Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast

Abstract

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.

Performance and robustness analysis reveals phenotypic trade-offs in yeast

“…Bet-hedging implies sometimes a reduced mean fitness ( Olofsson et al, 2009 ), which might explain the low performance of LBCM1079 and LBCM1095. Analysis of the Ethanol Red phylogeny has revealed its close relation to S288C, Y22-3, wine strains, and sugarcane strains, suggesting that its genetic hybridization might also be responsible for its robustness mechanisms ( Gronchi et al, 2022 ). The second mechanism is cross-protection ( Dhar et al, 2013 ), which arises when cells counteract new perturbations based on previous exposure to the same or different perturbations.…”

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools

“…The engineering strain showed significantly higher starch decomposition activity, which proved its ability as a good enzyme secretor. 156 Apart from that, a combination strategy was adopted to integrate the endogenous pathway genes for friedelin synthesis into the genome and knock out the suppressor genes by the CRISPR/ Cas9 system. As a result, all strains with genetic integration showed that the yield of friedelin increased by 3.0−6.8-fold compared with the original strain BY4741.…”

“…The strain L20 of S. cerevisiae was engineered by CRISPR/Cas9 system for the expression of glucoamylase glaA and α-amylase amyA genes. The engineering strain showed significantly higher starch decomposition activity, which proved its ability as a good enzyme secretor . Apart from that, a combination strategy was adopted to integrate the endogenous pathway genes for friedelin synthesis into the genome and knock out the suppressor genes by the CRISPR/Cas9 system.…”

Mimicked Mixing-Induced Heterogeneities of Industrial Bioreactors Stimulate Long-Lasting Adaption Programs in Ethanol-Producing Yeasts

“…A sequencing output in the form of .fastqsanger files was uploaded on a local Galaxy platform [29] followed by a quality check using FastQC v. 0.72 [30]. Sequence files were subsequently aligned with TopHat v. 2.1.1 [31] against the phylogenetically closely related S. cerevisiae S288C reference genome [32] (GCA 000146045.2-2011), which was accessed from the ENSEMBL database [33]. The overall alignment rate ranged between 83 and 92%.…”