Application of whole-genome transformation for enhancing thermotolerance in yeast

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.status: publishe

Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks.status: publishe

In-situ muconic acid extraction reveals sugar consumption bottleneck in a xylose-utilizing Saccharomyces cerevisiae strain

Abstract Background The current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification. Results We have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced. Conclusions This work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization

Diagnostic Allele-Specific PCR for the Identification of Candida auris Clades

Candida auris is an opportunistic pathogenic yeast that emerged worldwide during the past decade. This fungal pathogen poses a significant public health threat due to common multidrug resistance (MDR), alarming hospital outbreaks, and frequent misidentification. Genomic analyses have identified five distinct clades that are linked to five geographic areas of origin and characterized by differences in several phenotypic traits such as virulence and drug resistance. Typing of C. auris strains and the identification of clades can be a powerful tool in molecular epidemiology and might be of clinical importance by estimating outbreak and MDR potential. As C. auris has caused global outbreaks, including in low-income countries, typing C. auris strains quickly and inexpensively is highly valuable. We report five allele-specific polymerase chain reaction (AS-PCR) assays for the identification of C. auris and each of the five described clades of C. auris based on conserved mutations in the internal transcribed spacer (ITS) rDNA region and a clade-specific gene cluster. This PCR method provides a fast, cheap, sequencing-free diagnostic tool for the identification of C. auris, C. auris clades, and potentially, the discovery of new clades