Killer yeasts: expanding frontiers in the age of synthetic biology.

Killer yeasts secrete protein toxins that are selectively lethal to other yeast and filamentous fungi. These exhibit exceptional genetic and functional diversity, and have several biotechnological applications. However, despite decades of research, several limitations hinder their widespread adoption. In this perspective we contend that technical advances in synthetic biology present an unprecedented opportunity to unlock the full potential of yeast killer systems across a spectrum of applications. By leveraging these new technologies, engineered killer toxins may emerge as a pivotal new tool to address antifungal resistance and food security. Finally, we speculate on the biotechnological potential of re-engineering host double-stranded (ds) RNA mycoviruses, from which many toxins derive, as a safe and noninfectious system to produce designer RNA.

Engineering biology and climate change mitigation: Policy considerations.

Engineering biology (EngBio) is a dynamic field that uses gene editing, synthesis, assembly, and engineering to design new or modified biological systems. EngBio applications could make a significant contribution to achieving net zero greenhouse gas emissions. Yet, policy support will be needed if EngBio is to fulfil its climate mitigation potential. What form should such policies take, and what EngBio applications should they target? This paper reviews EngBio's potential climate contributions to assist policymakers shape regulations and target resources and, in so doing, to facilitate democratic deliberation on desirable futures.

Exploring future applications of the apiculate yeast Hanseniaspora.

As a metaphor, lemons get a bad rap; however the proverb 'if life gives you lemons, make lemonade' is often used in a motivational context. The same could be said of Hanseniaspora in winemaking. Despite its predominance in vineyards and grape must, this lemon-shaped yeast is underappreciated in terms of its contribution to the overall sensory profile of fine wine. Species belonging to this apiculate yeast are known for being common isolates not just on grape berries, but on many other fruits. They play a critical role in the early stages of a fermentation and can influence the quality of the final product. Their deliberate addition within mixed-culture fermentations shows promise in adding to the complexity of a wine and thus provide sensorial benefits. Hanseniaspora species are also key participants in the fermentations of a variety of other foodstuffs ranging from chocolate to apple cider. Outside of their role in fermentation, Hanseniaspora species have attractive biotechnological possibilities as revealed through studies on biocontrol potential, use as a whole-cell biocatalyst and important interactions with Drosophila flies. The growing amount of 'omics data on Hanseniaspora is revealing interesting features of the genus that sets it apart from the other Ascomycetes. This review collates the fields of research conducted on this apiculate yeast genus.

A spotlight on global collaboration in the Sc2.0 yeast consortium.

The Synthetic Yeast Genome Project (Sc2.0) is an international collaboration that aims to create and optimize synthetic versions of each Saccharomyces cerevisiae chromosome, with the ultimate goal of assembling a yeast organism with a synthetic with design features facilitating applications in synthetic biology and engineering projects. The consortium research groups are global, and, here, we highlight the work of the China-based Sc2.0 researchers and their thoughts on the future of Sc2.0 and synthetic biology.

Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects.

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Visioning synthetic futures for yeast research within the context of current global techno-political trends.

Yeast research is entering into a new period of scholarship, with new scientific tools, new questions to ask and new issues to consider. The politics of emerging and critical technology can no longer be separated from the pursuit of basic science in fields, such as synthetic biology and engineering biology. Given the intensifying race for technological leadership, yeast research is likely to attract significant investment from government, and that it offers huge opportunities to the curious minded from a basic research standpoint. This article provides an overview of new directions in yeast research with a focus on Saccharomyces cerevisiae, and places these trends in their geopolitical context. At the highest level, yeast research is situated within the ongoing convergence of the life sciences with the information sciences. This convergent effect is most strongly pronounced in areas of AI-enabled tools for the life sciences, and the creation of synthetic genomes, minimal genomes, pan-genomes, neochromosomes and metagenomes using computer-assisted design tools and methodologies. Synthetic yeast futures encompass basic and applied science questions that will be of intense interest to government and nongovernment funding sources. It is essential for the yeast research community to map and understand the context of their research to ensure their collaborations turn global challenges into research opportunities.

PCR-based gene targeting in Hanseniaspora uvarum.

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

The Influence of Pichia kluyveri Addition on the Aroma Profile of a Kombucha Tea Fermentation.

Traditional kombucha is a functional tea-based drink that has gained attention as a low or non-alcoholic beverage. The fermentation is conducted by a community of different microorganisms, collectively called SCOBY (Symbiotic Culture of Bacteria and Yeast) and typically consists of different acetic acid bacteria and fermenting yeast, and in some cases lactic acid bacteria that would convert the sugars into organic acids-mostly acetic acid. In this study, the effect of including a Pichia kluyveri starter culture in a kombucha fermentation was investigated. P. kluyveri additions led to a quicker accumulation of acetic acid along with the production of several acetate esters including isoamyl acetate and 2-phenethyl acetate. A subsequent tasting also noted a significant increase in the fruitiness of the kombucha. The significant contribution to the aroma content shows the promise of this yeast in future microbial formulations for kombucha fermentations.

Trimming the genomic fat: minimising and re-functionalising genomes using synthetic biology.

Naturally evolved organisms typically have large genomes that enable their survival and growth under various conditions. However, the complexity of genomes often precludes our complete understanding of them, and limits the success of biotechnological designs. In contrast, minimal genomes have reduced complexity and therefore improved engineerability, increased biosynthetic capacity through the removal of unnecessary genetic elements, and less recalcitrance to complete characterisation. Here, we review the past and current genome minimisation and re-functionalisation efforts, with an emphasis on the latest advances facilitated by synthetic genomics, and provide a critical appraisal of their potential for industrial applications.

Engineering a fermenting yeast able to produce the fragrant ß-ionone apocarotenoid for enhanced aroma properties in wine.

Wine is composed of multitudinous flavour components and volatile organic compounds that provide this beverage with its attractive properties of taste and aroma. The perceived quality of a wine can be attributed to the absolute and relative concentrations of favourable aroma compounds; hence, increasing the detectable levels of an attractive aroma, such as ß-ionone with its violet and berry notes, can improve the organoleptic qualities of given wine styles. We here describe the generation of a new grape-must fermenting strain of Saccharomyces cerevisiae that is capable of releasing ß-ionone through the heterologous expression of both the enzyme carotenoid cleavage dioxygenase 1 (CCD1) and its substrate, ß-carotene. Haploid laboratory strains of S. cerevisiae were constructed with and without integrated carotenogenic genes and transformed with a plasmid containing the genes of CCD1. These strains were then mated with a sporulated diploid wine industry yeast, VIN13, and four resultant crosses-designated MQ01-MQ04-which were capable of fermenting the must to dryness were compared for their ability to release ß-ionone. Analyses of their fermentation products showed that the MQ01 strain produced a high level of ß-ionone and offers a fermenting hybrid yeast with the potential to enhance the organoleptic qualities of wine.

Harnessing bioengineered microbes as a versatile platform for space nutrition.

Human enterprises through the solar system will entail long-duration voyages and habitation creating challenges in maintaining healthy diets. We discuss consolidating multiple sensory and nutritional attributes into microorganisms to develop customizable food production systems with minimal inputs, physical footprint, and waste. We envisage that a yeast collection bioengineered for one-carbon metabolism, optimal nutrition, and diverse textures, tastes, aromas, and colors could serve as a flexible food-production platform. Beyond its potential for supporting humans in space, bioengineered microbial-based food could lead to a new paradigm for Earth's food manufacturing that provides greater self-sufficiency and removes pressure from natural ecosystems.

Construction of a synthetic Saccharomyces cerevisiae pan-genome neo-chromosome.

The Synthetic Yeast Genome Project (Sc2.0) represents the first foray into eukaryotic genome engineering and a framework for designing and building the next generation of industrial microbes. However, the laboratory strain S288c used lacks many of the genes that provide phenotypic diversity to industrial and environmental isolates. To address this shortcoming, we have designed and constructed a neo-chromosome that contains many of these diverse pan-genomic elements and which is compatible with the Sc2.0 design and test framework. The presence of this neo-chromosome provides phenotypic plasticity to the Sc2.0 parent strain, including expanding the range of utilizable carbon sources. We also demonstrate that the induction of programmable structural variation (SCRaMbLE) provides genetic diversity on which further adaptive gains could be selected. The presence of this neo-chromosome within the Sc2.0 backbone may therefore provide the means to adapt synthetic strains to a wider variety of environments, a process which will be vital to transitioning Sc2.0 from the laboratory into industrial applications.

The film-forming Pichia spp. in a winemaker's toolbox: A simple isolation procedure and their performance in a mixed-culture fermentation of Vitis vinifera L. cv. Gewürztraminer must.

Certain yeast species belonging to the Pichia genus are known to form a distinctive film on grape must and wine. In a mixed-culture type fermentation, Pichia spp. (P. kluyveri in particular) are known to impart beneficial oenological attributes. In this study, we report on an easy isolation method of Pichia spp. from grape must by exploiting their film-forming capacity on media containing 10% ethanol. We isolated and identified two Pichia species, namely Pichia kudriavzevii and Pichia kluyveri, and subsequently co-inoculated them with Saccharomyces cerevisiae to ferment Gewürztraminer musts. Noteworthy differences included a significant increase in the 2-phenethyl acetate levels with the P. kluyveri co-fermentation and a general increase in ethyl esters with the P. kudriavzevii co-fermentation. Both Pichia co-inoculations yielded higher levels of glycerol in the final wines. Based on all the wine parameters we tested, the P. kluyveri strain that was isolated performed similarly to a commercial P. kluyveri strain.

Bioinformational trends in grape and wine biotechnology.

The creative destruction caused by the coronavirus pandemic is yielding immense opportunity for collaborative innovation networks. The confluence of biosciences, information sciences, and the engineering of biology, is unveiling promising bioinformational futures for a vibrant and sustainable bioeconomy. Bioinformational engineering, underpinned by DNA reading, writing, and editing technologies, has become a beacon of opportunity in a world paralysed by uncertainty. This article draws on lessons from the current pandemic and previous agricultural blights, and explores bioinformational research directions aimed at future-proofing the grape and wine industry against biological shocks from global blights and climate change.

Synthesis of Aroma Compounds as a Function of Different Nitrogen Sources in Fermentations Using Non-Saccharomyces Wine Yeasts.

Non-Saccharomyces yeasts are prevalent at the onset of grape must fermentations and can have a significant influence on the final wine product. In contrast to Saccharomyces cerevisiae, the biosynthetic pathways leading to aroma compound formation in these non-conventional yeasts, in particular those that are derived from amino acid metabolism, remains largely unexplored. Within a synthetic must environment, we investigated the amino acid utilization of four species (Hanseniaspora uvarum, Hanseniaspora osmophila, Zygosaccharomyces rouxii, Starmerella bacillaris) and S. cerevisiae. We report on the differential uptake preferences for amino acids with H. uvarum displaying the most rapid uptake of most amino acids. To investigate the fate of amino acids and their direct contribution to aroma synthesis in H. uvarum, H. osmophila and Z. rouxii, musts were supplemented with single amino acids. Aroma profiling undertaken after three days showed the synthesis of specific aroma compounds by the respective yeast was dependent on the specific amino acid supplementation. H. osmophila showed similarities to S. cerevisiae in both amino acid uptake and the synthesis of aroma compounds depending on the nitrogen sources. This study shows how the uptake of specific amino acids contributes to the synthesis of aroma compounds in wine fermentations using different non-Saccharomyces yeasts.

Synthetic biology for the engineering of complex wine yeast communities.

Wine fermentation is a representation of complex higher-order microbial interactions. Despite the beneficial properties that these communities bring to wine, their complexity poses challenges in predicting the nature and outcome of fermentation. Technological developments in synthetic biology enable the potential to engineer synthetic microbial communities for new purposes. Here we present the challenges and applications of engineered yeast communities in the context of a wine fermentation vessel, how this represents a model system to enable novel solutions for winemaking and introduce the concept of a 'synthetic' terroir. Furthermore, we introduce our vision for the application of control engineering.

Construction and Analysis of a Yeast for the Simultaneous Release and Esterification of the Varietal Thiol 3-Sulfanylhexan-1-ol.

Polyfunctional thiols like 3-sulfanylhexan-1-ol (3SH) and its ester 3-sulfanylhexyl acetate (3SHA) are important aroma determinants in wine with exceptionally low odor thresholds. 3SH is largely found in grape must bound to glutathione and cysteine and requires enzymatic action to be perceived sensorially. The wine yeast Saccharomyces cerevisiae is ineffective in releasing volatile thiols from their precursor configuration. For this purpose, a yeast strain was constructed that expresses the carbon-sulfur lyase encoding the tnaA gene from Escherichia coli and overexpresses its native alcohol acetyltransferase encoding genes, ATF1 and ATF2. The resulting yeast strain, which co-expresses tnaA and ATF1, showed elevated 3SH-releasing capabilities and the esterification of 3SH to its acetate ester 3SHA. Levels of over 7000 ng/L of 3SHA in Sauvignon blanc wines were achieved. Enhanced release and esterification of 3SH were also shown in the fermentation of guava and passionfruit pulp and three hop varieties. This study offers prospects for the development of flavor-enhancing yeast strains with optimized thiol-releasing and esterification capabilities in a diverse set of beverage matrices.

Yeast Synthetic Minimal Biosensors for Evaluating Protein Production.

The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. In addition to its relevance to human diseases like cancer, the induction of the UPR has a significant impact on the recombinant protein production in eukaryotic cell factories, including the industrial workhorseSaccharomyces cerevisiae. Being able to accurately detect and measure this ER stress response in single cells, enables the rapid optimization of protein production conditions and high-throughput strain selection strategies. Current methodologies to monitor the UPR in S. cerevisiae are often temporally and spatially removed from the cultivation stage or lack updated systematic evaluation. To this end, we constructed and systematically evaluated a series of high-throughput UPR sensors by different designs, incorporating either yeast native UPR promoters or novel synthetic minimal UPR promoters. The native promoters of DER1 and ERO1 were identified to have suitable UPR biosensor properties and served as an expression level guide for orthogonal sensor benchmarking. Our best synthetic minimal sensor is only 98 bp in length, has minimal homology to other native yeast sequences and displayed superior sensor characteristics. The synthetic minimal UPR sensor was able to accurately distinguish between cells expressing different heterologous proteins and between the different secretion levels of the same protein. This work demonstrated the potential of synthetic UPR biosensors as high-throughput tools to predict the protein production capacity of strains, interrogate protein properties hampering their secretion, and guide rational engineering strategies for optimal heterologous protein production.

Development of Genetic Modification Tools for Hanseniasporauvarum.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.