Ustiloxin biosynthetic machinery is not compatible between Aspergillus flavus and Ustilaginoidea virens

Highlights

• Synthetic genes of ustiloxin core peptides were expressed in Aspergillus flavus.

• Ustiloxin B was synthesized from the synthetic gene encoding YAIG.

• Ustiloxin biosynthetic machinery is optimized for native core peptide sequences.

• Hydroxyl group of Tyr in ustiloxin core peptide is indispensable for cyclization.

Abstract

Ustiloxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) first reported in Ascomycetes. Originally identified as metabolites of the rice pathogenic fungus Ustilaginoidea virens, they were recently identified among the metabolites of the mold Aspergillus flavus, along with their corresponding biosynthetic gene cluster. Ustilaginoidea virens produces ustiloxins A and B, whereas A. flavus produces only ustiloxin B. Correspondingly, in U. virens, the ustiloxin precursor peptide, from which the compound backbone is cleaved and cyclized, contains the core peptides Tyr(Y)-Val(V)-Ile(I)-Gly(G) and Tyr(Y)-Ala(A)-Ile(I)-Gly(G) for ustiloxins A and B, respectively, whereas that of A. flavus contains only the YAIG motif for ustiloxin B.

In this study, the gene that encodes the precursor peptide in A. flavus, ustA, was replaced with synthetic genes encoding the core peptides YVIG or FAIG, to investigate their compatibility with the ustiloxin biosynthetic machinery. We also examined the importance of the hydroxyl group on the aromatic ring of Tyr for cyclization of the YAIG core peptide. Against our expectation, the ustA variant possessing YVIG core peptides did not produce a detectable amount of ustiloxin A, even though the ustiloxin biosynthetic gene clusters of A. flavus and U. virens both contain 13 homologous genes. We confirmed that the lack of ustiloxin A production was not due to lack or insufficient expression of the substituted synthetic gene. This result, along with the differences between the primary sequences of UstYa and UstYb in A. flavus and U. virens, suggests that the ustiloxin biosynthetic machinery is optimized for the native core peptide sequences. The synthetic FAIG-encoding ustA did not yield any compounds specific to the FAIG core peptide, suggesting that the hydroxyl group on the aromatic ring of Tyr in the core peptide is indispensable for cyclization of the core peptide, even though it is not structurally involved in the cyclization.

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.