Functional analysis of two L-arabinose transporters from filamentous fungi reveals promising characteristics for improved pentose utilization in Saccharomyces cerevisiae

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±4.06mMand 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.