Fusion Enzymes Involved in Biosynthetic Tailoring Reactions in Fungi

Enzyme fusion, the fusion of enzymes with different domains to a single protein, has been widely recognized as a promising strategy in the development of biocatalysts. Nature has evolved gene fusion to combine different catalytic enzymes to function as a fusion enzyme, and this strategy is utilized in many natural product biosynthetic pathways. Owing to rapid advances in genome sequencing and biosynthetic pathway characterization, there is increasing interest in fusion enzymes from fungal biosynthetic pathways, particularly those involved in tailoring steps. This concept aims to provide an up-to-date overview of fusion enzymes that catalyze tailoring reactions in the biosynthesis of fungal secondary metabolites. Since fungal fusion enzymes are often associated with novel metabolites, this pioneering work may stimulate the exploration of the structural diversity of fungal natural products through genome mining of the untapped biosynthetic pathways involving fusion enzymes.     

Establishing a new methodology for genome mining and biosynthesis of polyketides and peptides through yeast molecular genetics

Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.     

Genomics-Guided Efficient Identification of 2,5-Diketopiperazine Derivatives from Actinobacteria

Secondary metabolites derived from microorganism constitute an important part of natural products. Mining of the microbial genomes revealed a large number of uncharacterized biosynthetic gene clusters, indicating their greater potential to synthetize specialized or secondary metabolites (SMs) than identified by classic fermentation and isolation approaches. Various bioinformatics tools have been developed to analyze and identify such gene clusters, thus accelerating significantly the mining process. Heterologous expression of an individual biosynthetic gene cluster has been proven as an efficient way to activate the genes and identify the encoded metabolites that cannot be detected under normal laboratory cultivation conditions. Herein, we describe a concept of genomics-guided approach by performing genome mining and heterologous expression to uncover novel CDPS-derived DKPs and functionally characterize novel tailoring enzymes embedded in the biosynthetic pathways. Recent works focused on the identification of the nucleobase-related and dimeric DKPs are also presented.     

Exploration and mining of the bacterial terpenome

Tens of thousands of terpenoids are present in both terrestrial and marine plants, as well as fungi. In the last 5-10 years, however, it has become evident that terpenes are also produced by numerous bacteria, especially soil-dwelling Gram-positive organisms such as Streptomyces and other Actinomycetes. Although some microbial terpenes, such as geosmin, the degraded sesquiterpene responsible for the smell of moist soil, the characteristic odor of the earth itself, have been known for over 100 years, few terpenoids have been identified by classical structure- or activity-guided screening of bacterial culture extracts. In fact, the majority of cyclic terpenes from bacterial species have only recently been uncovered by the newly developed techniques of "genome mining". In this new paradigm for biochemical discovery, bacterial genome sequences are first analyzed with powerful bioinformatic tools, such as the BLASTP program or Profile Hidden Markov models, to screen for and identify conserved protein sequences harboring a characteristic set of universally conserved functional domains typical of all terpene synthases. Of particular importance is the presence of variants of two universally conserved domains, the aspartate-rich DDXX(D/E) motif and the NSE/DTE triad, (N/D)DXX(S/T)XX(K/R)(D/E). Both domains have been implicated in the binding of the essential divalent cation, typically Mg(2+), that is required for cyclization of the universal acyclic terpene precursors, such as farnesyl and geranyl diphosphate. The low level of overall sequence similarity among terpene synthases, however, has so far precluded any simple correlation of protein sequence with the structure of the cyclized terpene product. The actual biochemical function of a cryptic bacterial (or indeed any) terpene synthase must therefore be determined by direct experiment. Two common approaches are (i) incubation of the expressed recombinant protein with acyclic allylic diphosphate substrates and identification of the resultant terpene hydrocarbon or alcohol and (ii) in vivo expression in engineered bacterial hosts that can support the production of terpene metabolites. One of the most attractive features of the coordinated application of genome mining and biochemical characterization is that the discovery of natural products is directly coupled to the simultaneous discovery and exploitation of the responsible biosynthetic genes and enzymes. Bacterial genome mining has proved highly rewarding scientifically, already uncovering more than a dozen newly identified cyclic terpenes (many of them unique to bacteria), as well as several novel cyclization mechanisms. Moreover, bioinformatic analysis has identified more than 120 presumptive genes for bacterial terpene synthases that are now ripe for exploration. In this Account, we review a particularly rich vein we have mined in the genomes of two model Actinomycetes, Streptomyces coelicolor and Streptomyces avermitilis, from which the entire set of terpenoid biosynthetic genes and pathways have now been elucidated. In addition, studies of terpenoid biosynthetic gene clusters have revealed a wealth of previously unknown oxidative enzymes, including cytochromes P450, non-heme iron-dependent dioxygenases, and flavin monooxygenases. We have shown that these enzymes catalyze a variety of unusual biochemical reactions, including two-step ketonization of methylene groups, desaturation-epoxidation of secondary methyl groups, and pathway-specific Baeyer-Villiger oxidations of cyclic ketones.     

Bioactivity‐Guided Genome Mining Reveals the Lomaiviticin Biosynthetic Gene Cluster in Salinispora tropica

The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico‐chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo‐ and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora tropica CNB‐440 by a DNA interference bioassay to isolate DNA‐targeting enediyne polyketides. An organic extract of S. tropica showed DNA‐interference activity that surprisingly was not abolished in genetic mutants of the targeted enediyne pathways, ST_pks1 and spo. Instead we showed that the product of the orphan type II polyketide synthase pathway, ST_pks2, is solely responsible for the DNA‐interfering activity of the parent strain. Subsequent comparative metabolic profiling revealed the lomaiviticins, glycosylated diazofluorene polyketides, as the ST_pks2 products. This study marks the first report of the 59 open reading frame lomaiviticin gene cluster (lom) and supports the biochemical logic of their dimeric construction through a pathway related to the kinamycin monomer.     

A genomic screening approach to the structure-guided identification of drug candidates from natural sources

The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence-guided genetic screening strategy. In this "genome mining" approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties.     

Biosynthetic Pathways of Dimeric Natural Products Containing Bisanthraquinone and Related Xanthones

Many dimeric natural products containing bisanthraquinone and related xanthones with diverse structures and versatile bioactivities have been isolated over the years. However, the complicated biosynthetic pathways of such natural products, which have remained elusive until recently, negatively impact their mass bioproduction and biosynthetic structural modification for drug discovery. In this concept, we summarize the recent progress in gene cluster mining and biosynthetic pathway elucidation of natural products containing bisanthraquinone and related xanthones. These pioneering works may pave the way for further biosynthetic pathway elucidation and structure modification of dimeric natural products through gene and protein engineering.     

Discovery of the Tiancilactone Antibiotics by Genome Mining of Atypical Bacterial Type II Diterpene Synthases

Although genome mining has advanced the identification, discovery, and study of microbial natural products, the discovery of bacterial diterpenoids continues to lag behind. Herein, we report the identification of 66 putative producers of novel bacterial diterpenoids, and the discovery of the tiancilactone (TNL) family of antibiotics, by genome mining of type II diterpene synthases that do not possess the canonical DXDD motif. The TNLs, which are broad‐spectrum antibiotics with moderate activities, are produced by both Streptomyces sp. CB03234 and Streptomyces sp. CB03238 and feature a highly functionalized diterpenoid skeleton that is further decorated with chloroanthranilate and γ‐butyrolactone moieties. Genetic manipulation of the tnl gene cluster resulted in TNL congeners, which provided insights into their biosynthesis and structure–activity relationships. This work highlights the biosynthetic potential that bacteria possess to produce diterpenoids and should inspire continued efforts to discover terpenoid natural products from bacteria.     

Natural Voltage-Gated Sodium Channel Ligands: Biosynthesis and Biology

Natural product biosynthetic pathways are composed of enzymes that use powerful chemistry to assemble complex molecules. Small molecule neurotoxins are examples of natural products with intricate scaffolds which often have high affinities for their biological targets. The focus of this Minireview is small molecule neurotoxins targeting voltage-gated sodium channels (VGSCs) and the state of knowledge on their associated biosynthetic pathways. There are three small molecule neurotoxin receptor sites on VGSCs associated with three different classes of molecules: guanidinium toxins, alkaloid toxins, and ladder polyethers. Each of these types of toxins have unique structural features which are assembled by biosynthetic enzymes and the extent of information known about these enzymes varies among each class. The biosynthetic enzymes involved in the formation of these toxins have the potential to become useful tools in the efficient synthesis of VGSC probes.     

Gene Discovery in Gelsemium Highlights Conserved Gene Clusters in Monoterpene Indole Alkaloid Biosynthesis

Genome mining is a routine technique in microbes for discovering biosynthetic pathways. In plants, however, genomic information is not commonly used to identify novel biosynthesis genes. Here, we present the genome of the medicinal plant and oxindole monoterpene indole alkaloid (MIA) producer Gelsemium sempervirens (Gelsemiaceae). A gene cluster from Catharanthus roseus, which is utilized at least six enzymatic steps downstream from the last common intermediate shared between the two plant alkaloid types, is found in G. sempervirens, although the corresponding enzymes act on entirely different substrates. This study provides insights into the common genomic context of MIA pathways and is an important milestone in the further elucidation of the Gelsemium oxindole alkaloid pathway.     

The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus**

The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4–10), temperature (up to 100 °C) and cosolvent space (up to 80 % (v/v) nonaqueous medium), and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 10⁶ for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.     

Characterization of a Dehydratase and Methyltransferase in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides in Lachnospiraceae

As a result of the exponential increase in genomic data, discovery of novel ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) has progressed rapidly in the past decade. The lanthipeptides are a major subset of RiPPs. Through genome mining we identified a novel lanthipeptide biosynthetic gene cluster (lah) from Lachnospiraceae bacterium C6A11, an anaerobic bacterium that is a member of the human microbiota and which is implicated in the development of host disease states such as type 2 diabetes and resistance to Clostridium difficile colonization. The lah cluster encodes at least seven putative precursor peptides and multiple post-translational modification (PTM) enzymes. Two unusual class II lanthipeptide synthetases LahM1/M2 and a substrate-tolerant S-adenosyl-l -methionine (SAM)-dependent methyltransferase LahS_B are biochemically characterized in this study. We also present the crystal structure of LahS_B in complex with product S-adenosylhomocysteine. This study sets the stage for further exploration of the final products of the lah pathway as well as their potential physiological functions in human/animal gut microbiota.     

微生物合成植物天然产物的细胞工厂设计与构建

植物天然产物结构多样,具有丰富的生理活性与功能。利用微生物细胞工厂生产来源稀缺、获取难度大的植物天然产物具有经济可行、环境友好等优势。本文系统介绍了萜烯、黄酮以及生物碱的生物合成途径及其关键酶,阐述了差异转录组学、同功酶挖掘等途径解析与重构的方法。指出关键酶改造、途径动态调控、代谢区室化与代谢网络再平衡是增大外源途径代谢通量、抑制副产物合成、降低产物毒性与菌株代谢负担、提高目标产物合成能力的有效策略。提出了解析合成植物天然产物关键酶在微生物中的催化特异性机制、开发外源途径的高效组装方法等进一步提高微生物细胞工厂生产效率的建议。     

Probing the compatibility of type II ketosynthase-carrier protein partners

Drug discovery often begins with the screening of large compound libraries to identify lead compounds. Recently, the enzymes that are involved in the biosynthesis of natural products have been investigated for their potential to generate new, diverse compound libraries. There have been several approaches toward this end, including altering the substrate specificities of the enzymes involved in natural product biosynthesis and engineering functional communication between enzymes from different biosynthetic pathways. While there exist assays to assess the substrate specificity of enzymes involved in these pathways, there is no simple method for determining whether enzymes from different synthases will function cooperatively to generate the desired product(s). Herein we report a method that provides insight into both substrate specificity and compatibility of protein-protein interactions between the acyl carrier protein (ACP) and ketosynthase (KS) domains involved in fatty acid and polyketide biosynthesis. Our technique uses a one-pot chemoenzymatic method to generate post-translationally modified ACPs that are capable of covalently interacting with KS domains from different biosynthetic systems. The extent of interaction between ACPs and KSs from different systems is easily detected and quantified by a gel-based method. Our results are consistent with previous studies of substrate specificity and ACP-KS binding interactions and provide new insight into unnatural substrate and protein interactions.     

Long Non-Coding RNA Epigenetics

Long noncoding RNAs exceeding a length of 200 nucleotides play an important role in ensuring cell functions and proper organism development by interacting with cellular compounds such as miRNA, mRNA, DNA and proteins. However, there is an additional level of lncRNA regulation, called lncRNA epigenetics, in gene expression control. In this review, we describe the most common modified nucleosides found in lncRNA, 6-methyladenosine, 5-methylcytidine, pseudouridine and inosine. The biosynthetic pathways of these nucleosides modified by the writer, eraser and reader enzymes are important to understanding these processes. The characteristics of the individual methylases, pseudouridine synthases and adenine–inosine editing enzymes and the methods of lncRNA epigenetics for the detection of modified nucleosides, as well as the advantages and disadvantages of these methods, are discussed in detail. The final sections are devoted to the role of modifications in the most abundant lncRNAs and their functions in pathogenic processes.     

The l-Alanosine Gene Cluster Encodes a Pathway for Diazeniumdiolate Biosynthesis

N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l -alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid l -2,3-diaminopropionic acid (l -Dap) is synthesized and loaded onto a free-standing peptidyl carrier protein (PCP) domain in l -alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.     

Accessing the hidden majority of marine natural products through metagenomics

Tiny marine animals represent an untapped reservoir for undiscovered, bioactive natural products. However, their small size and extreme chemical variability preclude traditional chemical approaches to discovering new bioactive compounds. Here, we use a metagenomic method to directly discover and rapidly access cyanobactin class natural products from these variable samples, and provide proof-of-concept for genome-based discovery and supply of marine natural products. We also address practical optimization of complex, multistep ribosomal peptide pathways in heterologous hosts, which is still very challenging. The resulting methods and concepts will be applicable to ribosomal peptide and other biosynthetic pathways.     

Type II Polyketide Synthases: A Bioinformatics-Driven Approach

Bioinformatics has become an indispensable tool for natural products research in the genomic era. One of the key challenges is to accurately convert sequence data of a biosynthetic gene cluster into chemical information such as the enzymatic function or the biosynthetic product structure. Type II polyketide synthase is the most bioinformatically well-studied class of non-modular biosynthetic machinery and represents a model system to showcase bioinformatic applications in natural products research. This review takes a bioinformatics-centered perspective and summarizes the past advances and future opportunities of bioinformatics-guided research on type II polyketide synthases. How bioinformatics has contributed to deepen the chemical understanding of type II PKSs will be discussed with the focus on enzymology, evolution, structural prediction of the biosynthetic products, genome mining, and the global analyses of their polyketide products.