Discovery of ER-localized sugar transporters for cellulase production with lac1 being essential

Abstract

Abstract Background In the process of cellulose hydrolysis, carbohydrate hydrolysates are transported into cells through membrane transporters, and then affect the expression of cellulase-encoding genes. Sugar transporters play a crucial role in cellulase production in lignocellulolytic fungi, of which relatively few have been functionally validated to date and are all reported to be on cell membrane. Result Through transcriptome analysis and qRT-PCR, three putative MFS sugar transporters GST, MFS, and LAC1 were found to display significantly higher mRNA levels in T. reesei grown on cellulose than on glucose. The individual deletion of these three genes compromised cellulase production and delayed sugar absorption by 24 h in T. reesei. Nevertheless, they transported pretty low level of sugars, including galactose, lactose, and mannose, and did not transport glucose, when expressed in yeast system. Meanwhile, all three transporters were unexpectedly found to be intracellular, being located in endoplasmic reticulum (ER). Particularly, the knockout of lac1 almost abolished cellulase production, and significantly inhibited biomass generation regardless of sugar types, indicating that lac1 is essential for cellulase production and biomass formation. The absence of lac1 upregulated genes involved in ribosome biogenesis, while downregulated genes in cellulase production, protein processing in ER (particularly protein glycosylation), and lipid biosynthesis. The inhibition of lac1 deletion on the transcriptional levels of genes related to cellulase biosynthesis was restored after 72 h, but the cellulase production was still inhibited, indicating lac1 might pose a post-transcription regulation on cellulase production that are independent on the known cellulase regulation mediated by CRT1 and XYR1. Conclusion For the first time, intracellular sugar transporters (mfs, gst, and lac1) facilitating cellulase production were identified, which was distributed in ER. Their sugar transporting ability was very weak, indicating that they might be related to sugar utilization inside cells rather than the cellular sugar uptake. More importantly, sugar transporter lac1 is first found to be essential for cellulase production and biomass formation by affecting protein processing in ER (particularly protein glycosylation) and lipid biosynthesis. The effect of LAC1 on cellulase production seems to be post-transcriptional at late stage of cellulase production, independent on the well-known cellulase regulation mediated by CRT1 and XYR1. These findings improve the understanding of intracellular sugar transporters in fungi and their important role in cellulase synthesis.

Keywords

• Filamentous fungi
• Trichoderma reesei
• Cellulase
• Nucleotide sugar transporter
• Endoplasmic reticulum