Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization

Abstract

Efficient and specific transporters may enhance pentose uptake and metabolism by Saccharomyces cerevisiae. Eight heterologous sugar transporters were characterized in S. cerevisiae. The transporter Mgt05196p from Meyerozyma guilliermondii showed the highest xylose transport activity among them. Several key amino acid residues of Mgt05196p were suggested by structural and sequence analysis and characterized by site-directed mutagenesis. A conserved aromatic residue-rich motif (YFFYY, position 332-336) in the seventh trans-membrane span plays an important role in D-xylose transport activity. The phenyl ring of the residue at position 336 may take the function to prevent D-xylose from escaping during uptake. F432A and N360S mutations enhanced the D-xylose transport activities of Mgt05196p. Furthermore, mutant N360F specifically transported D-xylose without any glucose-inhibition, high lighting its potential application in constructing glucose-xylose co-fermentation strains for biomass refining.

Combining C6 and C5 sugar metabolism for enhancing microbial bioconversion

“…A similar mutant transporter (Gal2p-N376V) that can transport both glucose and xylose without glucose repression was also identified [14 ]. Recently, two independent groups found that mutations Hxt36p-N367 [46] and Mgt05196p-N360F [35] in the arginine residue of other sugar transporters resulted in improved transport of xylose without glucose repression. The isolation of the mutant hexose transporters capable of transporting xylose without glucose inhibition was a crucial step towards simultaneous co-fermentation of glucose and xylose.…”

“…Heterologous transporters with higher affinities for xylose over glucose were identified. These transporters include Gxs1p from Candida intermedia [33], Xut3p from S. stipitis [34], and Mgt05196p from Meyerozyma guilliermondii [35]. Also, heterologous overexpression of STP2 from Arabidopsis thaliana and ARAT from S. stipitis in S. cerevisiae led to improved anaerobic L-arabinose fermentation, especially at low Larabinose concentrations.…”

Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production

“…Normally, xylose is absorbed non-specifically and insufficiently by hexose transporters and is competitively inhibited by glucose in S. cerevisiae (Subtil and Boles 2012). Therefore, to address this problem, both wild-type and mutated endogenous and heterologous transporters were screened (Diao et al 2013; Farwick et al 2014; Moon et al 2013; Nijland et al 2014; Runquist et al 2009; Shin et al 2015; Wang et al 2015, 2016). Moreover, with higher efficiency and/or specificity for xylose, a transporter lacking glucose inhibition may relieve the glucose repression effect in a xylose absorption node, thereby increasing xylose metabolism.…”

“…Moreover, with higher efficiency and/or specificity for xylose, a transporter lacking glucose inhibition may relieve the glucose repression effect in a xylose absorption node, thereby increasing xylose metabolism. Recently, we found the mutant MGT05196 N360F , which is derived from the Meyerozyma guilliermondii transporter gene MGT05196 and exhibits xylose-specific transport and no glucose inhibition (Wang et al 2015). …”

“…For rational strain design, two novel proprietary heterologous genes, MGT05196 N360F (ZL 2014105263569) (Wang et al 2015) and the Ru- xylA (US 8,586,336 B2, EP 2679686, ZL 201110042170.2) (Bao et al 2013; Hou et al 2016a), were integrated into the genome of diploid strain BSIF. Together with other strategies, including the overexpression of the genes XKS1 (xylulokinase), TAL1 (transaldolase), TKL1 (transketolase), RKI1 (ribose 5-phosphate isomerase) and RPE1 (ribulose 5-phosphate epimerase) in the non-oxidative PPP and the inactivation of GRE3 (aldose reductase) and PHO13 (alkaline phosphatase), a functional xylose metabolic pathway was constructed in BSIF.…”