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31 October 2021, Volume 2 Issue 5

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2021, 2(5):  0. 

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Engineered yeast facilitates rapid and systematic mining of fungal chimeric terpene synthases

Zhen FAN, Haixue PAN, Gongli TANG

2021, 2(5):  666-673.  doi:10.12211/2096-8280.2021-084

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Chimeric terpene synthases (PTTSs), which consist of C-terminal prenyltransferase (PT) and N-terminal ClassⅠterpene synthase (TS) domains, are unique to fungi for catalyzing the synthesis of structurally diverse diterpenes and sesterterpenes. Since the first PTTS was discovered in 2007, only about 20 PTTSs have been functionally verified, and understanding of the origin and functional evolution of PTTS genes is very limited. Recently, Professor Tiangang Liu's research team at Wuhan University and Professor Feng Chen's research team at the University of Tennessee in the United States have conducted in-depth exploration on the origin and functional evolution of PTTS with an efficient terpene precursor yeast chassis system combined with the high-throughput automatic screening platform. The research not only expands the chimeric terpene synthase family, but also provides an advanced method for mining more terpene synthases for the biosynthesis of terpenoids.

Invited Review



From chemical synthesis to biosynthesis: trends toward total synthesis of natural products

Faguang ZHANG, Ge QU, Zhoutong SUN, Jun′an MA

2021, 2(5):  674-696.  doi:10.12211/2096-8280.2021-039

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The complexity and diversity of natural products have made them a rich source for drug and agrochemical discovery. To overcome the supplying limitation of natural resources, tremendous effort has been made by the academic and industrial communities during the past two centuries for the total artificial synthesis of natural products. In this regard, total chemical synthesis has achieved significant progress, and numerous highly complex natural products have been synthesized through different chemical processes. Despite these great achievements in total chemical synthesis, there are still many challenges including expensive chemical reagents, harsh reaction conditions, difficult control on stereoselectivity, long synthetic route, and low product yield. Notably, the development of synthetic biology has allowed more and more natural products to be produced through biological cell factories, which provides a new and complementary strategy for the synthesis of natural products at a large scale. This review critically comments on the representative advances in total chemical synthesis of natural products (Section 1), and then highlight major progress and trends in the biosynthesis of pharmaceutically important natural products (Sections 2 and 3). In Section 2.1, we selected the production of penicillin, erythromycin, and avermectin as examples to analyze the modification and optimization of natural product biosynthetic pathways. The discovery and utilization of secondary metabolites from microorganisms has been a continuous driving force in the field of natural products. Notably, significant progress has been made in the total biosynthesis of natural products from secondary metabolism via the genetic manipulation of microbial cells. In Section 2.2, we selected Vitamin B₁₂ and Tropane alkaloids as examples to demonstrate the use of heterologous expression and biological production for natural product synthesis. In recent years, on the basis of analyzing the structure of natural products in animals, plants, and microorganisms, great advances have emerged in exploring their biochemical reaction mechanisms and synthetic routes. More importantly, expressing and regulating the relative genes in heterologous microbial cells have enabled the complete biosynthesis of many natural products. Furthermore, in Section 3, human insulin, artemisinin, saframycin, azaphilone, kainic acid, and podophyllotoxin were selected as examples to showcase the power of merging chemical and biological processes for the total synthesis of natural products. Although there are still many challenges in the total synthesis of new and complex natural products, biosynthesis will ultimately play a significant role in the construction of natural molecules and their relative analogues. By taking advantage of the merits with organic chemistry, synthetic biology, and artificial intelligence, the development of highly efficient and automatic biosynthesis could be a trend in this field.





Applications and prospects of genome mining in the discovery of natural products

Qian YANG, Botao CHENG, Zhijun TANG, Wen LIU

2021, 2(5):  697-715.  doi:10.12211/2096-8280.2021-012

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Natural products have been an abundant source of leader compounds for new drugs, but traditional isolation and analysis technologies to obtain novel natural products cannot satisfy the requirement for drug discovery. Genomic data have been utilized for identifying potential drug targets, or exploring biosynthesis pathways for natural products that were neglected before. Genome sequencing has unveiled a plethora of undeveloped chemical diversity in microorganisms and plants. From genome sequences, a large amount of information is available, from functional enzymes to conserved patterns/signatures, even potential structures and features that can be interpreted to hunt for new biocatalysts. With the advent of the genomic era, the computational mining of genomes has become an important part in the discovery of novel natural products as drug leads. Meanwhile, the development of high-throughput sequencing and the establishment of DNA database, genome mining methods and tools have contributed to the discovery and characterization of these natural products. In spite of the diversity of natural products, the biosynthetic rules and thus the biosynthetic machineries for many of these compounds are often remarkably conserved, which is highlighted in the high amino acid sequence similarity of the core biosynthetic enzymes, such as polyketides synthases (PKS), non-ribosomally peptides synthetases (NRPS), and many others. Besides, most of natural products are considered to be produced by the host to kill or limit the growth of competitors through the inhibition or inactivation of essential housekeeping enzymes. Therefore, accumulating knowledge on the self-resistance mechanisms, for instance, mining for SRE (self-resistance enzyme), have promoted research on natural products. Moreover, a phylogeny-guided mining approach provides a method to quickly screen a large number of microbial genomes or metagenomes to detect new biosynthetic gene clusters of interest, and many web tools and databases have been developed and utilized by researchers to mine for key enzymes. This paper reviews recent advances in the genome mining tools, databases and approaches, with a focus on the ways of mining biosynthetic gene clusters (BGCs) of natural products, from classical genome mining to resistance-based and phylogeny-guided mining, and also include a short overview on status and perspective in the discovery of novel natural products.





Advances and challenges in microbial production of benzylisoquinoline alkaloids

Zhi LIN, Zhiwei HU, Xudong QU, Shuangjun LIN

2021, 2(5):  716-733.  doi:10.12211/2096-8280.2021-058

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Plant secondary metabolites are an important source of drug discovery and development, but their low accumulation in plants and the long growth duration of the plants make their production costly. In recent years, an alternative route, microbial synthesis, has been developed for producing plant-derived secondary metabolites, which is cost-effective and sustainable compared to routes through plant cultivation and chemical synthesis. Benzylisoquinoline alkaloids (BIAs), representatives of plant-derived alkaloids, are a family of ～2500 alkaloids, which have become attractive for microbial synthesis owing to their pharmaceutical functions and potentials in this regard. Recent advances in the elucidation of biosynthetic pathways of BIAs, together with the discovery of a variety of enzymatic tools, have facilitated the assembly of the synthetic pathways of BIAs in microbial hostssuch as Escherichia coli and Saccharomyces cerevisiae. However, most production of BIAs remains at a laboratory scale, due to the long metabolic pathway and the broad substrate spectrum of those enzymes for their microbial synthesis with various side-products generated. To date, only the production of (S)-reticuline, a major precursor of BIAs, is closed to scalable production with a titer of 4.6 g/L, which was reported by Martin’s team in 2020. This review comments the development and current status in the microbial production of BIAs and highlights critical aspects on overcoming the bottlenecks. The broad substrate spectrum of key enzymes including methyltransferases, norcoclaurine synthase and codeinone reductase for the microbial synthesis of BIAs has been demonstrated in vitro, and thus this review also summarizes the possible effect of the catalytic properties of these enzymes on the metabolic flux, indicating importance of the selection and improvement of enzyme catalytic elements. At the end, the challenges for the microbial synthesis of BIAs are highlighted, and the importance of enzyme engineering and the design of new artificial microbial synthesis pathway to address these challenges are expected for more efficient production of BIAs through microbial synthesis.





Biosynthesis of alkyne moiety in natural products and application of alkyne biosynthetic machineries

Jianming LYU, Huan ZHAO, Dan HU, Hao GAO

2021, 2(5):  734-750.  doi:10.12211/2096-8280.2021-056

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Alkyne is a biologically significant moiety in many drugs and natural products, which is also a versatile building block in modern chemistry. Therefore, it is of great importance to efficiently synthesize alkyne-containing products in the fields of medicinal chemistry, organic chemistry, chemical biology and so on. Generally, alkyne-containing products are obtained via chemical synthesis, but this strategy often suffers from high cost, low efficiency and harsh reaction conditions. Alternatively, microbial biotransformation can be performed through feeding alkyne-containing precursors, but it is still challenging since these precursors are not easily accessible. Inspired by the advancement of synthetic biology, de novo biosynthesis is expected to be a promising approach for producing acetylenic products, which is environmentally friendly and industrially tractable. Great efforts have thus been devoted to elucidating the biosynthetic machinery of alkyne moiety in natural products so as to provide efficient enzymatic tools for the de novo biosynthesis of acetylenic products. In this review, we comment recent progress in biosynthesis of alkynes in different natural products. In unsaturated fatty acids, a special family of desaturases serve as acetylenases, converting olefinic bonds to triple bonds via O₂-dependent dehydrogenation with the use of a diiron active site. In polyketides, although lots of work has been done in revealing the biosynthetic routes of enediyne antibiotics, the genetic basis for synthesizing acetylenic bonds in their core backbones remains enigmatic. In polyketide-non ribosomal peptide hybrid molecules, the three-gene cassette encoding the ligase, acyl carrier protein (ACP) and acetylenase is responsible for the formation of the terminal alkyne-labeled fatty acyl-ACP, which is then used as the starting unit to be incorporated into the assembly line. In amino acids, the halogenase catalyzes the side-chain halogenation followed by the oxidase-mediated side-chain cleavage, and then the lyase catalyzes the elimination reaction to convert resulted alkene to the terminal triple bond. In meroterpenoids, the cytochrome P450 oxidase can consecutively catalyze two rounds of dehydrogenation to provide the internal alkyne in the prenyl chain. Moreover, we also introduce de novo biosynthesis of the terminal-alkyne tagged polyketides and proteins on the basis of the characterized biosynthetic machineries of alkynes. Despite the great progress in alkyne biosynthesis, it needs to be further strengthened in exploring the types of alkyne synthases as well as expanding their substrate specificity, so as to provide more enzymatic tools for the de novo biosynthesis of alkyne-containing products.





Biosynthesis and application of pyrethrins: a natural pesticide from plants

Fengjiao WANG, Haiyang XU, Jianbin YAN, Wei LI

2021, 2(5):  751-763.  doi:10.12211/2096-8280.2021-068

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How to produce safe pesticides in chassis through energy-saving and environmentally friendly methods is one of challenges for synthetic biology. As a natural botanical insecticide, Pyrethrins derived from Pyrethrum (Tanacetum cinerariifolium) have good insecticidal and deworming activities with broad spectrum. Compared with chemical synthetic counterparts (pyrethroids), pyrethrins are less toxic and harmful to environment and human. These characteristics make pyrethrins not only one of the most ideal biological pesticides so far with broad applications and great potentials, but also candidates for their production via synthetic biology in the future. In this article, we review the discovery and chemical and biological characteristics of pyrethrins as well as the elucidation of their biosynthetic pathway by summarizing the catalytic enzymes identified recently including cytochrome P450s, dehydrogenases, methyltransferase and phosphatase etc. The synthesis process is mainly involved three parts: the synthesis of acid moieties, the synthesis of alcohol moieties and the condensation of acids and alcohols to esters. At present, the synthesis of acid moieties has been largely deciphered, and genes encoding key enzymes for acid-alcohol condensation have also been determined, but the synthesis of alcohol moieties needs to be explored further. With the identification of a large number of gene elements and the function of related genes in the plants, the rise of synthetic biology and the progress of synthetic technology, it is possible to express heterologous genes in chassis, integrate the pathway of heterologous synthesis to produce targeted products. This also makes it possible to express the metabolic pathway for the production of pyrethrins and their derivatives in suitable microbial chassis. Therefore, this review also focuses on challenges that need to be addressed in this regard, including regulation, transportation and chassis adaptation for more efficient production, and forecasts future development. We hope that more research progress could provide a solid scientific basis and application guidance for the biosynthesis of the green and effective pesticide, and finally realize the large-scale production of pyrethrins.





Recombinant expression of milk proteins and biosynthesis of animal-free milk: analysis on related patents and trend for technology development

Zhengfu ZHOU, Yu PANG, Wei ZHANG, Jin WANG, Yongliang YAN, Yingying ZHENG, Min CHEN, Zhihua LIAO, Min LIN

2021, 2(5):  764-777.  doi:10.12211/2096-8280.2021-057

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Milk proteins are the major component in animal milk, which have various biological activities such as easy uptake and digestion, high nutrition, immunity enhancement and antioxidation. The efficient expression of various milk protein components derived from natural milk by genetic engineering and cell factories has become a hot spot and also a technical breakthrough in the field of animal-free milk biosynthesis. Animal-free milk, as a commercially-available high-tech food that is changing our world, has the same nutritional function as natural milk, but does not contain lactose, cholesterol, allergens, contaminated antibiotics and other undesirable components. The animal-free milk production process does not need to breed animals, which can effectively save resources and energy. It is a new model for future dairy production that could reform the traditional farming industry and lead the development of the food industry and cellular agriculture. In this review, we systematically summarize the current status of intellectual property protection of core technologies related to the metabolic reconstruction of model microbial chassis, efficient expression of recombinant milk proteins, and industrialization of animal-free milk products; discuss hot issues such as bottleneck technologies and biosafety challenges in the R&D of animal-free milk products; present the latest development in the combinatorial biosynthesis of milk proteins, man-made flavoring substance additives, sequence modifications for allergen and smart designs for cell factories. At present, the R&D of recombinant milk protein expression and animal-free milk biosynthesis technology in China is still lagging behind, and funds for R&D and venture capital investment for such an emerging business are insufficient. In order to cope with the intensive international competition, we should increase investment in R&D during the 14th Five-Year Plan Period, and strive to break through key technologies and product production processes. On the other hand, the formulation of biosynthetic food commercialization regulations and industrial policies needs to be facilitated. These efforts will provide irreplaceable scientific and technological support for promoting the development of future farming industry and achieving the agricultural "carbon peaking and neutralization" goal in China.





Construction of a light-controlled expression system and its application in Yarrowia lipolytica

Ping ZHANG, Wenping WEI, Ying ZHOU, Bangce YE

2021, 2(5):  778-791.  doi:10.12211/2096-8280.2021-018

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Light has the advantages of fast response, non-destructive, reversible, and unique temporal and spatial specificity as an inducing factor, which make it widely used in the metabolic engineering of biological chassis cells. In this study, a light-controlled expression system for recombinant Yarrowia lipolytica was developed, which successfully improved the synthesis efficiency and yield of p-coumaric acid and naringenin. Based on the green light response regulator CarH and the transcription activator VPR-HSF1, the light response complex CarH-VPRH, the core component of the light control system, was constructed, which cannot polymerize under green light irradiation conditions, and thus cannot regulate the transcription and expression of target genes. The results showed that the light-induced sensor based on CarH-VPRH, 2CarOTEF and mCherry could respond to green light significantly to inhibit the expression of target genes, while mCherry transcription was activated under dark conditions. The fluorescent signals of mCherry at 72 h and 120 h were 43 times and 143 times of that detected under green light irradiation conditions. Furthermore, the sensor was applied to induce the synthesis of p-coumaric acid and naringenin in Y. lipolytica successfully, and the yields of p-coumarin acid and naringenin under dark culture conditions were 2.0 times and 2.6 times of that produced under the induction of green light, reaching 99.1 mg/L, and 117.1 mg/L, respectively. In terms of cell growth, the growth of the green light irradiation group was better than that of the dark group, and especially when the dark condition was switched on after 24 h of green light irradiation, the cell growth was substantially improved, indicating the potential of the green light-induced system to regulate product synthesis and cell growth balance in Y. lipolytica. Therefore, it can be concluded that the green light-responsive gene regulation system can be applied to the transcription regulation of target genes in Y. lipolytica as an effective regulatory element with the advantages of low cost, nontoxicity, flexible insertion and removal. Therefore, it has a potential for the large-scale synthesis and production of target products.





Production of sesquiterpenoids α-neoclovene and β-caryophyllene by engineered Saccharomyces cerevisiae

Xiaodong LI, Chengshuai YANG, Pingping WANG, Xing YAN, Zhihua ZHOU

2021, 2(5):  792-803.  doi:10.12211/2096-8280.2021-014

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Sesquiterpenoids α-neoclovene and β-caryophyllene are major components in volatile oils from Panax ginseng, which have been demonstrated to play important roles in antibacteria, antitumor and cardiovascular protection. Moreover, they have attracted attentions for potential use as biofuels with high-energy-density. However, the industrial production of α-neoclovene and β-caryophyllene as well as other sesquiterpenoids are mainly relied on extraction from plant materials, which is too costly for applications at a large scale. Currently, this challenge could be addressed by advances in synthetic biology for natural product biosynthesis. Through heterologously assembling and integrating of their biosynthetic pathways into microbial chassis cells, targeted natural compounds from plants could be produced by microbial fermentation in a sustainable, low-cost and large-scale way. In this study, by comparing the production potential of sesquiterpenes between different Saccharomyces cerevisiae strainsand followed by enhancing the endogenous mevalonate pathway, a yeast sesquiterpene chassis strain (SQTBY03) with an increase of 458 times in farnesyl pyrophosphate production was constructed. Then by inserting the codon-optimized sesquiterpene synthase gene ec38-cs from the endophytic fungi Hypoxylon sp. EC38 and the codon-optimized caryophyllene synthase gene QHS1 from Artemisia annua into SQTBY03, respectively, we built yeast cell factories NCVBY01 and CPLBY01 for de novo production of α-neoclovene and β-caryophyllene at their titers of 25.8 mg/L and 250.4 mg/L, respectively, in shake flasks. Furthermore, fed-batch fermentation using NCVBY01 and CPLBY01 resulted in the de novo production of 487.1 mg/L α-neoclovene and 2949.1 mg/L β-caryophyllene from glucose. It is also possible to further chemically catalyze β-caryophyllene to produce α-neoclovene. Our work provides strategies for the sustainable production of α-neoclovene and β-caryophyllene from glucose through microbial fermentation, which would benefit their applications as medicine and other functional products. In addition, our yeast chassis for the sesquiterpene production could offer a platform for the sustainable production of other valuable sesquiterpenoids via synthetic biology approach.





Regulation on oxidation selectivity for β-amyrin by Class Ⅱ cytochrome P450 enzymes

Wentao SUN, Xinzhe ZHANG, Shengtong WAN, Ruwen WANG, Chun LI

2021, 2(5):  804-814.  doi:10.12211/2096-8280.2021-081

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Cytochrome P450 enzymes are tailoring enzymes for a key role in the biosynthesis of many natural products, and their catalytic promiscuity enables diverse structures to catalyze different reactions involved in the same biosynthetic pathway, such as the oxidization of same intermediates to form undesired analogues. The traditional strategies for regulating catalytic selectivity for substrate by P450 enzymes mainly focus on engineering their catalytic domains and/or redox partners. However, roles of the transmembrane domain, metabolism of membrane components and the expression ratio of P450 enzymes catalyzing different reactions in the catalytic specificity of membrane-bound Class Ⅱ P450 enzymes have been overlooked. To explore regulation mechanism underlying the catalytic selectivity for substrate by Class Ⅱ P450 enzymes to eliminate the formation of by-product 11-deoxyglycyrrhetinic acid from the uncontrolled catalytic selectivity of CYP72A63 (T338S) during the biosynthesis of glycyrrhetinic acid, the influences of these factors were studied in vivo throughthe Saccharomyces cerevisiae expression and verification platform with three strategies: remodeling the transmembrane structure of CYP72A63 (T338S) by domain swapping with the transmembrane domain of the yeast endogenous P450 enzymes and other endoplasmic reticulum located proteins, redirecting the membrane metabolism by reconstructing the metabolic pathway of host membrane components, and regulating the expression ratio of P450 enzymes upstream and downstream the glycyrrhetinic acid synthetic pathway. Our experimental results indicate that the remodeling of the transmembrane domain and the regulation of membrane metabolism significantly changed the substrate selectivity of CYP72A63 (T338S), and the fusion of N-terminal transmembrane domain with NTE1 and the knockout of sphinganine C4-hydroxylase encoding gene SUR2 significantly inhibited the oxidation selectivity for β-amyrin by CYP72A63 (T338S), but the overexpression of glucosylceramide synthase from Pichia pastoris remarkably enhanced its oxidation selectivity for β-amyrin. Moreover, by up-regulating the expression of Uni25647, and thus enhancing its competition ability for β-amyrin against CYP72A63 (T338S), the production of 11-deoxyglycyrrhetinic acid was completely eliminated. This study provides new ideas and methods for the catalytic regulation of P450 enzymes, especially for the membrane-bound Class Ⅱ P450 enzymes.





Potential biosynthesis of nonribosomal peptides by hypocrealean entomopathogenic fungi

Liwen ZHANG, István MOLNÀR, Yuquan XU

2021, 2(5):  815-825.  doi:10.12211/2096-8280.2021-005

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Nonribosomal peptide natural products synthesized by Hypocrealean entomopathogenic fungi have antibacterial, insecticidal, anticancer, immunomodulatory and other biological activities, with high potential for application in clinical or agricultural fields. These bioactive compounds are synthesized by nonribosomal peptide synthetases (NRPSs) and tailored by additional enzymes that are encoded by clustered genes. In addition to the 20 amino acids, non-canonical amino acids as well as α-hydroxy acids can also be incorporated into nonribosomal peptides as structural units, and these, together with further modifications, empower an almost unlimited structural diversity. The rapid increase in the number of sequenced fungal genomes shows that there exists a large number of nonribosomal peptide biosynthetic gene clusters with unknown functions. Accurate and effective prediction of the functions of these "orphan" biosynthetic gene clusters can help to select those clusters that have potential to synthesize novel natural products, and increase the efficiency of natural product genome mining. In this study, we systematically analyzed the genes encoding NRPSs and their gene clusters in the genomes of 40 strains from Hypocrealean entomopathogenic fungi. Genes encoding NRPSs were predicted and categorized based on the hidden Markov models for adenylation, condensation and thiolation domains to reveal 445 modular NRPSs and 1243 NRPS-like proteins. A sequence similarity network based on the amino acid sequences of adenylation domains of these synthetases was constructed. Using adenylation domains from known NRPSs as references, we analyzed the main categories of predicted nonribosomal peptide products using the Markov clustering algorithm. We identified several biosynthetic gene clusters that potentially yield known bioactive compounds or their congeners. In addition, biosynthetic gene clusters that may be able to produce new bioactive compounds belonging to the linear peptide, cyclic peptide, lipopeptide, and alkaloid structural classes were also discovered. The results reveal a great potential of Hypocrealean entomopathogenic fungi to synthesize nonribosomal peptides, and provide insight for genome mining to identify new products by activating silent gene clusters or modifying biosynthetic pathways by synthetic biology methods.





Study on the post-translational modification of RiPPs Xye catalyzed by CyFE PacB

Yuanjun HAN, Tianlu MO, Zixin DENG, Qi ZHANG, Wei DING

2021, 2(5):  826-836.  doi:10.12211/2096-8280.2021-080

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Cyclic ribosomally synthesized and post-translationally modified peptides (RiPPs) with macrocycles derived from three amino acid residues have attracted intense interest in recent years. Biosynthesis of these peptides usually involves a radical (S)-adenosylmethionine (rSAM) enzyme that catalyzes the formation of a C—C or C—O bond between an aromatic carbon and a side chain carbon of another residue. Such enzymes are named three-residue cyclophane forming enzymes (3-CyFEs). The rSAM enzyme family is known as one of the largest enzyme super families, which consists of more than 22000 members. rSAM enzymes are widely found in all three life domains as one of the earliest biocatalysts on earth. A large amount of microbial genomic information shows that many RiPPs biosynthesis gene clusters contain rSAM enzymes. Recently, several 3-CyFEs from Xye gene clusters, which form three cyclizations on the precursor peptides, were reported. In this paper, we report a new Xye RiPP pacpeptide and its key biosynthetic enzyme PacB from Photorhabdus australis DSM 17609. We coexpressed the pacA and pacB in Escherichia coli and reconstituted the activity of heterologously expressed protein to investigate the bioactivity of 3-CyFEs PacB. The high-resolution mass spectrometry assay indicates that PacB indeed led to forming all three cyclophanes of pacpeptide. Additionally, the mutation of Asn/Argto Gly on the precursor peptide resulted in only two cyclophane was found, which demonstrated that three cyclization reactions were generated independently with different efficiency. PacB also catalyzed all three cyclophanes formation on the precursor peptide with Asn/Arg mutated to Ala. These results expand our understanding of 3-CyFEs chemistry in natural product biosynthesis and provide insights into the post-modification of cyclic RiPPs with three-residue cyclophanes.





Genome mining for novel natural products in Sorangium cellulosum So0157-2 by heterologous expression

Haibo ZHOU, Qiyao SHEN, Hanna CHEN, Zongjie WANG, Yuezhong LI, Youming ZHANG, Xiaoying BIAN

2021, 2(5):  837-849.  doi:10.12211/2096-8280.2021-024

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Myxobacteria are an important source for natural products. Sorangium cellulosum So0157-2 produces anticancer epothilone, and its genome of 14.78 Mb is the largest prokaryotic genome sequenced to date. Bioinformatic analysis indicated that the genome harbors 35 biosynthetic gene clusters (BGCs). In addition to the known epothilone BGC and another two terpene BGCs with 100% similarity to their predicted BGCs, there are 17 BGCs for polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and PKS-NRPS hybrids, which mean this strain has great potential to produce novel natural products. However, limits on slow growth, difficult culture, and the absence of efficient genetic manipulation tools for So0157-2 impede the deep mining of its metabolic potential. Therefore, transferring its BGCs to a simple heterologous host and using heterologous expression strategy to achieve genome mining would be an effective way for discovering novel natural products produced by this strain. In this work, a PKS-NRPS hybrid BGC (BGC18) was directly cloned into an Escherichia coli expression vector and transferred into the heterologous host Schlegelella brevitalea DSM 7029. Fractionation was conducted by using repeated column chromatography (HPLC-MS) over a RP-C₁₈ column packing with silica gel, and three pure products were obtained, which were identified by extensive NMR analysis and the Marfey's method as new compounds: (1) cyclo(N-Me-L-Leu-L-Val), (2) cyclo(N-Me-L-Leu-L-Leu), and (3) cyclo(N-Me-L-Leu-L-Ile). Analysis of BGC18 reveals that the lack of thiolation domain in the PKS module might lead to the skipping of this PKS module, and only three cyclodipeptides were synthesized by the two NRPS modules, which might be a strategy for structural diversity in the bacterial NRPS-PKS BGCs. A NRPS-PKS hybrid BGC in S. cellulosum So0157-2 was successfully cloned, and expressed by using direct cloning and heterologous expression strategy with three corresponding products isolated and identified. This study lays a solid foundation for subsequent discovery of more active natural products in S. cellulosum So0157-2, and also provides theoretical guidance for mining secondary metabolites from other microbes which are difficult for culture.