Potential biosynthesis of nonribosomal peptides by hypocrealean entomopathogenic fungi

doi:10.12211/2096-8280.2021-005

Abstract

Abstract:

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.

Key words: hypocrealean entomopathogenic fungi, nonribosomal peptide synthetase (NRPS), nonribosomal peptide (NRP), biosynthetic gene clusters (BGCs), sequence similarity network analysis (SSN)

摘要：

肉座菌目虫生真菌合成的非核糖体多肽类天然产物具有抗菌、杀虫、抗癌、调节免疫等生物活性，在临床和农业等领域有重要应用价值。近年来，随着真菌基因组测序数量的迅速增加、注释和分析工具的不断进步，人们发现真菌基因组中存在大量产物未知的天然产物合成基因。准确有效地预测这些基因的功能，筛选最有潜力合成新颖天然产物的基因簇，可以提高天然产物挖掘的效率。本研究选取40株肉座菌目虫生真菌基因组，系统分析了编码非核糖体多肽合成酶的基因及基因簇。基于隐马尔可夫模型，预测了445个模块型非核糖体多肽合成酶和1243个类非核糖体多肽合成酶；通过提取腺苷酰化结构域，构建序列相似性网络，以已知功能的非核糖体多肽合成酶作为标签，利用马尔可夫聚类算法，分析了非核糖体多肽产物的主要类别，发现了可能合成线性多肽、环缩肽、脂多肽、生物碱等新型结构化合物的基因簇。研究结果不仅揭示了肉座菌目虫生真菌合成非核糖体多肽活性产物的巨大潜力，而且为通过激活沉默基因簇挖掘新产物、利用合成生物学手段改造合成途径提供参考。

关键词: 肉座菌目虫生真菌, 非核糖体多肽合成酶, 非核糖体多肽, 生物合成基因簇, 序列相似性网络分析

CLC Number:

Liwen ZHANG, István MOLNÀR, Yuquan XU. Potential biosynthesis of nonribosomal peptides by hypocrealean entomopathogenic fungi[J]. Synthetic Biology Journal, 2021, 2(5): 815-825.

张礼文, MOLNÁR István, 徐玉泉. 虫生真菌非核糖体多肽活性产物生物合成潜力预测[J]. 合成生物学, 2021, 2(5): 815-825.

Figures/Tables 5

Tab. 1 Annotion for NRPS domains in hypocrealean entomopathotenic fungi (HEF)

Description	Function with HMM	Source	Cutoff (Score)	Length	Domains (SwissProt)	Domains (HEF)
Adenylation domain	AMP-binding	PFAM00501.21	55	418	280	2297
Condensation domain	Condensation	PFAM00668.13	50	301	255	1483
Thiolation domain	PP-binding	PFAM0050.20	30	67	312	2775

Tab. 2 Statistic summary of NRPSs in hypocrealean entomopathotenic fungi (HEF)

Subtype	Definition (Proteins that contain…)	Number(SwissProt)	Number(HEF)
Multi-modular	more than one A AND C domains	57	280
Single-modular	ONE A AND C domains	21	165
Pseudo	A OR C domain	86	1243

Fig. 2 Analysis of representative biosynthetic gene clusters in hypocrealean entomopathogenic fungi

Fig. 1 Network for the subgroups of multi-modular NRPSs in Hypocrealean Entomopathogenic fungi(Nodes in the network represent A domains, and the line boldness is proportional to the identity. The nodes are colored according to the domain composition of NRPS. Referenced A domains from voucher NRPSs with known products are indicated by arrowheads. The predicted products/functions of the enzymes are listed below the clades. Boxed groups show A domains from the group of enzymes that also contain DtxS1, or superclades that are formed by closely related A domain clades such as clades #52～64 (see text for details). Sub-clades of A domains are indicated with a number-letter combination, for example 52a. NRPSs of the BGCs that contain the voucher A domains include: #27a～32a, destruxin synthetase DtxS1[9]; #32b, cyclosporine synthetase[29]; #33～39, serinocyclin synthetase[13-14]; #44～45, ACVS: N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthetase in penicillin/cephalosporin biosynthesis; #46～51, Tex1: a peptaibol synthetase from Trichoderma virens[30]; #75～76, beauvericin and bassianolide synthetase[11-12].)

Fig. 3 Overview of the A domain distance network for monomodular, bimodular or siderophore-like NRPSs in Hypocrealean entomopathogenic fungal species(Nodes in the network represent A domains, and the line boldness is proportional to the identity. Nodes are colored according to the domain composition of NRPS. Referenced A domains from voucher NRPSs with known products are indicated by arrowheads. The predicted products/functions of the enzymes are listed below the clades. NRPSs that contain the voucher A domains include: AAR, L-aminoadipate-semialdehyde dehydrogenase[24]; LpsC and LpsB: D-lysergyl-peptide synthetase subunit 1 and 2, respectively, involved in ergot alkaloid biosynthesis[32]; ChNPS10[33-34]: an NRPS-like protein involved in morphological development; NPS2/SidC[35-36] and SidI[37]: synthetases for intracellular siderophores; SidN[38] and NPS6[39]: synthetases for extracellular siderophores. Numbers in parentheses indicate the position of the A domain in the voucher NRPS, for example (1/3) indicates the first A domain out of the three housed in the NRPS.)

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