Beginning the Mission Work in Alaska by the Presbyterian Church

"The Presbyterian Church is indebted to the late Rev. A. L. Lindsley, D. D., pastor of the First Prebyterian Church, Portland, Oregon, for eighteen years, for opening mission work in Alaska.

Bioflavoring by non-conventional yeasts in sequential beer fermentations

Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S.\ua0cerevisiae to complete the fermentation. For measurement of phenolic compoundsand their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P.\ua0kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomycesspecies and enhancing 4-vinyl guaiacol (clove-like aroma) with T.\ua0delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S.\ua0cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species

Intragenic repeat expansion in the cell wall protein gene HPF1 controls yeast chronological aging

Aging varies among individuals due to both genetics and environment, but the underlying molecular mechanisms remain largely unknown. Using a highly recombined Saccharomyces cerevisiae population, we found 30 distinct quantitative trait loci (QTLs) that control chronological life span (CLS) in calorie-rich and calorie-restricted environments and under rapamycin exposure. Calorie restriction and rapamycin extended life span in virtually all genotypes but through different genetic variants. We tracked the two major QTLs to the cell wall glycoprotein genes FLO11 and HPF1. We found that massive expansion of intragenic tandem repeats within the N-terminal domain of HPF1 was sufficient to cause pronounced life span shortening. Life span impairment by HPF1 was buffered by rapamycin but not by calorie restriction. The HPF1 repeat expansion shifted yeast cells from a sedentary to a buoyant state, thereby increasing their exposure to surrounding oxygen. The higher oxygenation altered methionine, lipid, and purine metabolism, and inhibited quiescence, which explains the life span shortening. We conclude that fast-evolving intragenic repeat expansions can fundamentally change the relationship between cells and their environment with profound effects on cellular lifestyle and longevity

Identification of novel alleles conferring superior production of rose flavor phenylethyl acetate using polygenic analysis in yeast.

Flavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side activities of enzymes having their main function in other areas of metabolism. We have applied pooled-segregant whole-genome sequence analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcoholic beverages imparting rose- and honey-like aromas, with production of high 2-PEAc levels considered a superior trait. Four quantitative trait loci (QTLs) responsible for high 2-PEAc production were identified, with two loci each showing linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation. Exchange of both superior alleles in the ER18 parent strain increased 2-PEAc production 70%, nearly to the same level as in the best superior segregant. Our results show that polygenic analysis combined with CRISPR/ Cas9-mediated allele exchange is a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages

Molecular basis for ethyl acetate production and sulfonate transport in the yeast Saccharomyces cerevisiae

Flavor production is an essential property of brewer’s yeast and a driving force for consumer’s choice. Here, we have focused on identification of superior mutations responsible for low ethyl acetate in beer production. Ethyl acetate is a commonly used organic solvent that gives an undesirable solvent-like off-flavor in beer brewing. We have isolated a superior haploid strain (with a low ethyl acetate production) and an inferior haploid strain (with a high ethyl acetate production), which were used in pooled segregant whole genome sequence analysis experiments to identify the genomic areas linked to ethyl acetate production in Saccharomyces cerevisiae. In two strong quantitative trait loci (QTLs), appearing in both low and high ethyl acetate pools, we have identified the causative mutations responsible for 72% of its production in strains without ATF1. Overexpression of one of the causative genes, encoding for a putative mitochondrial enzyme, led to an increase with 85 mg/l ethyl acetate, without affecting other aroma compounds. Surprisingly, engineering of the mutations in the causative genes led to an increase in ethyl acetate in the presence of an active ATF1 gene. It was due to an increase in the ATF1-derived AATase activity, as isoamyl acetate, which is formed by the ATF1 gene, was also significantly increased. Isoamyl acetate is a ‘banana’ like ester, that provides an essential fruitiness in beer. Future industrial valorization will therefore identify whether reduction in the ethyl acetate levels or an increase in the isoamyl acetate production can be obtained by engineering of the mutations into commercial brewing strains. In addition to investigating engineering of low ethyl acetate production in S. cerevisiae, we have explored the potential usage of non-conventional yeast species to enhance the flavor enhancement in beer production. We first developed a novel methodology to facilitate quantification of phenolic compounds (4-vinyl guaiacol, 4-ethylguaiacol, and 4-ethylphenol) and their hydroxycinnamic acid precursors (trans-ferulic and p-coumaric acid), using HPLC coupled with simultaneous fluorescence and UV detection. This HPLC-UV/fluorescence method was used together with gas chromatography in screening for interesting flavor metabolite profiles (esters, alcohols, and phenolic compounds) in a collection of 17 different non-Saccharomyces species. All the Pichia kluyverii strains produced a high level of isoamyl acetate of 10 mg/L and above after 48 hours of fermentation, while the three Brettanomyces strains were unique for production of the ethyl phenolic compounds 4-ethylguaiacol and 4-ethylphenol. Seven strains with potentially desirable flavor profiles were chosen for validation in sequential fermentations with inoculation of conventional brewing yeast to complete the alcoholic fermentation. The results show that the flavor production by non-conventional yeasts in sequential fermentations with S. cerevisiae is not additive, but appears to occur in a species/strain dependent manner. In conclusion, we have taken the first steps towards development of brewing protocol with bioflavoring of beer by non-conventional yeast species. The second major research line explored in this PhD, is based on the previous identification of the transceptor function of Sul1 and Sul2 in the MCB laboratory. These experiments had shown that the protein kinase A (PKA) nutrient sensing pathway was still activated by low levels of sulfite in a sul1Dsul2D strain, highlighting the presence of yet unidentified transporters or transceptors in the S. cerevisiae genome. In this thesis, we first assayed triple, quadruple, and quintuple deletion strains of Sul1 and Sul2 plus two homologs of SUL1 and SUL2 (namely YGR125W, YPR003C) and the putative MFS transporter gene SOA1 (YIL166C), which is highly upregulated under sulfur starvation, for growth with sulfite and high sulfate. The additional deletion of YIL166C in the sul1Dsul2Dyil166cD triple deletion strain was auxotrophic for sulfur-containing amino acids, even in the presence of high levels of sulfate (40 mM). We thus confirmed Yil166c to be the last remaining transporter of inorganic sulfur compounds. Single deletion of the Yil166c transporter led to lack of growth on range of organic sulfur compounds, including sulfonate compounds taurine and isethionate which are found in ecological niches were yeasts and fungi are frequently isolated. The transporter was therefore named Soa1 for Sulfonate transporter 1. The Soa1 gene product is remarkably conserved with transporter orthologs found in the three fungal domains and with multiple paralogs found in many fungi. S. uvarum, S. arboricola, and S. eubayanus also contained a paralog, Soa2, which complemented Soa1 for sulfonate transport. In conclusion, we have identified a novel family of inorganic sulfur, sulfonate, and sulfate ester transporters that previously had remained elusive.nrpages: 209status: publishe

Bioflavoring by non-conventional yeasts in sequential beer fermentations

Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S. cerevisiae to complete the fermentation. For measurement of phenolic compounds and their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P. kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomyces species and enhancing 4-vinyl guaiacol (clove-like aroma) with T. delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S. cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species.status: publishe