Author:

Abstract:

The global awareness regarding the environmental impact of fossil fuel-derived products requires fuel alternatives for the energy and automotive industry. Bioethanol has been proposed as a gasoline alternative and historically has been produced from sugar cane, corn and wheat. However, because these are food crops, alternative materials in the form of lignocellulose waste products and high energy grasses have been proposed. Second-generation bioethanol production using these waste materials is currently non-competitive with fossil fuel-derived ethanol/gasoline due to its high processing costs. In large, this is the result of the different structure of this so-called lignocellulosic biomass that requires expensive enzyme cocktails to liberate free sugars from sugar polymers in plant biomass necessary for microbial fermentation. Consolidated bio-processing (CBP), which is currently projected to be the most cost-optimal production set-up, requires a micro-organism that secretes lignocellulolytic enzymes and ferments the released sugars into the desired end product. Saccharomyces cerevisiae, commonly known as bakers' yeast, is a preferred micro-organism for industrial ethanol production due to its high fermentation rate, high inhibitor tolerance (particularly ethanol) and ethanol production rate. However, in a CBP approach, the discrepancy between the optimal hydrolysis temperature of lignocellulolytic enzymes (45°C-60°C) and the optimum fermentation temperature of most S. cerevisiae strains (32°C-35°) causes an undesirable trade-off leading to lower enzyme activity and thus higher enzyme loads resulting in a higher final bioethanol cost. The use of yeast strains that are able to ferment at temperatures of 42°C and higher are expected to reduce both capital and operational expenditures, resulting in a lower final ethanol cost. Most yeast strain improvement methods are time-consuming and identification of causative (genetic) factors is often difficult. We implemented a technique called "whole-genome transformation" (WGT) to both rapidly acquire superior strains and identify causative elements. In WGT the genomic DNA of a tolerant species is isolated and transformed into the host strain. The DNA of the thermotolerant yeast species Kluyveromyces marxianus and Ogataea (Hansenula) polymorpha, which are able to grow and ferment at 45°C and higher, was transformed into an industrial, haploid S. cerevisiae strain ER18A HPH. Screening of the transformants on solid nutrient plates at non-permissive temperatures allowed the selection of transformants of which many outperformed ER18A HPH in fermentations at 42°C. To identify the causative genetic changes in the transformants, we submitted three transformants (KEA17, KEA24 and OEA28) together with the parent to whole-genome sequence analysis. This revealed the presence of a surprisingly low number of putative causative variants in the transformants when the sequence was compared with that of ER18A HPH. Using the CRISPR/Cas9 technology we showed that an anticodon mutation in KEA17 and KEA24 in a lysine and methionine tRNA, respectively, and an anticodon stem mutation in a threonine tRNA in OEA28 were the causative mutations. An in-depth investigation of why these tRNA mutations were causative for improved high-temperature fermentation revealed the crucial role of TRT2, an essential gene encoding tRNAThrCGU. In ER18A HPH, TRT2 contains an anticodon stem loop mutation resulting in loss of base pairing, likely destabilizing the tRNA at high temperature. OEA28 acquired a complementary mutation in TRT2, restoring its tRNA's thermostability. Instead of Trt2 stabilization in KEA17 and KEA24, the anticodon of other tRNAs (tK(CUU)K and EMT2) was altered to that of TRT2, providing an alternative source of tRNAThrCGU. We demonstrated that these new tRNA variants are functional alternatives for Trt2 and provided evidence with evolutionary sequence trees that such anticodon-switching events have apparently occurred regularly during evolution. Since this was the first time that causative elements were identified after WGT of eukaryotic species, we wanted to compare this result with that of an existing identification method. Pooled-segregant whole-genome sequence analysis has been used frequently to map quantitative trait loci (QTLs) and identify causative variants in the parent strain(s). Surprisingly, QTLs identified by pooled-segregant whole-genome sequence analysis with OEA28 as one of the parents did not overlap with mutations identified after comparative analysis of the genome sequences of OEA28 and ER18A HPH. Instead, six independent QTLs linked to OEA28 and two linked to BTC.1D, the second parent, were identified. In QTL1 on chromosome XV, the IRA2 allele of BTC.1D has been linked to superior thermotolerance. The OEA28 allele contained an 8bp frameshift resulting in a non-functional Ira2 protein. Allele switching in both parents using the CRISPR/Cas9 technology indicated that the functioning of this gene had an impact on general fermentation performance rather than on high-temperature fermentation specifically and, in addition, negatively affected propagation. Furthermore, we identified an intergenic region in QTL2 on chromosome VII linked to BTC.1D. The presence of several transcription factor binding sites regulates the expression of VRG4 and OST5, two genes involved in protein mannosylation and glycosylation. Lower expression of both genes resulted in an improved fermentation rate and ethanol yield at 42°C. This is one of the first reported cases where the causative element in an intergenic region has been identified using pooled-segregant whole-genome sequence analysis. Identification of causative elements in other QTLs could help further understand the polygenic nature of thermotolerance. We believe that further use and development of WGT could lead to rapid improvements of industrial S. cerevisiae strains, e.g. for bio-based chemicals production. The combination of straightforward generation of superior strains and the subsequent rapid identification of causative elements has been demonstrated for thermotolerance in this work. The construction of highly thermotolerant S. cerevisiae strains that ferment efficiently at temperatures similar to those of lignocellulolytic enzymes in a CBP approach will result in cost-efficient production of chemicals and support an accelerated shift towards a completely bio-based economy.