Identification and Functional Characterisation of Novel Aspergillus Niger and Trichoderma Reesei Sugar Transporters

J Sloothaak¹, JAT Tamayo-Ramos¹, DI Odoni¹, T Laothanachareon^1,2, VAP Martins dos Santos¹, PJ Schaap¹

Abstract

Microbial cell factories can be used to replace the petrochemical production of chemicals and fuels. The use of second generation feedstocks as substrate avoids competition with the demand for food and feed. Effective transport of the constituent sugars into the cell factories is considered a bottleneck, so molecular engineering designs must consider sugar porters. The filamentous fungi Aspergillus niger and Trichoderma reesei are able to utilise a large range of sugars found in plant waste biomass, and are among the most important workhorses of today’s industrial biotechnology. Both fungi have over 200 predicted sugar porter proteins, but the characteristics of these remain largely unknown. We aim to identify and characterise the most important transporter proteins for the uptake of the constituent sugars in second generation feedstocks.

We used a combination of in silico and in vivo approaches to select likely transporter candidates. Hidden Markov models, trained with previously characterised transporters, were used to predict transporter function in the A. niger and T. reesei proteomes in silico. Quantitative membrane proteomics, performed on Aspergillus niger growing on various carbon sources, was used to obtain abundance profiles of putative sugar transporters in vivo. Likely transporter candidates were selected and expressed in a suitable S. cerevisiae host to study the function ex vivo. Plate and liquid growth profiles were obtained and uptake characteristics were determined using radioactively labeled sugars.

Several novel Aspergillus niger and Trichoderma reesei sugar porters of the main constituents of second generation feedstocks were characterized. These transporters can be applied in the design of improved and novel cell factories for the sustainable production of biochemicals and biofuels. 

Affiliations

¹ Laboratory of Systems and Synthetic Biology, Wageningen University, Stippeneng 4, 6708WE, Wageningen, The Netherlands

² Enzyme Technology Laboratory and Integrative Biorefinery Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Science Park, 113 Pahonyothin Road, Pathumthani 12120, Thailand