Consolidated Bioprocess for Bioethanol Production from Raw Flour of Brosimum alicastrum Seeds Using the Native Strain of Trametes hirsuta Bm-2

Consolidated bioprocessing (CBP), which integrates biological pretreatment, enzyme production, saccharification, and fermentation, is a promising operational strategy for cost-effective ethanol production from biomass. In this study, the use of a native strain of Trametes hirsuta (Bm-2) was evaluated for bioethanol production from Brosimum alicastrum in a CBP. The raw seed flour obtained from the ramon tree contained 61% of starch, indicating its potential as a raw material for bioethanol production. Quantitative assays revealed that the Bm-2 strain produced the amylase enzyme with activity of 193.85 U/mL. The Bm-2 strain showed high tolerance to ethanol stress and was capable of directly producing ethanol from raw flour at a concentration of 13 g/L, with a production yield of 123.4 mL/kg flour. This study demonstrates the potential of T. hirsuta Bm-2 for starch-based ethanol production in a consolidated bioprocess to be implemented in the biofuel industry. The residual biomass after fermentation showed an average protein content of 22.5%, suggesting that it could also be considered as a valuable biorefinery co-product for animal feeding.

Overexpression of endogenous stress-tolerance related genes in Saccharomyces cerevisiae improved strain robustness and production of heterologous cellobiohydrolase

ABSTRACT To enable Saccharomyces cerevisiae to produce renewable fuels from lignocellulose in a consolidated bioprocess, a heterologous cellulase system must be engineered into this yeast. In addition, inherently low secretion titers and sensitivity to adverse environmental conditions must be overcome. Here, two native S. cerevisiae genes related to yeast stress tolerance, YHB1 and SET5, were overexpressed under transcriptional control of the constitutive PGK1 promoter and their effects on heterologous secretion of Talaromyces emersonii cel7A cellobiohydrolase was investigated. Transformants showed increased secreted enzyme activity that ranged from 22% to 55% higher compared to the parental strains and this did not lead to deleterious growth effects. The recombinant strains overexpressing either YHB1 or SET5 also demonstrated multi-tolerant characteristics desirable in bioethanol production, i.e. improved tolerance to osmotic and heat stress. Quantitative reverse transcriptase PCR analysis in these strains showed decreased transcription of secretion pathway genes. However, decreased unfolded protein response was also observed, suggesting novel mechanisms for enhancing enzyme production through stress modulation. Overexpression of YHB1 in an unrelated diploid strain also enhanced stress tolerance and improved ethanol productivity in medium containing acetic acid. To our knowledge, this is the first demonstration that improved heterologous secretion and environmental stress tolerance could be engineered into yeast simultaneously.