Xin-Ai Chen

Asperpyridone A represents an unusual class of pyridone alkaloids with demonstrated potential for hypoglycemic activity, primarily by promoting glucose consumption in HepG2 cells. Trichodin A, initially isolated from the marine fungus Trichoderma sp. strain MF106, exhibits notable antibiotic activities against Staphylococcus epidermidis. Despite their pharmacological significance, the regulatory mechanisms governing their biosynthesis have remained elusive. In this investigation, we initiated the activation of a latent gene cluster, denoted as “top”, through the overexpression of the Zn₂Cys₆ transcription factor TopC in Tolypocladium ophioglossoides. The activation of the top cluster led to the biosynthesis of asperpyridone A, pyridoxatin, and trichodin A. Our study also elucidated that the regulator TopC exerts precise control over the biosynthesis of asperpyridone A and trichodin A through the detection of protein–nucleic acid interactions. Moreover, by complementing these findings with gene deletions involving topA and topH, we proposed a comprehensive biosynthesis pathway for asperpyridone A and trichodin A in T. ophioglossoides.

As a potential protein kinase C inhibitor, the fungus metabolite balanol has become more attractive in recent decades. In our previous work, we revealed its biosynthetic pathway through overexpression of the cluster-situated regulator gene blnR in Chinese herb fungus Tolypocladium ophioglossoides. However, information on the regulation of blnR is still largely unknown. In this study, we further investigated the regulation of balanol biosynthesis by BlnR through the analysis of affinity binding using EMSA and RNA-seq analysis. The results showed that BlnR positively regulates balanol biosynthesis through binding to all promoters of bln gene members, including its own promoter. Microscopic observation revealed blnR overexpression also affected spore development and hypha growth. Furthermore, RNA-seq analysis suggested that BlnR can regulate other genes outside of the balanol biosynthetic gene cluster, including those involved in conidiospore development. Finally, balanol production was further improved to 2187.39 mg/L using the optimized medium through statistical optimization based on response surface methodology.

The azoxy compounds with an intriguing chemical bond [-N=N⁺(-O⁻)-] are known to have broad applications in many industries. Our previous work revealed that a nonheme diiron N-oxygenase AzoC catalyzed the oxidization of amino-group to its nitroso analogue in the formation of azoxy bond in azoxymycins biosynthesis. However, except for the reported pyridine alkaloid azoxy compounds, most azoxy bonds of nitrogen heterocycles have not been biosynthesized so far, and the substrate scope of AzoC is limited to p-aminobenzene-type compounds. Therefore, it is very meaningful to use AzoC to realize the biosynthesis of azoxy nitrogen heterocycles compounds. In this work, we further studied the catalytic potential of AzoC toward nitrogen heterocycle substrates including 5-aminopyrimidine and 5-aminopyridine compounds to form new azoxy compounds through directed evolution. We constructed a double mutant L101I/Q104R via molecular engineering with improved catalytic efficiency toward 2-methoxypyrimidin-5-amine. These mutations also proved to be beneficial for N-oxygenation of methyl 5-aminopyrimidine-2-carboxylate. The structural analysis showed that relatively shorter distance between the substrate and the diiron center and amino acid residues of the active center may be responsible for the improvement of catalytic efficiency in L101I/Q104R. Our results provide a molecular basis for broadening the AzoC catalytic activity and its application in the biosynthesis of azoxy six-membered nitrogen catenation compounds.

Background: Large-scale genome reduction has been performed to significantly improve the performance of microbial chassis. Identification of the essential or dispensable genes is pivotal for genome reduction to avoid synthetic lethality. Here, taking Streptomyces as an example, we developed a combinatorial strategy for systematic identification of large and dispensable genomic regions in Streptomyces based on multi-omics approaches. Results: Phylogenetic tree analysis revealed that the model strains including S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 were preferred reference for comparative analysis of candidate genomes. Multiple genome alignment suggested that the Streptomyces genomes embodied highly conserved core region and variable sub-telomeric regions, and may present symmetric or asymmetric structure. Pan-genome and functional genome analyses showed that most conserved genes responsible for the fundamental functions of cell viability were concentrated in the core region and the vast majority of abundant genes were dispersed in the sub-telomeric regions. These results suggested that large-scale deletion can be performed in sub-telomeric regions to greatly streamline the Streptomyces genomes for developing versatile chassis. Conclusions: The integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to generate more miscellaneous and versatile chassis with minimized genome for drug discovery.

Streptomyces chattanoogensis L10 is the industrial producer of natamycin and has been proved a highly efficient host for diverse natural products. It has an enormous potential to be developed as a versatile cell factory for production of heterologous secondary metabolites. Here we developed a genome-reduced industrial Streptomyces chassis by rational ‘design-build-test’ pipeline. To identify candidate large non-essential genomic regions accurately and design large deletion rationally, we performed genome analyses of S. chattanoogensis L10 by multiple computational approaches, optimized Cre/loxP recombination system for high-efficient large deletion and constructed a series of universal suicide plasmids for rapid loxP or loxP mutant sites inserting into genome. Subsequently, two genome-streamlined mutants, designated S. chattanoogensis L320 and L321, were rationally constructed by depletion of 1.3 Mb and 0.7 Mb non-essential genomic regions, respectively. Furthermore, several biological performances like growth cycle, secondary metabolite profile, hyphae morphological engineering, intracellular energy (ATP) and reducing power (NADPH/NADP+) levels, transformation efficiency, genetic stability, productivity of heterologous proteins and secondary metabolite were systematically evaluated. Finally, our results revealed that L321 could serve as an efficient chassis for the production of polyketides. Here we developed the combined strategy of multiple computational approaches and site-specific recombination system to rationally construct genome-reduced Streptomyces hosts with high efficiency. Moreover, a genome-reduced industrial Streptomyces chassis S. chattanoogensis L321 was rationally constructed by the strategy, and the chassis exhibited several emergent and excellent performances for heterologous expression of secondary metabolite. The strategy could be widely applied in other Streptomyces to generate miscellaneous and versatile chassis with minimized genome. These chassis can not only serve as cell factories for high-efficient production of valuable polyketides, but also will provide great support for the upgrade of microbial pharmaceutical industry and drug discovery.

A cryptic gene cluster, bln, was activated by genome mining in Tolypocladium ophioglossoides. This activation led to the production of balanol and eight other metabolites. Gene disruption and metabolite profile analysis showed that the biosynthesis of balanol involved the convergence of independent PKS and NRPS pathways, and a biosynthetic pathway for balanol was proposed.