The introduction of the fungal d-galacturonate pathway enables the consumption of d-galacturonic acid by Saccharomyces cerevisiae

Abstract

BackgroundPectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. One possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. However, the ethanol-producing organism of choice, Saccharomyces cerevisiae, is not able to catabolize d-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, namely, 18 % (w/w) from citrus pulp and 16 % (w/w) sugar beet pulp.ResultsIn the current work, we describe the construction of a strain of S. cerevisiae in which the five genes of the fungal reductive pathway for d-galacturonic acid catabolism were integrated into the yeast chromosomes: gaaA, gaaC and gaaD from Aspergillus niger and lgd1 from Trichoderma reesei, and the recently described d-galacturonic acid transporter protein, gat1, from Neurospora crassa. This strain metabolized d-galacturonic acid in a medium containing d-fructose as co-substrate.ConclusionThis work is the first demonstration of the expression of a functional heterologous pathway for d-galacturonic acid catabolism in Saccharomyces cerevisiae. It is a preliminary step for engineering a yeast strain for the fermentation of pectin-rich substrates to ethanol.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0544-1) contains supplementary material, which is available to authorized users.

Engineering Saccharomyces cerevisiae for co-utilization of d-galacturonic acid and d-glucose from citrus peel waste

“…We found that d -galacturonate reductase from Trichoderma reesei (GAR1) and 2-keto-3-deoxy-L-galactonate aldolase from Aspergillus niger (GAAC) have activities near the rate-limiting specific activity of glycolysis: 0.1 µmol min −1 mg −1 protein (Supplementary Table 2 ) 32 . However, consistent with previous reports 26 , 28 , we observed very low activity for the three L-galactonate dehydratase (LGD1) enzymes tested. To allow for facile detection of dehydratase expression, we N-terminally tagged the T. reesei LGD1 with a yellow fluorescent protein (Venus).…”

“…Several attempts at engineering S. cerevisiae as a host for d -galUA fermentations have been published, yet a strain capable of growing on this sugar as the primary carbon source or co-consuming d -galUA with d -glucose have not yet been reported 24 – 27 . Efforts to enable growth on d -galUA using a fungal catabolic pathway have been impaired by low specific activity of enzymes within the pathway, particularly the l -galactonate dehydratase step 26 , 28 . Additionally, S. cerevisiae lacks a dedicated transport system for d -galUA and import inhibition of d -galUA by d -glucose has not been described prior to this report.…”

Valorisation of pectin-rich agro-industrial residues by yeasts: potential and challenges

“…b Engineered S. cerevisiae strains expressing D-GalA membrane transporter Gat1 from Neurospora crassa and the uronate dehydrogenase (UDH) from Agrobacterium tumefaciens and D-galacturonic acid reductase (GAAA) from Aspergillus niger to convert D-GalA into the metabolites meso-galactaric acid and L-galactonate, (Benz et al 2014). c Engineered S. cerevisiae strain with Dgalacturonic acid plasma membrane transporters from N. crassa (GAT1) and enzymes of the D-GalA catabolic pathway GaaA, GaaB, GaaC and GaaD from A. niger (in green) and LGD1 from Trichoderma reesei (in purple); D-Fructose was used as co-substrate (Biz et al 2016). d Engineered S. cerevisiae strains with the non-glucose repressible plasma membrane D-galacturonic acid transporter GatA from A. niger (GATA) and D-GalA catabolic pathway as in c); D-glucose was used as co-substrate (Protzko et al 2018) dependent enzymes: the D-galacturonate reductase and the Lglyceraldehyde reductase, for the catabolisation of Dgalacturonic acid into glycerol (Biz et al 2016) (Fig.…”

“…c Engineered S. cerevisiae strain with Dgalacturonic acid plasma membrane transporters from N. crassa (GAT1) and enzymes of the D-GalA catabolic pathway GaaA, GaaB, GaaC and GaaD from A. niger (in green) and LGD1 from Trichoderma reesei (in purple); D-Fructose was used as co-substrate (Biz et al 2016). d Engineered S. cerevisiae strains with the non-glucose repressible plasma membrane D-galacturonic acid transporter GatA from A. niger (GATA) and D-GalA catabolic pathway as in c); D-glucose was used as co-substrate (Protzko et al 2018) dependent enzymes: the D-galacturonate reductase and the Lglyceraldehyde reductase, for the catabolisation of Dgalacturonic acid into glycerol (Biz et al 2016) (Fig. 3c) leading to intracellular cofactor imbalance.…”

“…Endogenously, S. cerevisiae can only use a very limited range of carbon sources. For this reason, genetically modified strains have been developed to also utilise pentoses and D-galacturonic acid for synthesis of novel compounds (Hong and Nielsen 2012;Benz et al 2014;Biz et al 2016;Yaguchi et al 2018;Rebello et al 2018;Protzko et al 2018;Nielsen 2019). However, there are non-conventional species considered advantageous alternatives to S. cerevisiae since they can express highly interesting metabolic pathways (Rebello et al 2018), efficiently assimilate a wider range of carbon sources (Do et al 2019) or exhibit higher tolerance to relevant bioprocess-related stresses, such as the presence of a wide range of inhibitory compounds and supraoptimal temperatures (Radecka et al 2015;Kręgiel et al 2017;Mukherjee et al 2017).…”