Aspergillus niger as a Secondary Metabolite Factory

Aspergillus niger, one of the most common and important fungal species, is ubiquitous in various environments. A. niger isolates possess a large number of cryptic biosynthetic gene clusters (BGCs) and produce various biomolecules as secondary metabolites with a broad spectrum of application fields covering agriculture, food, and pharmaceutical industry. By extensive literature search, this review with a comprehensive summary on biological and chemical aspects of A. niger strains including their sources, BGCs, and secondary metabolites as well as biological properties and biosynthetic pathways is presented. Future perspectives on the discovery of more A. niger-derived functional biomolecules are also provided in this review.


INTRODUCTION
Aspergillus, one sizeable genus belonging to Aspergillaceae family, comprises as many as 492 species registered on the database of the National Center for Biotechnology Information (NCBI) to date. Its section Nigri is an important group of species, and the A. niger aggregate represents its most complicated taxonomic subgroup with eight morphologically indistinguishable taxa (Perrone et al., 2011). Owing to superior adaptability and survivability, A. niger is ubiquitous in nature, including in terrestrial soil (Xie et al., 2006), ocean (Li et al., 2016;Uchoa et al., 2017), the Arctic (Singh et al., 2011), and space. It also occupies a wide spectrum of habitats in plants and animals such as herb (Shreelalitha and Sridhar, 2015;Manganyi et al., 2018), shrub (Kaur et al., 2015;Liu et al., 2016), tree (Soltani and Moghaddam, 2014;Wang et al., 2019), lichen (Elissawy et al., 2019), shrimp (Liu et al., 2013;Fang et al., 2016), and marine sponge Hiort et al., 2004). A. niger strain grows well in various media with different carbon sources, including glucose, bran, maltose, xylan, xylose, sorbitol, and lactose (Toghueo et al., 2018). However, its metabolism is remarkably affected by culture conditions, such as medium composition and fermentation mode.
The genome features of strain L14 are summarized in a polycyclic graph (Figure 1), which consists of in-paralog pair, GC skew, widely, SM biosynthetic gene cluster (BGC), ncRNA, repeat, strand coding sequence (CDS) annotation, and scaffold. There are some in-paralog pairs between different scaffolds, and SM BGCs and CDS distributed widely in genome. As shown in Table 1, genome sizes of WT A. niger strains range from 33.8 to 36.1 Mb. Their G + C% and gene numbers are closely similar, while the numbers of scaffolds are different owing to various sequencing and assembling manners. The antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) results indicated that each WT A. niger strain harbors at least 20 cryptic SM BGCs, including PKS, NRPS, NRPS-like, and their hybrids (Figure 2 and Supplementary Table S1) (Blin et al., 2019). These BGCs involving in indole and terpene biosynthesis are ubiquitous and have great potential to synthesize therapeutical agents and pesticides, such as AbT1, azanigerone A, fusarin, ferrichrome, nidulanin A, melanin, TAN-1612, yanuthone D, and aflavarin (Supplementary Table S2).
It is a matter of controversy that some A. niger isolates are renowned for biosynthesis of valuable natural products of nutritional, agrochemical, and pharmaceutical interest, while others are reputed to cause the "black mold" disease (Hayden et al., 1994;Ozer and Koycu, 2006) and produce a plethora of mycotoxins (Sanchez et al., 2012). A. niger possesses a bulk warehouse of prolific genes, which involve in regulation of primary and secondary metabolisms (Pel et al., 2007). A genome-scale metabolic network for A. niger has been established on account of its high efficiency in rational metabolic design and systems biology studies, such as strain improvement and process optimization (Sun et al., 2007;Lu FIGURE 1 | General genome features of marine strain Aspergillus niger L14 (From the inside out: In-paralog pairs; GC skew, the green part represent positive value while the orange part represent negative value; G + C%; SMs gene clusters; ncRNA; repeat; minus strand coding sequence (CDS) annotation; plus strand CDS annotation; scaffolds).
Frontiers in Chemistry | www.frontiersin.org July 2021 | Volume 9 | Article 701022 2 et al., 2017). Numerous A. niger strains have been applied in many fields for a long time. For instance, citric acid as one of incredible organic acids in food industry had been produced on a large scale by A. niger 100 years ago (Cairns et al., 2018;. It is important that A. niger is one of the excellent producers of valuable proteases, which had been widely used as detergents and food ingredients and additives, such as acetylesterase, amylase, fucosidase, glucose oxidase, glucosidase, mannanase, phospholipase, phytase, prolyl endopeptidase, triacylglycerol lipase, trehalase, and xylanase. In addition, numerous chemical studies have indicated that A. niger is one of the rich sources of bioactive SMs, with great potential application in agriculture and medicine. Moreover, endoxylanase isozymes of A. niger have great potential transforming lignocellulose in pulp and paper industry as industrial bleaching aids (Duarte and Costaferreira, 1994). Furthermore, A. niger is also able to deal with the phenolic contaminants in waste water of fermentation broth from industry (Duarte and Costaferreira, 1994). Since genetic engineering is inefficient for fully exploiting in the filamentous fungi industry, a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system had been developed (Nødvig et al., 2015;Nødvig et al., 2018). Based on these genome-editing toolbox, gene inactivation and knockout, gene insertion, base editing, promoter replacement, and regulation of gene expression in A. niger have come true. In the future, more importance may be focused on traceless gene editing, multiple gene editing and fine regulation of gene expression in A. niger.

SECONDARY METABOLITES FROM ASPERGILLUS NIGER
By extensive search on the database of Dictionary of Natural Products (DNP), as many as 166 A. niger-derived secondary metabolites (1-166) were detected till 2020. On the basis of chemical structures, these chemicals are grouped into five types: pyranone, alkaloid, cyclopentapeptide, polyketide, and sterol and, respectively, introduced as follows. (More detailed information about these substances is provided in the Supplementary Materials (Supplementary Table S3).)

c-Pyranones
To the best of our knowledge, only four γ-pyranone derivatives (57-60) had been detected in SM of A. niger. Among these substances, kojic acid (57) is the most common product with weak antimicrobial property (Liu et al., 2011;Happi et al., 2015;Padhi et al., 2020). In addition to carbonarone A (59) and tensidol B (60), one new benzyl γ-pyranone nigerpyrone (58) was discovered from a mutant strain FGSC A1279 ΔgcnE and was found to have potent and selective activity against Candida parapsilosis Padhi et al., 2020).

CONCLUSION AND FUTURE PROSPECTS
A. niger strains are ubiquitous in nature and occupy a wide spectrum of habitats in animal and plant environments, and they are economically important both as harmful or beneficial microorganisms. Numerous chemical studies suggest that A. niger is one of the prolific sources of functional biomolecules, including organic acids, vitamins, pesticides, valuable proteases, and therapeutic agents, which have potential Frontiers in Chemistry | www.frontiersin.org July 2021 | Volume 9 | Article 701022 application in various fields including agriculture, food industry, and medicine. However, the number of new bioactive compounds from A. niger has been decreasing for the past 5 years. This deteriorating trend will result in a negative impact on discovery and development of new A. niger-derived valuable substances, such as new drug leads. Therefore, more efforts should be made to explore more sources for isolation of new A. niger strains and to awaken their silent BGCs to manufacture novel functional biomolecules using new strategies, such as one strain many compounds (OSMAC) approach (Hemphill et al., 2017;Pan et al., 2019) and genetic mining combined with metabolic engineering Wei et al., 2021). Moreover, functional genomics should allow for an in-depth understanding of the underlying biosynthetic logic of A. niger-derived SMs (He et al., 2018). In order to accelerate development of valuable products from A. niger, construction and breeding of robust strains as well as optimization of their cultivation and fermentation processes should be intensively conducted at various levels (Zou et al., 2015;Xu et al., 2019).

AUTHOR CONTRIBUTIONS
RY made a draft of this review; JL and YW searched and collected all references; HW helped in critical assessing this manuscript; and HZ conceived and revised this review.

FUNDING
Financial supports from the National Key R&D Program of China (2018YFC0311004), the National Natural Science Foundation of China (41776139) and the Fundamental Research Fund for the Provincial Universities of Zhejiang (RF-C2019002) were greatly appreciated.