Microorganisms can biosynthesize a vast array of active natural products, which have been a major source for drug development for humans and are closely related to human health and social safety. They also play a crucial role in agriculture, animal husbandry, food industry, and other fields. The biosynthetic process of microbial natural products is intricate and greatly influenced by both internal and external factors. Especially under laboratory conditions, a large number of biosynthetic genes for natural products are in a "silent" state, and their products are referred to as the "dark matter of life" in microbes (Nature 2015; Nature Reviews Drug Discovery 2023). How to effectively activate and mine these microbial "dark matter of life" has always been a bottleneck restricting the discovery of new natural products and a global challenge in this field. Currently, research in this area mainly focuses on regulating the genetic material, living environment, or metabolites of individual strains to activate and discover microbial "dark matter of life" under laboratory conditions. However, the complexity of the synthesis and regulation process of natural products, differences in physiology and metabolism among different strains, and long cycles of strain cultivation and genetic manipulation greatly limit the role of these methods in advancing the field. With the popularization of gene sequencing technology, a wealth of genomic data and increasingly mature genomic analysis techniques are expected to bypass cumbersome modification procedures, break through the individual differences of strains, and provide ideas for studying the universal laws of natural product synthesis and developing modification tools.
On April 12th, Beijing time, the collaborative team led by Luo Xiaozhou/Jay D. Keasling/Tang Xiaoyu published a research paper titled "Elucidation of Genes Enhancing Natural Product Biosynthesis Through Co-evolution Analysis" in the journal Nature Metabolism, along with research notes titled "Co-evolved Genes Improve the Biosynthesis of Secondary Metabolites." Through co-evolution analysis, the research team identified 597 genes that co-evolved with polyketide synthases in the genus Streptomyces. These genes belong to different functional families such as transcription, transport, coenzymes, fatty acid synthesis, metal ion transport, etc. Taking the pyrroloquinoline quinone (PQQ) gene from the "coenzyme" family as an example, its application in multiple actinomycetes revealed that introducing the PQQ biosynthesis pathway significantly increased the yield of natural products in Streptomyces and produced various new compounds with potential pharmaceutical prospects. This is of great significance for developing new antibiotics and improving the yield of natural products.
Screenshot of the article published online
Link of the article: https://www.nature.com/articles/s42255-024-01024-9
Streptomyces are among the microorganisms known to have the richest clusters of natural product biosynthetic genes, widely distributed in soils, water sources, and on plant surfaces. Due to nutrient scarcity and lack of motility in their natural environments, Streptomyces have evolved with a large number of secondary metabolism genes organized in the form of gene clusters, producing various bioactive substances to resist external threats and suppress competitors. Previous studies have found that Streptomyces encode significantly more secondary metabolism gene clusters compared to other species, with considerable variation in the capacity for encoding these clusters even among different genera and species within this group. Therefore, Streptomyces strains possessing more secondary metabolism gene clusters may have also evolved with a variety of genes beneficial for the synthesis of various secondary metabolites during their long-term evolution to maintain the production of multiple secondary metabolites. By identifying these strains with abundant secondary metabolism gene clusters (Figure 1) and uncovering these co-evolved genes, it will expand our toolkit for exploring the "dark matter of life" in natural metabolism and develop new methods for modifying Streptomyces to enhance the synthesis of secondary metabolites and activate silent gene clusters.
Figure 1. Phyletic evolution and secondary metabolism gene cluster analysis of the Streptomyces genus. Streptomyces are divided into 14 families, with Family 12 encoding significantly more PKS (polyketide synthase) gene clusters than other families
Based on this observation, the team utilized pan-genome analysis techniques to identify 597 genes that co-evolved with polyketide gene clusters (Figure 2), focusing their research on the gene cluster involved in the synthesis of the coenzyme pyrroloquinoline quinone (PQQ), suggesting a key role of PQQ in the biosynthesis of natural products in Streptomyces.
The research team introduced the PQQ biosynthesis pathway into 11 Streptomyces strains and two other industrial actinomycete strains, finding that this modification significantly increased the natural product yield of Streptomyces, enhancing the production of at least 16,385 metabolites, including 36 known natural products (Figure 3). These natural products have a wide range of applications, including as antibacterials, antifungals, and anticancer agents.
Figure 3. Introduction of the PQQ gene cluster promotes the synthesis of various known natural products
In addition to enhancing the yield of natural products, the research team also observed that the transcriptional activity of some silent natural product biosynthetic gene clusters was upregulated after introducing the PQQ gene cluster. This means that PQQ can not only directly affect the biosynthesis of known natural products, but may also activate previously undiscovered metabolic pathways in Streptomyces, enriching the variety and diversity of natural products. Further experiments confirmed this idea, as new metabolites were discovered in Streptomyces strains with the introduced PQQ gene cluster, some of which have potential antibiotic activity, even against clinical infection strains (Figure 4). This provides important clues and candidate compounds for the discovery of novel antibiotics and future drug development.
In addition, through in-depth proteomic and metabolomic analyses, the study revealed the mechanism by which PQQ enhances the efficiency of natural product synthesis. It was found that the introduction of the PQQ gene cluster can increase ATP levels and improve the ratio of key cofactors, while also enhancing the degradation of triglycerides to produce the key precursor acetyl-CoA, thereby increasing the synthesis of various natural products.
Currently, there are mainly two engineering strategies used in Streptomyces to enhance the production of natural products: one is to enhance the biosynthetic pathway of precursors or delete competitive pathways through metabolic engineering; the other is to modify regulatory factors to enhance the expression level of synthetic pathway genes. However, given the wide diversity of natural product producers and biosynthetic pathways, there are still few universal strategies available to date to increase the yield of different natural products. This study utilized pan-genome analysis technology to systematically analyze the genomes of the entire Streptomyces genus, searching for a universal modification method in a bottom-up manner, bypassing the time and labor costs associated with traditional mechanism-based research. This provides new insights into developing modification methods for different types of natural products in various plant and microbial species, accelerating the pace of understanding the "dark matter" in the natural product synthesis process.
The corresponding authors of this article are Luo Xiaozhou, a researcher with the Institute of Synthetic Biology of the SIAT, Chinese Academy of Sciences, Jay D. Keasling, a professor with the University of California, Berkeley, and Tang Xiaoyu, a researcher with the Shenzhen Bay Laboratory’s Institute of Chemical Biology. Wang Xinran, assistant researcher, Chen Ningxin, research assistant, and Pablo Cruz-Morales, a researcher from the Technical University of Denmark, are co-first authors of the article. Professor Bai Linquan from Shanghai Jiao Tong University and Professor Sun Yuhui from Huazhong University of Science and Technology also contributed to the research. This project was supported by the National Key R&D Program, the National Natural Science Foundation of China, the Shenzhen Science and Technology Plan Project, the Shenzhen Medical Research Project, and platforms such as the Shenzhen Institute of Synthetic Biology. Additionally, thanks go to Wei Zhenqin, a research assistant with the Institute of Synthetic Biology of the SIAT, for assisting in organizing meetings and discussions during the project implementation.
Expert Review:
Academician Deng Zixin of Chinese Academy of Sciences:
The co-evolution strategy has revealed key genes that enhance the biosynthesis of natural products. This achievement reflects the use of synthetic biology as a driver and integration disciplines such as bioinformatics, microbial genetics, genomics, and metabolomics to provide a new successful example of interdisciplinary collaboration in the field of synthetic biology.
The biosynthetic process of microbial natural products is exceptionally complex and delicate, influenced by both internal and environmental factors. Under laboratory conditions, many microbial natural product biosynthetic genes are often silent, making these "dark matters" a new source of natural products awaiting further exploration. Supported by the National Key R&D Program for Synthetic Biology, the team identified 597 genes co-evolved with polyketide synthases in the genus Streptomyces through co-evolution analysis. They discovered that by introducing the PQQ biosynthetic pathway, it could significantly increase the natural product yield of Streptomyces and produce various new compounds with drug potential, providing important clues and material basis for new drug discovery.
Notably, this study employed pan-genome analysis technology, bypassing cumbersome modification procedures and breaking through individual strain differences, offering new ideas for studying the universal laws of natural product biosynthesis. Academician Deng Zixin, an advisory expert in the field of synthetic biology under the National Key R&D Program, pointed out: "This achievement not only sparks our common interest in restarting the 'dark matter' biosynthesis of microbial natural products, but also provides new strategies for the application of synthetic biology in drug development and other fields. I look forward to seeing more similar research results in the future, making greater contributions to human health and social development."
Ge Huiming, Vice Dean of the College of Life Sciences, Nanjing University:
Natural products are a major source of small molecule drugs. Although genome sequencing and bioinformatics analysis have suggested the presence of numerous "silent" biosynthetic gene clusters, how to activate these "dark matters of life" under laboratory conditions has been a significant challenge in the field for a long time. In this study, the research team from the SIAT led by Luo Xiaozhou, Jay Keasling, and Tang Xiaoyu from Shenzhen Bay Laboratory used pan-genome analysis methods to search for genes co-evolved with natural product gene clusters. Through experiments, they revealed that the pyrroloquinoline quinone cofactor (PQQ) is closely related to the expression of polyketide gene clusters and further clarified that overexpressing the PQQ gene cluster activates the synthesis of a large number of new natural product molecules universally. The unique perspective and innovative viewpoint of this study not only provide a new approach for mining new natural products, but also help increase the yield of existing important natural products.
It is worth noting that this study not only focuses on the synthesis of natural products, but also emphasizes the interaction and co-evolution of genes on the biosynthetic process. This comprehensive research method helps us fully understand the complexity of natural product biosynthesis, offering new ideas and strategies for designing more efficient biosynthetic pathways in the future. Overall, this study has advanced the development of natural product mining and synthetic biology of natural products, providing a brand-new perspective and method for us to deeply understand the biosynthetic process of natural products.
