Synthetic genome engineering forging new frontiers for wine yeast
Over the past 15 years, the seismic shifts caused by the convergence of biomolecular, chemical, physical, mathematical, and computational sciences alongside cutting-edge developments in information technology and engineering have erupted into a new field of scientific endeavor dubbed Synthetic Biology. Recent rapid advances in high-throughput DNA sequencing and DNA synthesis techniques are enabling the design and construction of new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways), and the redesign of biological systems (cells and organisms) for useful purposes. In 2014, the budding yeast Saccharomyces cerevisiaebecame the first eukaryotic cell to be equipped with a fully functional synthetic chromosome. This was achieved following the synthesis of the first viral (poliovirus in 2002 and bacteriophage Phi-X174 in 2003) and bacterial (Mycoplasma genitalium in 2008 and Mycoplasma mycoides in 2010) genomes, and less than two decades after revealing the full genome sequence of a laboratory (S288c in 1996) and wine (AWRI1631 in 2008) yeast strain. A large international project – the Synthetic Yeast Genome (Sc2.0) Project – is now underway to synthesize all 16 chromosomes (∼12 Mb carrying ∼6000 genes) of the sequenced S288c laboratory strain by 2018. If successful,S. cerevisiae will become the first eukaryote to cross the horizon of in silico design of complex cells through de novo synthesis, reshuffling, and editing of genomes. In the meantime, yeasts are being used as cell factories for the semi-synthetic production of high-value compounds, such as the potent antimalarial artemisinin, and food ingredients, such as resveratrol, vanillin, stevia, nootkatone, and saffron. As a continuum of previously genetically engineered industrially important yeast strains, precision genome engineering is bound to also impact the study and development of wine yeast strains supercharged with synthetic DNA. The first taste of what the future holds is the de novo production of the raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one, in a wine yeast strain (AWRI1631), which was recently achieved via metabolic pathway engineering and synthetic enzyme fusion. A peek over the horizon is revealing that the future of “Wine Yeast 2.0” is already here. Therefore, this article seeks to help prepare the wine industry – an industry rich in history and tradition on the one hand, and innovation on the other – for the inevitable intersection of the ancient art practiced by winemakers and the inventive science of pioneering “synthetic genomicists”. It would be prudent to proactively engage all stakeholders – researchers, industry practitioners, policymakers, regulators, commentators, and consumers – in a meaningful dialog about the potential challenges and opportunities emanating from Synthetic Biology. To capitalize on the new vistas of synthetic yeast genomics, this paper presents wine yeast research in a fresh context, raises important questions and proposes new directions.
Pretorius IS (2016) Critical Reviews in Biotechnology (in press) Published online 18 Aug 2016)
DOI: 10.1080/07388551.2016.1214945
Proteins differentially expressed during limonene biotransformation by Penicillium digitatum DSM 62840 were examined using iTRAQ labeling coupled with 2D-LC–MS/MS
This study focused on the differences in protein expression at various periods during limonene biotransformation by Penicillium digitatum DSM 62840. A total of 3644 protein-species were quantified by iTRAQ during limonene biotransformation (0 and 12 h). A total of 643 proteins were differentially expressed, 316 proteins were significantly up-regulated and 327 proteins were markedly down-regulated. GO, COG, and pathway enrichment analysis showed that the differentially expressed proteins possessed catalytic and binding functions and were involved in a variety of cellular and metabolic process. Furthermore, the enzymes involved in limonene transformation might be related to cytochrome P-450. This study provided a powerful platform for further exploration of biotransformation, and the identified proteins provided insight into the mechanism of limonene transformation. This study focused on the differences in protein expression at various periods during limonene biotransformation by Penicillium digitatum DSM 62840. A total of 3644 protein-species were quantified by iTRAQ during limonene biotransformation (0 and 12 h). A total of 643 proteins were differentially expressed, 316 proteins were significantly up-regulated and 327 proteins were markedly down-regulated. GO, COG, and pathway enrichment analysis showed that the differentially expressed proteins possessed catalytic and binding functions and were involved in a variety of cellular and metabolic process. Furthermore, the enzymes involved in limonene transformation might be related to cytochrome P-450. This study provided a powerful platform for further exploration of biotransformation, and the identified proteins provided insight into the mechanism of limonene transformation.
Zhang LL et al. (2016) Journal of Industrial Microbiology & Biotechnology 43:1481-1495
Impact of D-limonene synthase up- or down-regulation on sweet orange fruit and juice odor perception
Citrus fruits are characterized by a complex mixture of volatiles making up their characteristic aromas, being the D-limonene the most abundant one. However, its role on citrus fruit and juice odor is controversial. Transgenic oranges engineered for alterations in the presence or concentration of few related chemical groups enable asking precise questions about their contribution to overall odor, either positive or negative, as perceived by the human nose. Here, either down- or up-regulation of a D-limonene synthase allowed us to infer that a decrease of as much as 51 times in D-limonene and an increase of as much as 3.2 times in linalool in juice were neutral for odor perception while an increase of only 3 times in ethyl esters stimulated the preference of 66% of the judges. The ability to address these questions presents exciting opportunities to understand the basic principles of selection of food.
Rodriguez A et al. (2016) Food Chemistry 217:139-150
Use of vine-trimming wastes as carrier for Amycolatopsis sp. to produce vanillin, vanillyl Alcohol, and vanillic acid
Raw vine-trimming wastes or the solid residues obtained after different fractionation treatments were evaluated for their suitability as Amycolatopsis sp. immobilization carriers during the bioconversion of ferulic acid into valuable phenolic compounds such as vanillin, vanillyl alcohol, and vanillic acid, the main flavor components of vanilla pods. Previously, physical–chemical characteristics of the materials were determined by quantitative acid hydrolysis and water absorption index (WAI), and microbiological characteristics by calculating the cell retention in the carrier (λ). Additionally, micrographics of carrier surface were obtained by field emission-scanning electron microscopy to study the influence of morphological changes during pretreatments in the adhesion of cells immobilized. The results point out that in spite of showing the lowest WAI and intermediate λ, raw material was the most appropriated substrate to conduct the bioconversion, achieving up to 262.9 mg/L phenolic compounds after 24 h, corresponding to 42.9 mg/L vanillin, 115.6 mg/L vanillyl alcohol, and 104.4 mg/L vanillic acid. The results showed the potential of this process to be applied for biotechnological production of vanillin from ferulic acid solutions; however, further studies must be carried out to increase vanillin yield. Additionally, the liquors obtained after treatment of vine-trimming wastes could be assayed to replace synthetic ferulic acid.
Castañón-Rodríguez JF et al. (2016) Current Microbiology 73 :561-568
Okara (soybean residue) biotransformation by yeast Yarrowia lipolytica
Okara, or soybean residue, is a soy food processing by-product from the manufacture of soymilk and soybean curd (tofu). In this study, solid-state fermentation of okara was conducted over 5 days using yeast Yarrowia lipolytica, and the changes in proximate composition, antioxidant capacity, non-volatiles and volatiles were investigated. Yeast metabolism of okara significantly increased the amounts of lipid, succinate and free amino acids and enhanced the antioxidant capacity. In particular, there was a marked increase in important umami tastants after fermentation, with 3-fold increase in succinate and a 20-fold increase in glutamate. The final fermented okara contained 3.37 g succinate and 335 mg glutamate/100 g dry matter. Aldehydes and their derived acids in the fresh okara were catabolised by Y. lipolytica mainly to methyl ketones, leading to a reduced grassy off-odour and a slightly pungent, musty and cheese-like odour in the fermented okara. Amino acid-derived volatiles, such as 3-methylbutanal and 2-phenylethanol, were also produced. Overall, the okara fermented by Y. lipolytica had a greater amount of umami-tasting substances, a cheese-like odour, improved digestibility and enhanced antioxidant capacity. These changes highlight the potential of Yarrowia-fermented okara as a more nutritious, savoury food product or ingredient. Y. lipolyticawas thus demonstrated to be suitable for the biovalorisation of this soy food processing by-product.
Vong WC et al. (2016) International Journal of Food Microbiology 235:1-9
Impact of solid state fermentation on nutritional, physical and flavor properties of wheat bran
To improve the nutritional, physical and flavor properties of wheat bran, yeast and lactic acid bacteria (LAB) were used for fermenting wheat bran in solid state. Appearance properties, nutritional properties, microstructure, hydration properties and flavor of raw bran and fermented bran were evaluated. After treatments, water extractable arabinoxylans were 3–4 times higher than in raw bran. Total dietary fiber and soluble dietary fiber increased after solid state fermentation. Over 20% of phytic acid was degraded. Microstructure changes and protein degradation were observed in fermented brans. Water holding capacity and water retention capacity of fermented brans were improved. Results suggest that solid state fermentation is an effective way to improve the properties of wheat brans.
Zhao HM et al. (2017) Food Chemistry 217:28-36
Selected dehydrogenases in Yarrowia lipolytica JMY 861: their role in the synthesis of flavor compounds
The presence of selected dehydrogenases, including alcohol dehydrogenase (ADH-YL) and aldehyde dehydrogenase (ALDH-YL), in Yarrowia lipolytica JMY 861, and their potential role in flavor synthesis were investigated. The experimental findings showed that using reduced form of nicotinamide adenine dinucleotide (NADH) as cofactor, the ADH-YL activity in vitro was 6-fold higher than that with reduced form of nicotinamide adenine dinucleotide phosphate (NADPH); however, under the experimental conditions used in this study, an ALDH-YL activity was not detected. The in situ hexanal reduction reaction was found to be instantaneous; however, when the yeast cells suspension was diluted 150 times, the initial relative hexanal concentration was increased by 84.1%. The chromatographic analyses indicated the conversion, in situ, of linoleic acid hydroperoxides (HPODs) into volatile C6-compounds after 60 min of HPODs addition to the yeast cells suspension.
Aziz M et al (2016) Bioscience, Biotechnology, and Biochemistry 80: 2184-2191