Butyrolactone I Quantification from Lovastatin Producing Aspergillus terreus Using Tandem Mass Spectrometry—Evidence of Signalling Functions

Abstract

Aspergillus terreus is an industrially important filamentous fungus producing a wide spectrum of secondary metabolites, including lovastatin and itaconic acid. It also produces butyrolactone I which has shown potential as an antitumour agent. Additionally, butyrolactone I has been implicated to have a regulating role in the secondary metabolism and morphology of A. terreus. In this study, a quantitative time-course liquid chromatography—electrospray ionisation—tandem mass spectrometry (LC-ESI-MS-MS) analysis of butyrolactone I is reported for the first time in nine-day long submerged cultures of A. terreus. Butyrolactone I was fragmented in the mass analysis producing a reproducible fragmentation pattern of four main daughter ions (m/z 307, 331, 363 and 393) in all the samples tested. Supplementing the cultures with 100 nM butyrolactone I caused a statistically significant increase (up to two-fold) in its production, regardless of the growth stage but was constitutive when butyrolactone I was added at high cell density during the stationary phase. Furthermore, the extracellular butyrolactone I concentration peaked at 48 h post inoculation, showing a similar profile as has been reported for bacterial quorum sensing molecules. Taken together, the results support the idea of butyrolactone I as a quorum sensing molecule in A. terreus.

Transcriptomic Complexity of Aspergillus terreus Velvet Gene Family under the Influence of Butyrolactone I

“…In A. terreus , butyrolactone I has been demonstrated to induce features that suggest a regulative role of the quorum-sensing phenomenon. These include autoinduction, an increase in secondary metabolite biosynthesis (lovastatin and sulochrin) and sporulation [ 18 , 19 , 20 ]. However, the regulatory role of butyrolactone I on the velvet family genes has not been previously reported.…”

“…A. terreus MUCL 38669 was cultured under shaken, submerged growth conditions in three biological replicates for nine days. The growth conditions were the same as in our previous studies of secondary metabolism [ 19 , 20 ]. Briefly, A. terreus MUCL 38669 spores were maintained on yeast and malt extract (YME) agar slants.…”

“…The filamentous fungus Aspergillus terreus is commonly isolated from soil rhizospheres [ 1 , 2 ], as well as from decaying organic matter [ 3 ] and has been reported to produce a plenitude of secondary metabolites, such as itaconic acid, putative lipases and cellulases [ 3 , 4 , 5 , 6 ], with potential industrial importance, lovastatin with medical significance as a serum cholesterol lowering agent [ 7 ] and toxins citrinin, emodin and gliotoxin [ 8 , 9 , 10 , 11 ], as well as other compounds, including aspulvinones, asterriquinones, butyrolactones and (+)-geodin [ 12 , 13 , 14 , 15 , 16 , 17 ]. The exogenous addition of butyrolactone I (methyl 4-hydroxy-2-[[4-hydroxy-3-(3-methylbut-2-enyl)phenyl]methyl]-3-(4-hydroxyphenyl)-5- oxofuran-2-carboxylate) has been observed to enhance hyphal branching and sporulation, increase the produced amount of secondary metabolites lovastatin, sulochrin and itself and was thus suggested to play a quorum-sensing role in this fungus, i.e., implicating autoregulatory cell-to-cell communication in order to adapt to the environmental conditions [ 18 , 19 , 20 ]. In addition to the pharmaceutically- and industrially-important increase in secondary metabolism regarding lovastatin, the observed effect on morphological development is of significance, as well.…”

“…In this study, we aim to further illuminate the effect of exogenous addition of butyrolactone I on transcriptional level. The transcriptome of A. terreus strain MUCL 38669 was sequenced to discover and clarify transcriptional differences between the annotated strain NIH2624 and strain MUCL 38669, which was used in our previous studies [ 19 , 20 ]. In this study, we discovered butyrolactone I to upregulate laeA gene expression, confirming the suggested role for butyrolactone I in secondary metabolism.…”

Induction of secondary metabolism of Aspergillus terreus ATCC 20542 in the batch bioreactor cultures

“…Another molecule, butyrolactone I (Fig. 6c ), was described in a several strains of A. terreus (Nitta et al 1983 ; Palonen et al 2014 ; Rao et al 2000 ), including a reisolate (Schimmel et al 1998 ) and mutant (Schimmel and Parsons 1999 ) of A. terreus ATCC 20542. Its formation was proposed previously to originate from 4-hydroxyphenylpyruvic acid and involved the catalytic activity of a non-ribosomal peptide synthetase-like enzyme (Guo et al 2013 ).…”