Increased production of inosine and guanosine by means of metabolic engineering of the purine pathway in Ashbya gossypii

Abstract

BackgroundInosine and guanosine monophosphate nucleotides are convenient sources of the umami flavor, with attributed beneficial health effects that have renewed commercial interest in nucleotide fermentations. Accordingly, several bacterial strains that excrete high levels of inosine and guanosine nucleosides are currently used in the food industry for this purpose.ResultsIn the present study, we show that the filamentous fungus Ashbya gossypii, a natural riboflavin overproducer, excretes high amounts of inosine and guanosine nucleosides to the culture medium. Following a rational metabolic engineering approach of the de novo purine nucleotide biosynthetic pathway, we increased the excreted levels of inosine up to 27-fold.ConclusionsWe generated Ashbya gossypii strains with improved production titers of inosine and guanosine. Our results point to Ashbya gossypii as the first eukaryotic microorganism representing a promising candidate, susceptible to further manipulation, for industrial nucleoside fermentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0234-4) contains supplementary material, which is available to authorized users.

Increased riboflavin production by manipulation of inosine 5′-monophosphate dehydrogenase in Ashbya gossypii

“…Physiological GMP inhibition has been also questioned for the E. coli enzyme (Pimkin and Markham 2008). Moreover, we have recently determined that, in the wild-type A. gossypii A4 strain, the intracellular concentrations of XMP are about 8 times higher than those of GMP and about 40 times larger than IMP (Ledesma-Amaro et al 2015), further supporting the hypothesis that XMP-rather than GMP-feedback inhibition might be a main regulator of IMPDH activity in vivo. Therefore, by solving the highresolution crystallographic structures in complex with IMP and XMP, we have settled a reliable basis for computational modeling as well as protein engineering strategies towards enzyme optimization and/or product inhibition removal which we are at present developing in our laboratory.…”

“…Additionally, we also studied the production of the nucleosides guanosine and inosine, given their interest as precursors of the foodstuff additives IMP and GMP with flavoring, nutritional and pharmaceutical properties (Ledesma-Amaro et al 2013). To this respect, we have recently shown that both nucleosides are excreted to the extracellular media of A. gossypii's flask fermentations at milligrams-per-liter levels and have proposed this fungus as a promising candidate for nucleoside industrial production (Ledesma-Amaro et al 2015). Given that overexpression of IMPDH causes the disruption of the STE12 locus (by the insertion of the P-GPD -IMPDH construct), we previously demonstrated that the disruption of STE12 does not affect inosine, guanosine, riboflavin production, or growth rate (Table S2 in the Supplementary Material).…”

“…They are not only precursors of nucleic acids but they also represent the energy source for translation and microtubule polymerization and are implied in signal transduction, angiogenesis (Chong et al 2006), and axon guidance (Long et al 2006), among many other cellular functions. From a biotechnological point of view, guanine nucleotides are very significant as they are the metabolic precursors of a variety of metabolites with biotechnological interest, such as tetrahydrobiopterin (Gross and Levi 1992), guanosine 5′-monophosphate (GMP) flavor enhancer (Ledesma-Amaro et al 2015), folates (vitamin B9; (Cossins and Chen 1997)), or riboflavin (vitamin B2; (Bacher et al 2000)). …”

Ashbya gossypii beyond industrial riboflavin production: A historical perspective and emerging biotechnological applications

“…GTP, the other riboflavin precursor, is synthesized through the purine pathway, which starts with the formation of phosphoribosyl pyrophosphate (PRPP) from ribose 5-P and ATP. Interestingly, A. gossypii was recently shown to secrete high amounts of inosine and guanosine, which are purine nucleosides produced via this same pathway (Ledesma-Amaro et al, 2015). PRPP is not only required for the de novo and salvage pathways of purine, pyrimidine and pyridine (NAD + , NADP + ) nucleotides, but also as a precursor of histidine ( Fig.…”

“…However, since the beginning of this decade, we have witnessed the increasingly stronger shift of the A. gossypii biotechnological paradigm from riboflavin to other commercially interesting high-value compounds, such as recombinant proteins (Ribeiro et al, 2010), single cell oil (SCO; Ledesma-Amaro et al, 2014a) and flavour compounds (Ravasio et al, 2014;Ledesma-Amaro et al, 2015). In this review, we will focus on the emerging biotechnological potential of this promising cell factory that nowadays goes far beyond riboflavin production ( Fig.…”