Total biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase α: heterologous expression of four biosynthetic genes in Aspergillus oryzae

Clustering of biosynthetic genes for producing fungal secondary metabolites, which frequently consist of less than ten genes, has been recognized with numerous genomes. The heterologous expression of whole genes in the clusters will therefore produce various types of natural products when using a suitable fungal host. We introduced the whole gene cluster for the biosynthesis of diterpene aphidicolin into the fungal quadruple auxotrophic host, Aspergillus oryzae, by using four different vectors (pTAex3, pPTRI, pUSA and pAdeA) which harbor a starch-inducible promoter/terminator to examine the expression conditions. The resulting quadruple transformant carrying the genes of geranylgeranyl diphosphate synthase PbGGS, terpene synthase PbACS, and two monooxygenases (PbP450-1 and PbP450-2) produced aphidicolin. The double and triple transformants also respectively produced aphidicolan-16β-ol and 3-deoxyaphidicolin. Alternative host Saccharomyces cerevisiae carrying the genes, PbGGS and PbACS, produced key intermediate aphidicolan-16β-ol. This is the first example of a total biosynthesis of terpenoids using fungal hosts.     

Subcellular localization of aphidicolin biosynthetic enzymes heterologously expressed in Aspergillus oryzae

The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.     

真菌二萜环化酶的研究进展

二萜类化合物广泛存在于植物和真菌中,是一类具有重要商业价值的天然产物。二萜环化酶作为催化牻牛儿牻牛儿焦磷酸(geranylgeranyl diphosphate,GGPP)形成二萜的关键生物合成酶,在不同生物中的特异性决定了二萜化合物的结构多样性和生物活性多样性。对不同物种中二萜环化酶基因的分离、克隆和表达特征的分析有利于二萜类化合物的生物合成及调控研究。相比植物,真菌二萜化合物和二萜环化酶的研究刚刚起步。本文综述了近几年真菌二萜环化酶的研究进展,重点叙述了真菌二萜化合物的生物合成途径、二萜环化酶的特征及其克隆策略,并对二萜环化酶的代谢工程作了简要概述。     

Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.     

Recent advances regarding diterpene cyclase genes in higher plants and fungi

Cyclic diterpenoids are commonly biosynthesized from geranylgeranyl diphosphate (GGDP) through the formation of carbon skeletons by specific cyclases and subsequent chemical modifications, such as oxidation, reduction, methylation, and glucosidation. A variety of diterpenoids are produced in higher plants and fungi. Rice produces four classes of diterpene phytoalexins, phytocassanes A to E, oryzalexins A to F, oryzalexin S, and momilactones A and B. The six diterpene cyclase genes involved in the biosynthesis of these phytoalexins were identified and characterized. Fusicoccin A was produced by the phytopathogenic Phomopsis amygdali and served as a plant H(+)-ATPase activator. A PaFS, encoding a fungal diterpene synthase responsible for fusicoccin biosynthesis, was isolated. The PaFS is an unusual chimeric diterpene synthase that possesses not only terpene cyclase activity (the formation of fusicoccadiene, a biosynthetic precursor of fusicoccin A), but also prenyltransferase activity (the formation of GGDP). Thus, we identified a unique multifunctional diterpene synthase family in fungi.     

Nonribosomal peptides in fungal cell factories: from genome mining to optimized heterologous production

Fungi are notoriously prolific producers of secondary metabolites including nonribosomal peptides (NRPs). The structural complexity of NRPs grants them interesting activities such as antibiotic, anti-cancer, and anti-inflammatory properties. The discovery of these compounds with attractive activities can be achieved by using two approaches: either by screening samples originating from various environments for their biological activities, or by identifying the related clusters in genomic sequences thanks to bioinformatics tools. This genome mining approach has grown tremendously due to recent advances in genome sequencing, which have provided an incredible amount of genomic data from hundreds of microbial species. Regarding fungal organisms, the genomic data have revealed the presence of an unexpected number of putative NRP-related gene clusters. This highlights fungi as a goldmine for the discovery of putative novel bioactive compounds. Recent development of NRP dedicated bioinformatics tools have increased the capacity to identify these gene clusters and to deduce NRPs structures, speeding-up the screening process for novel metabolites discovery. Unfortunately, the newly identified compound is frequently not or poorly produced by native producers due to a lack of expression of the related genes cluster. A frequently employed strategy to increase production rates consists in transferring the related biosynthetic pathway in heterologous hosts. This review aims to provide a comprehensive overview about the topic of NRPs discovery, from gene cluster identification by genome mining to the heterologous production in fungal hosts. The main computational tools and methods for genome mining are herein presented with an emphasis on the particularities of the fungal systems. The different steps of the reconstitution of NRP biosynthetic pathway in heterologous fungal cell factories will be discussed, as well as the key factors to consider for maximizing productivity. Several examples will be developed to illustrate the potential of heterologous production to both discover uncharacterized novel compounds predicted in silico by genome mining, and to enhance the productivity of interesting bio-active natural products.     

Characterization of a Gene Cluster Responsible for the Biosynthesis of Anticancer Agent FK228 in Chromobacterium violaceum No. 968

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.     

真菌聚酮化合物生物合成研究进展

具有广泛生物活性的真菌聚酮化合物因具有复杂的化学结构,其生物合成途径一般包含多样且新颖的酶催化反应。文中主要综述了2013-2016年来源于还原性聚酮合成酶(HR-PKSs)、非还原性聚酮合成酶(NR-PKSs)、聚酮-非核糖体多肽合成酶(PKS-NRPSs)和还原性-非还原性聚酮合成酶(HR-NR PKSs)杂合型等四大类型的真菌聚酮类化合物的生物合成研究进展。众多真菌聚酮类化合物生物机理的阐明,为未来新型真菌聚酮类天然产物生物合成基因簇的挖掘、新结构化合物的发现及其类似物的研究提供了方向和理论基础。     

Identification and functional analysis of brassicicene C biosynthetic gene cluster in Alternaria brassicicola

The biosynthetic gene cluster of brassicicene C was identified in Alternaria brassicicola strain ATCC 96836 from genome database search. In vivo and in vitro study clearly revealed the function of Orf8 and Orf6 as a fusicoccadiene synthase and methyltransferase, respectively. The understanding toward the biosynthetic pathway promises construction of this type of diterpene compounds with genetic engineering.     

Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis

Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.     

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.     

Characterization of a gene cluster responsible for the biosynthesis of anticancer agent FK228 in Chromobacterium violaceum No. 968

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.     

An unexpected diterpene cyclase from rice: functional identification of a stemodene synthase

We have cloned a novel diterpene synthase (OsKSL11) from rice that produces stemod-13(17)-ene from syn-copalyl diphosphate. Notably, this gene sequence was not predicted from the extensive sequence information available for rice, nor, despite extensive phytochemical investigations, has this diterpene or any derived natural product previously been reported in rice plants. OsKSL11 represents the first identified stemodene synthase, which catalyzes the committed step in biosynthesis of the stemodane family of diterpenoid natural products, some of which possess antiviral activity. In addition, OsKSL11 is highly homologous to the mechanistically similar stemarene synthase recently identified from rice, making this pair of diterpene cyclases an excellent model system for investigating the enzymatic determinants for differential product outcome. The unexpected nature of this cyclase and its product parallels recent observations of previously unrecognized natural products metabolism in Arabidopsis thaliana, suggesting that many, if not all, plant species will prove to have extensive biosynthetic capacity.     

CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochrome P450 (CYP) mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNA interference double knock-down of this pair of closely related CYPs reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which was ultimately achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al, and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al, and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene-derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis.     

Strategy for efficient cloning of biosynthetic gene clusters from fungi

Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites(SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters(BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this.Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension(SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.     

Climate-specific biosynthetic gene clusters in populations of a lichen-forming fungus

Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen-forming fungus Umbilicaria pustulata. We collected a total of 600 individuals from the Mediterranean and cold-temperate climates. Population genomic analyses indicate that U. pustulata contains three clusters that are highly differentiated between the Mediterranean and cold-temperate populations. One entire cluster is exclusively present in cold-temperate populations, and a second cluster is putatively dysfunctional in all cold-temperate populations. In the third cluster variation is fixed in all cold-temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate-associated BGCs and BGC variations in lichen-forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.     

基于CRISPR/Cas系统的天然产物生物合成基因簇克隆和编辑技术

合成生物学和基因组测序技术的快速发展使挖掘和高效合成天然产物进入了一个全新的时代。由于多数原始菌株生长缓慢、难以培养及遗传改造困难等问题,导致天然产物生物合成基因簇的激活和高效表达受到严重制约。基于此,将原始菌株来源的基因簇转移到操作简便、遗传背景清晰的模式宿主中进行异源表达成为天然产物发现和产量提高的一种有效手段。其中,基因簇的克隆与编辑是实现天然产物异源表达的一个主要限速步骤。CRISPR/Cas技术的应用极大地提高了大型基因簇克隆和编辑的效率,有效促进了微生物来源新药的发现。本文针对基于CRISPR/Cas开发的基因簇克隆和编辑技术进行了系统梳理和全面总结,探讨相关技术在天然产物挖掘和高效合成中的应用及其重要意义。