Physiological characterization and transcriptome analysis of Pichia pastoris reveals its response to lignocellulose-derived inhibitors.

The negative effects of lignocellulose-derived inhibitors such as acetic acid and furaldehydes on microbial metabolism constitute a significant drawback to the usage of biomass feedstocks for the production of fuels and chemicals. The yeast Pichia pastoris has shown a great biotechnological potential for producing heterologous proteins and renewable chemicals. Despite its relevance, the performance of P. pastoris in presence of lignocellulose-derived inhibitors remains unclear. In this work, our results show for the first time the dose-dependent response of P. pastoris to acetic acid, furaldehydes (HMF and furfural), and sugarcane biomass hydrolysate, both at physiological and transcriptional levels. The yeast was able to grow in synthetic media with up to 6 g.L-1 acetic acid, 1.75 g.L-1 furaldehydes or hydrolysate diluted to 10% (v/v). However, its metabolism was completely hindered in presence of hydrolysate diluted to 30% (v/v). Additionally, the yeast was capable to co-consume acetic acid and glucose. At the transcriptional level, P. pastoris response to lignocellulose-derived inhibitors relays on the up-regulation of genes related to transmembrane transport, oxidoreductase activities, RNA processing, and the repression of pathways related to biosynthetic processes and central carbon metabolism. These results demonstrate a polygenetic response that involves detoxification activities, and maintenance of energy and cellular homeostasis. In this context, ALD4, OYE3, QOR2, NTL100, YCT1, and PPR1 were identified as target genes to improve P. pastoris tolerance. Altogether, this work provides valuable insights into the P. pastoris stress tolerance, which can be useful to expand its use in different bioprocesses.

Fast spectroscopic monitoring of inhibitors in the 2G ethanol process.

One of the main challenges of second generation (2G) ethanol production is the high quantities of phenolic compounds and furan derivatives generated in the pretreatment of the lignocellulosic biomass, which inhibit the enzymatic hydrolysis and fermentation steps. Fast monitoring of these inhibitory compounds could provide better control of the pretreatment, hydrolysis, and fermentation processes by enabling the implementation of strategic process control actions. We investigated the feasibility of monitoring these inhibitory compounds by ultraviolet-visible (UV-Vis) spectroscopy associated with partial least squares (PLS) regression. Hydroxymethylfurfural, furfural, vanillin, and ferulic and p-coumaric acids generated during different severities of liquid hot water pretreatment of sugarcane bagasse were quantified with highly accuracy. In cross-validation (leave-one-out), the PLS-UV-Vis method presented root mean square error of prediction (RMSECV) of around only 5.0%. The results demonstrated that the monitoring performance achieved with PLS-UV-Vis could support future studies of optimization and control protocols for application in industrial processes.