Genome Sequencing and Analysis Reveal the Mechanism of Terpenoids Biosynthesis in Deep-See-Derived Fungus Aspergillus Tubingensis

In recent years, deep-sea-derived fungi have become an important source of marine natural products. Aspergillus tubingensis is extremely rich in secondary metabolites, which is a valuable deep-sea-derived fungus needs to be further explored. So far, many small molecular compounds with novel structure and signicant biological activity have been discovered from A. tubingensis, among which terpenoids account for about 20%, showing great research potential in anti-tumor, antiviral and antibacteria. Although more and more new terpenoids have been discovered from A. tubingensis and their structures have been identied, few studies have investigated the biosynthetic pathway of terpenoid. In order to further elucidate the mechanism of terpenoid biosynthesis, the key genes and enzymes involved in terpenoid biosynthesis were successfully mined and further analyzed based on genome sequencing analysis. Subsequently, hydroxymethylglutaryl-CoA synthase, mevalonate kinase, phosphomevalonate kinase as well as diphosphomevalonate decarboxylase were annotated, which played important roles in terpenoid biosynthesis of A. tubingensis. Furthermore, the biosynthetic pathway of terpenoid in A. tubingensis has been constructed, which could be applied to develop the metabolic regulation of A. tubingensis. This study would provide more sucient scientic basis and new ideas for the genetic regulation of terpenoid biosynthesis in A. tubingensis.


Introduction
The deep sea is one of the most mysterious and unexplored extreme environments in the world, characterized by lack of light, low temperatures, anaerobic conditions, and hydrostatic pressures that could reach more than 1000 atmospheres (Thornburg et al. 2010). Over the past 50 years, there have been about 24,000 natural products of marine origin, but less than 2% of them come from deep-sea sources. Generally, deep-sea microorganisms have unique physiological metabolic processes by the adaptation to extreme environmental conditions, and thus exhibit the chemical structure diversity, novelty, and signi cant biological activity of their metabolites (Skropeta and Wei 2014). It has been reported that over 75% of natural products from deep-sea sources are bioactive, and more than half show signi cant cytotoxic activity against human tumor cell lines (Skropeta 2008). As an important group of deep-sea microorganisms, deep-sea fungi are well known for their diversity of secondary metabolites, which have the potential to treat human diseases and nd new biological targets, providing important support for marine drug research and development (Xu et al. 2018).
Aspergillus tubingensis, as a valuable deep-sea-derived fungus needs to be further explored, is extremely rich in secondary metabolites (Koch et al. 2014). At present, anthraquinones, alkaloids, terpenoids and other compounds have been isolated from A. tubingensis, and many of the secondary metabolites have the activities of antibacterial, anti-in ammatory, anti-cancer and antiviral, which have a certain potential as drugs (Carboue et al. 2019;Ottoni et al. 2019).
Terpenoids refer to derivatives with (C 5 H 8 ) n general formula, oxygen content and different saturation levels, which can be regarded as a class of natural compounds linked in various ways by isoprene or isopentane (Hillier and Lathe 2019). Although terpenoids are not the dominant components in A. tubingensis, they play an important role among the active ingredients because of their anti-tumor, antiviral and anti-in ammatory activities. It was reported that ve terpenoids of 20-norisopimarane were isolated from Aspergillus from deep-sea sediments in the South China Sea, which showed signi cant antibacterial activity against Fusarium graminearum (Li et al. 2016). Furthermore, a new indole diterpene Penicindopene A was isolated from Penicillium YPCMAC1 from deep sea of western paci c, which showed moderate cytotoxicity to A549 and HeLa cell lines ). Subsequently, two unique phenol-sesquiterpenoids, phomeroid A-B, were isolated from Phomopsis tersa FS441 from deep-sea sediment samples in the Indian Ocean, which showed moderate inhibitory activity against human glioblastoma SF-268, breast cancer MCF-7, liver cancer HepG-2 and lung cancer A549 . The above studies indicated that more and more novel chemical structures derived from deep-sea microorganisms have been discovered, with the development of deep-sea microbial sample collection, separation and puri cation technology and compound structure analysis technology, which is bene cial to the excavation of deep-sea fungal resources (Li et al. 2015).
Even so, few studies have investigated the biosynthetic pathway of terpenoid, and it is becoming more and more urgent to investigate the hereditary information or functional genes of A. tubingensis by omics sequencing technology (Lin et al. 2019). Therefore, the functional genes involved in the biosynthetic pathway can be obtained by genome sequencing analysis, and the biological processes in different states can be revealed (Xiong and Zhao 2018). In the present study, to better understand the molecular factors and their regulatory genes involved in accumulation of terpenoid, the genomic pro les of A. tubingensis were analyzed. We gain insights into the terpenoid accumulation mechanism of A. tubingensis, particularly the functional genes and enzymes involved in terpenoid biosynthesis. Physiological observations such as growth and terpenoid biosynthesis were linked to genomic data obtained by genome sequencing. These results would provide novel insight into understanding the molecular mechanisms of terpenoid accumulation and aid in understanding its biosynthesis, and developing future studies on the metabolic regulation of A. tubingensis.

Strains and genomic data
The Draft Genome Sequence of A. tubingensis WU-2223L, a Citric Acid-producing Filamentous Fungus Belonging to Aspergillus Section Nigri, has been submitted to NCBI (BLWE00000000). The Genome Coverage was 163x, which was applied by HiSeq X; Nanopore MinION. The Assembly Method was MaSuRCA v. 3.3.3. The genomic pro les of A. tubingensis were downloaded for further bioinformatics analysis.

Functional gene mining of terpenoid biosynthesis in A. tubingensis
The les of genome sequencing results were downloaded on computer. Subsequently, the annotation result les of assembled Unigenes from the genome sequencing results were obtained. Noteworthy, paired-end reads were used again for gap lling of scaffolds to obtain sequences with least Ns and cannot be extended on either end. Such sequences were de ned as Unigenes. Unigene annotation provides information of expression and functional annotation of Unigene. The "annotation les" path was opened, and "map 00900" in the search bar was entered. Then, terpenoid backbone biosynthesis (map00900) in the KEGG classi cation of annotation les was searched. Furthermore, the functional genes such as hydroxymethylglutaryl-CoA synthase, mevalonate kinase, phosphomevalonate kinase and diphosphomevalonate decarboxylase, were mined and analyzed to con rm whether annotated in A. tubingensis genome.

Construction of biosynthetic pathway of terpenoid in A. tubingensis
Based on the KEGG Pathway annotation information by genome sequencing and short read sequence assembly, the biosynthetic pathways of terpenoid in A. tubingensis were analyzed. The metabolic pathways of terpenoid were searched and screened from KEGG PATHWAY Database (http://www.genome.jp/kegg/pathway.html). Subsequently, the KEGG Pathway annotation Database was used to nd the annotation pathways corresponding to the Pathway numbers obtained from the KEGG Pathway Database. The online KEGG Pathway database and genomic KEGG Pathway annotation information were compared and analyzed to study the biosynthetic pathways of terpenoids, as well as the corresponding Unigene sequences of related enzymes.

Phylogenetic tree analysis of 18S rRNA
The deep-sea fungi have the characteristics of species diversity. Phylogenetic tree analysis of the genetic relationships could be carried out to classify the characteristics at a higher level based on molecular biology and ITS region sequencing. The phylogenetic tree of A. tubingensis and other strains in Genbank was shown in Fig. 1, which indicated that A. tubingensis is closely related to Aspergillus magaliesburgensis (MK450649.1) and Aspergillus alliaceus (EF661548.1) in evolutionary relationship, forming a relatively independent branch. These diverse species of deep-sea fungi provided abundant secondary metabolites for humans.

Draft genome sequence of A. tubingensis
The A. tubingensis whole genome shotgun (WGS) project has the project accession BLWE00000000, which has been submitted to NCBI (Yoshioka et al. 2020). This version of the project has the accession number BLWE01000000, and consists of sequences BLWE01000001-BLWE01000015. We downloaded the genomic pro les from NCBI for further bioinformatics analysis, and the genome features of A. tubingensis were shown in Table 1. Mining functional proteins involved in terpenoid biosynthesis of A. tubingensis Paired-end reads were used for gap lling of scaffolds to obtain Unigenes with least Ns that cannot be extended on either end. Furthermore, the les of annotation result of assembled Unigenes from the genome sequencing were obtained for further analysis. Subsequently, the functional proteins involved in terpenoid biosynthesis of A. tubingensis were shown in Table 2. Among them, hydroxymethylglutaryl-CoA synthase, mevalonate kinase, phosphomevalonate kinase as well as diphosphomevalonate decarboxylase were annotated, which played important roles in terpenoid biosynthesis of A. tubingensis. Subsequently, bioinformatics analysis of the above proteins was developed, and the molecular weight and theoretical pI were shown in Table 2, respectively. Among them, diphosphomevalonate decarboxylase (GFN12862.1) was alkaline protein, mevalonate kinase (GFN12941.1) was neutral protein, and the rest were acidic proteins.

Secondary and tertiary structures of functional proteins involved in terpenoid biosynthesis
In order to further understand the function of these proteins, secondary and tertiary structures of the functional proteins involved in terpenoid biosynthesis of A. tubingensis were predicted. The predicted secondary structure composition of functional proteins was shown in Table 3, which showed the ratio of strand to helix in each protein, respectively. Subsequently, the predicted tertiary structures of functional proteins involved in terpenoid biosynthesis were shown in Fig. 2, which could indicate the protein family to which the protein belongs and the preference of the substrate. Constructing the predicted biosynthetic pathway of terpenoid in A. tubingensis Subsequently, terpenoid backbone biosynthesis (map00900) was applied to construct the biosynthetic pathway of terpenoid in A. tubingensis. The functional proteins involved in terpenoid biosynthesis of A. tubingensis were shown, as well as the predicted biosynthetic pathway of terpenoid in A. tubingensis was shown in Fig. 3, which might provide more su cient scienti c basis and new ideas for the genetic regulation of terpenoid biosynthesis in A. tubingensis. In this pathway, the biosynthesis up to terpenoid in A. tubingensis is different from that in other organisms. Following acetyl-CoA was converted to 3hydroxy-3-methyl-glutaryl-CoA by hydroxymethylglutaryl-CoA synthase, and 3-hydroxy-3-methyl-glutaryl-CoA was converted to mevalonate by hydroxymethylglutaryl-CoA reductase, mevalonate was subsequently converted to mevalonate-5P by mevalonate kinase, and mevalonate-5P was further converted to mevalonate-5PP by phosphomevalonate kinase. Moreover, mevalonate-5PP was converted to isopentenyl-PP by diphosphomevalonate decarboxylase, and isopentenyl-PP was used as precusor substance for monoterpenoid biosynthesis, diterpenoid biosynthesis and carotenoid biosynthesis and so on.

Discussion
Deep-sea environment is an extreme environment characterized by high pressure, low oxygen, high salt and no illumination. In order to adapt to the survival and reproduction in the deep-sea environment, deepsea fungi have evolved unique physiological structure and metabolic pathway, and can produce novel structure and signi cant variety of secondary metabolites with biological activities, which become an important resource for the development of new drugs (Zain Ul Arifeen et al. 2019). However, limited sampling techniques have limited the study of deep-sea fungi and their metabolites (Daletos et al. 2018).
In addition, there are hitherto unknown fungal lineages in the deep sea, which have unique ecological and physiological characteristics, and are di cult to grow through conventional culture methods. Fortunately, with the rapid development of deep-sea sampling and multiomics, abundant active natural products will be mined and contribute to the sustainable use of marine microbial resources and drug development. Therefore, it is more and more urgent to investigate the active ingredients of deep-sea fungi.
There are several studies focused on bacteria and archaea in deep sea, involving a wide range of species, including population distribution, separation of new active metabolites and so on (Carroll et al. 2021;Shin 2020). As marine eukaryotic microorganisms, marine fungi are the main decomposers of low organic matter, which are characterized by diverse species, wide distribution and complex metabolic pathways. In recent years, more than half of the new compounds derived from marine microbes were come from marine fungi (Carroll et al. 2021;Shin 2020). Secondary metabolites isolated from marine fungi have been found to have many important biological activities, such as anti-tumor, antibacterial, antioxidant, antiviral, anti-adhesion and so on (Moghadamtousi et al. 2015). Although terpenoids account for about 20% of the total active ingredients in A. tubingensis, they play an important role because of their antitumor, antiviral and anti-in ammatory activities (Khizar et al. 2020). Unfortunately, few studies have investigated the biosynthetic pathway of terpenoid, and the hereditary information or functional genes of A. tubingensis by omics sequencing technology is becoming more and more urgent to be investigated. In this study, we investigated the functional genes as well as proteins involved in the biosynthetic pathway of terpenoid by omics sequencing technology for the rst time, which could gain insights into the terpenoid accumulation mechanism of A. tubingensis, particularly the functional genes in terpenoid biosynthesis.
So far, there were several studies investigating the pharmacological effects of terpenoid. However, the studies on the functional genes as well as the enzymes involved in terpenoid biosynthesis of deep-sea fungi were few. For instance, the fungal terpenoid biosynthesis from biochemical, genetic, and genomic viewpoints were investigated, as well as the enzymes involved in synthesizing, transferring, and cyclizing the prenyl chains were systematically discussed, which provided the genomic information and functional evidence for biosynthetic mechanism of terpenoid (Schmidt-Dannert 2015). Furthermore, the characterization and evolution of gene clusters for terpenoid phytoalexin biosynthesis in tobacco were investigated, 1181 metabolic gene clusters with 34 of them potentially being involved in terpenoid biosynthesis were identi ed, which demonstrated that phytoalexins in tobacco can arise from operon-like gene clusters ). Unfortunately, the key genes involved in terpenoid biosynthesis of deepsea fungi were lacking in-depth genomic mining and analysis. Therefore, it is certainly needed to conduct the study of genome analysis of the key genes and enzymes involved in terpenoid biosynthesis of deepsea fungi.
To our knowledge, genome sequencing and analysis could provide information of gene expression and infer the gene functions (Liu et al. 2015;Wu et al. 2020). Next-generation sequencing technology of genome could systematically provide a complete view of expressed genes and their expression levels (Gu et al. 2019). The availability of a draft genome sequence opens new avenues for new exploration, application and improvements of marine fungi (Kumar et al. 2018). It will lead to the identi cation and manipulation of candidate genes or genomic regions to generate the new ways to synthesize new compounds with potentials in pharmaceuticals (Wang et al. 2010). In order to obtain a comprehensive insight into the biosynthesis of terpenoid in A. tubingensis, functional genes screened from the submitted genome database of A. tubingensis on NCBI, and their evolutionary relationships has been demonstrated. The availability of genome will facilitate the development of new products, and improve the e cient processes of production. The work present here would aid in understanding and carrying out future research on the genetic basis of biology of this organism and contribute to the further production and application of A. tubingensis. Figure 1 Phylogenetic tree of 18S rRNA of A. tubingensis.