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PubMed2025

Genome-based exploration of volatile flavor diversity from food yeast species.

Yeast shares a longer than 10 000-year history with humans in food fermentation by producing various volatile flavor compounds that contribute to the final taste and aroma of foods. Yeast-associated volatile flavor compounds include esters, benzenoids, sulfur compounds, and phenolic derivatives, which enhance the sensory complexity of fermented foods and beverages. Genome-scale technologies have advanced and transformed our understanding of the genetic and evolutionary drivers of volatile flavor diversity. The conventional approach to aroma enrichment and flavor balancing through single-strain optimization has been redefined through yeast cofermentation strategies, such as the pairing of Saccharomyces cerevisiae with nonconventional yeast species. This minireview summarizes the latest genomic insights into volatile flavor compound formation through ester, benzenoid, sulfur, and phenolic pathways in various yeast species and highlights the shaping of the next generation of food fermentation innovation via cofermentation combined with omics analysis, followed by a future perspective on synthetic biology for industrial applicability.

Volatile Organic Compounds↗

PubMed1999

Successful design and development of genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol.

Ethanol is an effective, environmentally friendly, nonfossil, transportation biofuel that produces far less pollution than gasoline. Furthermore, ethanol can be produced from plentiful, domestically available, renewable, cellulosic biomass. However, cellulosic biomass contains two major sugars, glucose and xylose, and a major obstacle in this process is that Saccharomyces yeasts, traditionally used and still the only microorganisms currently used for large scale industrial production of ethanol from glucose, are unable to ferment xylose to ethanol. This makes the use of these safest, most effective Saccharomyces yeasts for conversion of biomass to ethanol economically unfeasible. Since 1980, scientists worldwide have actively been trying to develop genetically engineered Saccharomyces yeasts to ferment xylose. In 1993, we achieved a historic breakthrough to succeed in the development of the first genetically engineered Saccharomyces yeasts that can effectively ferment both glucose and xylose to ethanol. This was accomplished by carefully redesigning the yeast metabolic pathway for fermenting xylose to ethanol, including cloning three xylose-metabolizing genes, modifying the genetic systems controlling gene expression, changing the dynamics of the carbon flow, etc. As a result, our recombinant yeasts not only can effectively ferment both glucose and xylose to ethanol when these sugars are present separately in the medium, but also can effectively coferment both glucose and xylose present in the same medium simultaneously to ethanol. This has made it possible because we have genetically engineered the Saccharomyces yeasts as such that they are able to overcome some of the natural barrier present in all microorganisms, such as the synthesis of the xylose metabolizing enzymes not to be affected by the presence of glucose and by the absence of xylose in the medium. This first generation of genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts relies on the presence of a high-copy-number 2 mu-based plasmid that contains the three cloned genetically modified xylose-metabolizing genes to provide the xylose-metabolizing capability. In 1995, we achieved another breakthrough by creating the super-stable genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts which contain multiple copies of the same three xylose-metabolizing genes stably integrated on the yeast chromosome. This is another critical development which has made it possible for the genetically engineered yeasts to be effective for cofermenting glucose and xylose by continuous fermentation. It is widely believed that the successful development of the stable glucose-xylose-cofermenting Saccharomyces yeasts has made the biomass-to-ethanol technology a step much closer to commercialization. In this paper, we present an overview of our rationales and strategies as well as our methods and approaches that led to the ingenious design and successful development of our genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose to biofuel ethanol.

Biomass↗

PubMed1998

Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose.

Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However, Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transform Saccharomyces spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2 microns-based high-copy-number yeast-E. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae). These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.

Escherichia coli↗

PubMed2000

Continuous fermentation studies with xylos-utilizing recombinant Zymomonas mobilis.

This study examined the continuous cofermentation performance characteristics of a dilute-acid "prehydrolysate-adapted" recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the cofermentation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5 cents/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (Dmax), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted Dmax from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), Dmax was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the Dmax was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the Dmax for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L.h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell.h).

Biotechnology↗

PubMed1998

Improving fermentation performance of recombinant Zymomonas in acetic acid-containing media.

In the production of ethanol from lignocellulosic biomass, the hydrolysis of the acetylated pentosans in hemicellulose during pretreatment produces acetic acid in the prehydrolysate. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process that uses a proprietary metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose. Acetic acid toxicity represents a major limitation to bioconversion, and cost-effective means of reducing the inhibitory effects of acetic acid represent an opportunity for significant increased productivity and reduced cost of producing fermentation fuel ethanol from biomass. In this study, the fermentation performance of recombinant Z. mobilis 39676:pZB4L, using a synthetic hardwood prehydrolysate containing 1% (w/v) yeast extract, 0.2% KH2PO4, 4% (w/v) xylose, and 0.8% (w/v) glucose, with varying amounts of acetic acid was examine. To minimize the concentration of the inhibitory undissociated form of acetic acid, the pH was controlled at 6.0. The final cell mass concentration decreased linearly with increasing level of acetic acid over the range 0-0.75% (w/v), with a 50% reduction at about 0.5% (w/v) acetic acid. The conversion efficiency was relatively unaffected, decreasing from 98 to 92%. In the absence of acetic acid, batch fermentations were complete at 24 h. In a batch fermentation with 0.75% (w/v) acetic acid, about two-thirds of the xylose was not metabolized after 48 h. In batch fermentations with 0.75% (w/v) acetic acid, increasing the initial glucose concentration did not have an enhancing effect on the rate of xylose fermentation. However, nearly complete xylose fermentation was achieved in 48h when the bioreactor was fed glucose. In the fed-batch system, the rate of glucose feeding (0.5 g/h) was designed to simulate the rate of cellulolytic digestion that had been observed in a modeled SSCF process with recombinant Zymomonas. In the absence of acetic acid, this rate of glucose feeding did not inhibit xylose utilization. It is concluded that the inhibitory effect of acetic acid on xylose utilization in the SSCF biomass-to-ethanol process will be partially ameliorated because of the simultaneous saccharification of the cellulose.

Acetic Acid↗