Ustiloxin biosynthetic machinery is not compatible between Aspergillus flavus and Ustilaginoidea virens
Introduction
Ustiloxins are cyclic peptides originally isolated from the rice pathogenic fungus, Ustilaginoidea virens (Suwa, 1915). Ustiloxins A and B include the tetrapeptides Tyr(Y)-Val(V)-Ile(I)-Gly(G) and Tyr(Y)-Ala(A)-Ile(I)-Gly(G), respectively, which are cyclized at the sidechains of Tyr and Ile and modified with the non-proteinogenic amino acid norvaline at Tyr (Koiso et al., 1992, Koiso et al., 1994) (Fig. 1). Because norvaline is present, the compounds were believed to be synthesized by a non-ribosomal peptide synthetase until the biosynthetic gene cluster (BGC) for ustiloxin B was identified in the filamentous fungus Aspergillus flavus (Umemura et al., 2013, Umemura et al., 2014). As the ustiloxin BGC contains a gene encoding a 16-fold repeated sequence that contains the YAIG motif, which is the exact structure of the ustiloxin B backbone, ustiloxin B was revealed not to be a non-ribosomal peptide but a ribosomally synthesized and post-translationally modified peptide (RiPP), first reported in Ascomycetes (Umemura et al., 2014).
RiPPs form a relatively new class of secondary metabolites first discovered in the bacterium Streptococcus lactis (Kaletta and Entian, 1989). The backbone of these peptides is directly encoded in the gene encoding the respective precursor peptides. This precursor undergoes modifications such as cyclization and methylation by tailoring enzymes (Arnison et al., 2013). In the case of ustiloxin B, the precursor peptide, UstA, is translocated to the endoplasmic reticulum (ER) via an N-terminal signal peptide, and cleaved at dibasic Lys(K)-Arg(R) residues at the end of each repetitive sequence by the KexB protease localized at the Golgi apparatus (Yoshimi et al., 2016). Each cleaved sequence containing a YAIG peptide is cyclized at the side chains of Tyr and Ile by three proteins (UstYa, UstYb, and UstQ), methylated at the trimmed N-terminus of Tyr by the methyl transferase UstM, and then modified with norvaline at Tyr by the cytochrome P450 enzyme (UstC), two flavin-containing monooxygenases (UstF1 and UstF2), and the pyridoxal phosphate-dependent enzyme UstD (Fig. S1) (Ye et al., 2016). UstYa and UstYb are homologous and both possess the DUF3328 domain, which is crucial for cyclization of ustiloxin (Nagano et al., 2016, Umemura et al., 2014, Ye et al., 2016). UstYa plays a more dominant role in the cyclization reaction than UstYb, because the deletion mutant for the gene encoding UstYa cannot produce ustiloxin B, whereas that for the gene encoding UstYb retains some ustiloxin B production (less than10% of the level in the wild type strain) (Nagano et al., 2016).
The segment of RiPP precursor peptides that is transformed into the compound is called the core peptide. The A. flavus ustiloxin precursor peptide, UstA, contains only the YAIG core peptide, which corresponds to ustiloxin B. On the other hand, the U. virens precursor, Uv_UstA, contains two different core peptide sequences, YVIG and YAIG, which correspond to ustiloxins A and B, respectively (Tsukui et al., 2015) (Fig. 1, right). In accordance with this, A. flavus produces only ustiloxin B (Umemura et al., 2014), whereas U. virens produces both ustiloxin A and B (Koiso et al., 1992). Although UstA and Uv_UstA differ in core peptide sequences and their repeat counts, they both possess an ER signal peptide at the N terminus and Kex2 protease recognition sites at the end of each repeated sequence (Tsukui et al., 2015). In addition to the genes that encode the precursor peptide, the ustiloxin BGCs of A. flavus and U. virens share 12 genes, which are highly homologous, with E-values less than 4E-78 by BlastP analysis (Fig. S2) (Tsukui et al., 2015).
Based on the similarity of the A. flavus and U. virens ustiloxin BGCs, we hypothesized that ustiloxin A (YVIG) might also be produced by A. flavus if the precursor peptide contained the YVIG core peptide motif. In other words, the A. flavus ustiloxin biosynthetic machinery might recognize the YVIG core peptide sequence, in addition to YAIG. Additionally, we considered that the hydroxyl group in Tyr’s aromatic ring might be indispensable for the cyclization reaction via the ether bond catalyzed by UstYa and UstYb, although the hydroxyl group is not directly involved in the ether bond. To elucidate these two points, in this study, we constructed ustA variants of A. flavus in which the gene encoding UstA was replaced by synthetic genes encoding the YVIG (ustA_yvig) or FAIG (ustA_faig) core peptides instead of YAIG, or retaining YAIG (ustA_yaig) as a control, and analyzed their production of ustiloxins A, B, and analogous compounds.
Section snippets
Fungal strains
The A. flavus strains used and constructed in this study are listed in Table 1. Aspergillus flavus A1421 (CA14 Δku70 ΔpyrG) was distributed from the Fungal Genetics Stock Center (McCluskey et al., 2010). The ustA variants constructed from A1421, in which native ustA was replaced by ustA_yaig, ustA_yvig, and ustA_faig, were named ustAYAIG, ustAYVIG, and ustAFAIG, respectively; the pyrG marker revertant was named pyrGrev. As described below, the marker-rescued strain, ustROE pyrG−, in which the
Construction of A. flavus ustA variants
The native ustA gene in A. flavus A1421 was replaced with synthetic genes, retaining the native core peptide YAIG (ustA_yaig) as a positive control, or altering it to YVIG (ustA_yvig) or FAIG (ustA_faig) (Fig. 2) by homologous recombination. The YVIG motif is contained in the U. virens ustiloxin-precursor peptide for ustiloxin A production, and the FAIG motif lacks the hydroxyl group present on the aromatic ring of Tyr in the YAIG motif, which might be important for cyclization of the core
Conclusions
We replaced the gene encoding the ustiloxin precursor peptide, ustA, with synthetic genes encoding the core peptides YAIG, YVIG, or FAIG, in A. flavus, and measured the production of compounds corresponding to the core peptide sequences. The ustA variant possessing the YAIG core peptide produced ustiloxin B, whereas that possessing YVIG did not produce ustiloxin A. This result was against our expectation because 13 of the genes in the A. flavus ustiloxin BGC are homologous with those in U.
CRediT authorship contribution statement
Maiko Umemura: Conceptualization, Validation, Investigation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Funding acquisition. Kaoru Kuriiwa: Investigation, Writing - review & editing. Koichi Tamano: Investigation, Visualization, Writing - review & editing. Yutaka Kawarabayasi: Writing - review & editing.
Acknowledgements
This work was financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant 17H05456), and the Medical Mycology Research Center, Chiba University, Japan.
References (20)
- et al.
Clustered Genes Involved in Cyclopiazonic Acid Production Are next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus
Fungal Genet. Biol.
(2009) Ustiloxin: A phytotoxin and a mycotoxin from false smut balls on rice panicles
Tetrahedron Lett.
(1992)Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes
J. Mol. Biol.
(2001)Class of cyclic ribosomal peptide synthetic genes in filamentous fungi
Fungal Genet. Biol.
(2016)Characterization of the biosynthetic gene cluster for the ribosomally synthesized cyclic peptide ustiloxin B in Aspergillus flavus
Fungal Genet. Biol.
(2014)Biomolecular chemistry heterologous production of asperipin-2a: proposal for sequential oxidative macrocyclization
Org. Biomol. Chem.
(2019)Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature
Nat. Prod. Rep.
(2013)Biosynthetic investigation of phomopsins reveals a widespread pathway for ribosomal natural products in Ascomycetes
Proc. Natl. Acad. Sci. USA
(2016)- et al.
Nisin, a peptide antibiotic: cloning and sequencing of the nisA gene and posttranslational processing of its peptide product
J Bacteriol
(1989) Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens
J. Antibiot.
(1994)
Cited by (4)
Tandem repeats in precursor protein stabilize transcript levels and production levels of the fungal ribosomally synthesized and post-translationally modified peptide ustiloxin B
2022, Fungal Genetics and BiologyCitation Excerpt :The same precursor protein with the 16-fold tandem-repeat segment was translated from both native ustA and the synthetic gene, ustA_yaig, but in the case of ustROE ustAYAIG, which had ustA_yaig as the precursor gene, there was much less ustiloxin B production and a much lower relative transcript level of the precursor gene than in the case of the control ustROE pyrGrev (Figs. 2, 3). ustROE ustAYAIG differs genetically from ustROE pyrGrev in three ways: intron absence in ustA_yaig, marker insertion downstream of ustA, and codon-usage bias in ustA_yaig (Umemura et al., 2020a). To investigate the influence of the former two factors, we constructed transformants without the intron in native ustA or with intact ustA, both having the pyrG marker downstream of ustA as in the other ustA variants of ustROE pyrG-.
Stereo-selective synthesis of non-canonical γ-hydroxy-α-amino acids by enzymatic carbon-carbon bond formation
2021, Catalysis Science and Technology