Functional Analysis of Two l -Arabinose Transporters from Filamentous Fungi Reveals Promising Characteristics for Improved Pentose Utilization in Saccharomyces cerevisiae

Abstract

ABSTRACT Limited uptake is one of the bottlenecks for l -arabinose fermentation from lignocellulosic hydrolysates in engineered Saccharomyces cerevisiae . This study characterized two novel l -arabinose transporters, LAT-1 from Neurospora crassa and MtLAT-1 from Myceliophthora thermophila . Although the two proteins share high identity (about 83%), they display different substrate specificities. Sugar transport assays using the S. cerevisiae strain EBY.VW4000 indicated that LAT-1 accepts a broad substrate spectrum. In contrast, MtLAT-1 appeared much more specific for l -arabinose. Determination of the kinetic properties of both transporters revealed that the K m values of LAT-1 and MtLAT-1 for l -arabinose were 58.12 ± 4.06 mM and 29.39 ± 3.60 mM, respectively, with corresponding V max values of 116.7 ± 3.0 mmol/h/g dry cell weight (DCW) and 10.29 ± 0.35 mmol/h/g DCW, respectively. In addition, both transporters were found to use a proton-coupled symport mechanism and showed only partial inhibition by d -glucose during l -arabinose uptake. Moreover, LAT-1 and MtLAT-1 were expressed in the S. cerevisiae strain BSW2AP containing an l -arabinose metabolic pathway. Both recombinant strains exhibited much faster l -arabinose utilization, greater biomass accumulation, and higher ethanol production than the control strain. In conclusion, because of higher maximum velocities and reduced inhibition by d -glucose, the genes for the two characterized transporters are promising targets for improved l -arabinose utilization and fermentation in S. cerevisiae .