Microorganisms possess the ability to synthesize a variety of natural products, making them a treasure trove for human drug development. However, during the synthesis of natural products by microorganisms, a large number of biosynthetic genes remain in a "silent" state, and their products are referred to as microbial "dark matter of life." How to effectively activate and explore these "dark matter of life" is a bottleneck in discovering new natural products.
With the popularization of genome sequencing technology and the maturity of genome analysis methods, there is hope to bypass the cumbersome modification processes, break through the individual differences of strains, and provide insights into revealing the universal rules of natural product synthesis and developing modification tools.
On April 12, researcher Luo Xiaozhou from the Shenzhen Advanced Institute of the Chinese Academy of Sciences, Professor Jay D. Keasling from the University of California, Berkeley, and researcher Tang Xiaoyu from the Shenzhen Bay Laboratory of Chemical Biology published their latest research results in "Nature Metabolism."
The research team used pan-genome analysis technology to identify 597 genes in the genus Streptomyces and discovered a key pathway that can significantly increase the natural product yield of Streptomyces, producing new compounds with pharmaceutical potential, which is of great significance for developing novel antibiotics and increasing natural product yield.
"This achievement not only ignites our common interest in rebooting the biosynthesis of microbial natural products 'dark matter' but also provides new strategies for the application of synthetic biology in drug development and other fields," commented Deng Zixin, academician of the Chinese Academy of Sciences and director of the National Key Laboratory of Microbial Metabolism.
Establishing a New Strategy for Yield Enhancement in "Cell Factories"
Actinomycetes are one of the main microorganisms producing antibiotics, and Streptomyces is the most typical genus in the class Actinomycetes. It is one of the microorganisms with the richest biosynthetic gene clusters known for natural product biosynthesis and is called a "cell factory."
"To survive in the natural environment, Streptomyces can evolve a large number of secondary metabolism genes to produce various bioactive substances in the form of gene clusters to resist enemies and suppress competitors. Compared with other microorganisms, Streptomyces has more secondary metabolism gene clusters, and there are also significant differences in capabilities among different strains," explained Wang Xinran, co-first author of the paper and assistant researcher at the Shenzhen Advanced Institute.
If strains of Streptomyces with differential abilities to produce bioactive substances can be found, and which genes may have co-evolved with high production of bioactive substances can be studied, it may be possible to develop new methods to promote product synthesis in Streptomyces, activate silent gene clusters, and uncover the microbial metabolism "dark matter of life."
Compared with microorganisms such as Escherichia coli and yeast, genetic modification technology for actinomycetes is not mature. On the one hand, the growth and fermentation time of modified strains are long, and individual strains are heterogeneous. Currently, research in this field mainly focuses on regulating the genetic material, living environment, or metabolites of individual strains; on the other hand, a large number of genome analyses require professionals to develop corresponding algorithms, and the cost of trial and error is high, with few universal strategies to increase the yield of different natural products.
In response, the team led by Luo Xiaozhou spent nearly four years using pan-genome analysis technology to systematically analyze the genomes of the entire Streptomyces genus, bypassing the study of individual Streptomyces strains and focusing on population rules. They established a common operating platform encompassing more than 20 different Streptomyces strains and developed universal modification methods through a "bottom-up" approach.
"This method bypasses the time and manpower costs traditionally spent on mechanistic research, provides new ideas for developing natural product production modification technologies applicable to various plants and microorganisms, and accelerates the process of uncovering unknown areas of natural product synthesis," said Luo Xiaozhou, co-corresponding author of the paper and researcher at the Shenzhen Advanced Institute.
Identifying Key Pathways for Yield Enhancement
In this study, the research team identified 597 genes that co-evolved with polyketide compounds through pan-genome analysis technology and found that the gene cluster responsible for the synthesis of coenzyme pyrroloquinoline quinone (PQQ) played a key role in Streptomyces natural product synthesis.
Collaborating with Professor Bai Linqun from Shanghai Jiao Tong University and Professor Sun Yuhui from Huazhong University of Science and Technology, the research team introduced the PQQ biosynthetic pathway into Streptomyces strains and industrial actinomycetes. They found that the production of at least 16,385 metabolites increased significantly, including 36 known natural products such as oxytetracycline and ansamycin, which could be used in fungicides, antifungals, and anticancer agents.
It is worth noting that the research team also observed the production of new metabolites, some of which even exhibited potential antibiotic activity and activity against clinical strains of infection. "This confirms that the introduction of this new pathway has 'awakened' some silent gene clusters in Streptomyces, activating potential metabolic pathways that have not been discovered in Streptomyces and providing important clues for the development of novel antibiotics and other drugs," said Luo Xiaozhou.
Furthermore, the study found that the introduction of the PQQ biosynthetic pathway enhanced the synthesis efficiency of various natural products through in-depth proteomic and metabolomic analysis.
"In the future, we will use major synthetic biology research infrastructure to automate the analysis of the 597 common genes found in Streptomyces. As more gene functions are characterized, we will further explore the mechanisms of gene enhancement and activation of natural products and their connections with antibiotics," Luo Xiaozhou explained. The team will also continue to advance the development and modification of strains, exploring the industrial applications of Streptomyces in producing antibiotics and natural products.
Related paper information: https://www.nature.com/articles/s42255-024-01024-9
Extracting useful information from vast genetic databases to facilitate the synthesis of various types of natural products. (Image provided by the research team)
