Cross-Module Enoylreduction in the Azalomycin†F Polyketide Synthase.

The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross-modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly-line catalysis.

An Iterative Module in the Azalomycin†F Polyketide Synthase Contains a Switchable Enoylreductase Domain.

Detailed analysis of the modular Type I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycin F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension-module 1 showed that this module catalyzes two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module can apparently be "toggled" off and on : it functions in only the second of these two cycles. This novel mechanism expands our understanding of PKS assembly-line catalysis and may explain examples of apparent non-colinearity in other modular PKS systems.

Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system.

The current diminishing returns in finding useful antibiotics and the occurrence of drug-resistant bacteria call for the need to find new antibiotics. Moreover, the whole genome sequencing revealed that the biosynthetic potential of Streptomyces, which has produced the highest numbers of approved and clinical-trial drugs, has been greatly underestimated. Considering the known gene editing toolkits were arduous and inefficient, novel and efficient gene editing system are desirable. Here, we developed an engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) combined with the counterselection system CodA(sm), the D314A mutant of cytosine deaminase, to rapidly and effectively edit Streptomyces genomes. In-frame deletion of the actinorhodin polyketide chain length factor gene actI-ORF2 was created in Streptomyces coelicolor M145 as an illustration. This CRISPR/Cas9-CodA(sm) combined system strikingly increased the frequency of unmarked mutants and shortened the time required to generate them. We foresee the system becoming a routine laboratory technique for genome editing to exploit the great biosynthetic potential of Streptomyces and perhaps for other medically and economically important actinomycetes.

[Cloning and sequence characterization of replication region in plasmid pJTU112 from Micoromonospora sp. 40027].

UNLABELLED: Micoromonospora sp. 40027, the producer of fortimicin A, harbors two plasmids pJTU101 and pJTU112. OBJECTIVE: Cloning and sequencing of replication region of pJTU12, analyzing replication region sequence of pJTU112. METHODS: Different fragments of pJTU112 were cloned and introduced by conjugation into Micromonospora sp. LXH20. The replication region of pJTU112 was identified. RESULTS: The replication region of pJTU112 was located in the 4.7 kb SacI-KpnI DNA fragment. DNA sequencing and analysis revealed that the 4.7 kb SacI-KpnI DNA fragment encoded five open reading frames. The pJTU112.1 and pJTU112.2 were related to plasmid conjugation, pJTU112.3, pJTU112.4 and pJTU112.5 were related to plasmid conjugation. CONCLUSION: The replication region of pJTU112 was located in the 4.7kb SacI-KpnI DNA fragment.