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Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings.

Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings

Motivation: Bioproduction of value-added compounds is frequently achieved by utilizing enzymes from other species. However, expression of such heterologous enzymes can be detrimental due to unexpected interactions within the host cell. Recently, an alternative strategy emerged, which relies on recruiting side activities of host enzymes to establish new biosynthetic pathways. Although such low-level ‘underground’ enzyme activities are prevalent, it remains poorly explored whether they may serve as an important reservoir for pathway engineering. Results: Here, we use genome-scale modeling to estimate the theoretical potential of underground reactions for engineering novel biosynthetic pathways in Escherichia coli. We found that biochemical reactions contributed by underground enzyme activities often enhance the in silico production of compounds with industrial importance, including several cases where underground activities are indispensable for production. Most of these new capabilities can be achieved by the addition of one or two underground reactions to the native network, suggesting that only a few side activities need to be enhanced during implementation. Remarkably, we find that the contribution of underground reactions to the production of value-added compounds is comparable to that of heterologous reactions, underscoring their biotechnological potential. Taken together, our genome-wide study demonstrates that exploiting underground enzyme activities could be a promising addition to the toolbox of industrial strain development. Availability and implementation: The data and scripts underlying this article are available on GitHub at https://github.com/pappb/Kovacs-et-al-Underground-metabolism.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- A nd off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: The length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- A nd off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: The length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro., BN/Stan Brouns Lab

We assessed indels resulting from Cas9-induced genome editing in CML cells in the presence of guide-complementary DNA oligonucleotides intended to inhibit Cas9 activity. We amplified pre-determined loci that were target with the supplied guides or were expected to be off-target sites for these guides. The on-target loci are EMX1 and FANCF., We assessed indels resulting from Cas9-induced genome editing in CML cells in the presence of guide-complementary DNA oligonucleotides intended to inhibit Cas9 activity. We amplified pre-determined loci that were target with the supplied guides or were expected to be off-target sites for these guides. The on-target loci are EMX1 and FANCF.

Background: Metabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms, including Saccharomyces cerevisiae. Although yeast physiology in laboratory strains is well-studied, the metabolic states under industrially relevant scenarios such as winemaking are still not sufficiently understood, especially as there is considerable variation in metabolism between commercial strains. To study the potential causes of strain-dependent variation in the production of volatile compounds during enological conditions, random flux sampling and statistical methods were used, along with experimental extracellular metabolite flux data to characterize the differences in predicted intracellular metabolic states between strains. Results: It was observed that four selected commercial wine yeast strains (Elixir, Opale, R2, and Uvaferm) produced variable amounts of key volatile organic compounds (VOCs). Principal component analysis was performed on extracellular metabolite data from the strains at three time points of cell cultivation (24, 58, and 144 h). Separation of the strains was observed at all three time points. Furthermore, Uvaferm at 24 h, for instance, was most associated with propanol and ethyl hexanoate. R2 was found to be associated with ethyl acetate and Opale could be associated with isobutanol while Elixir was most associated with phenylethanol and phenylethyl acetate. Constraint-based modeling (CBM) was employed using the latest genome-scale metabolic model of yeast (Yeast8) and random flux sampling was performed with experimentally derived fluxes at various stages of growth as constraints for the model. The flux sampling simulations allowed us to characterize intracellular metabolic flux states and illustrate the key parts of metabolism that likely determine the observed strain differences. Flux sampling determined that Uvaferm and Elixir are similar while R2 and Op

Genetic background and environmental conditions affect the production of sensory impact compounds by Saccharomyces cerevisiae. The relative importance of the strain-specific metabolic capabilities for the production of volatile organic compounds (VOCs) remains unclear. We investigated which amino acids contribute to VOC production and whether amino acid-VOC relations are conserved among yeast strains. Amino acid consumption and production of VOCs during grape juice fermentation was investigated using four commercial wine yeast strains: Elixir, Opale, R2, and Uvaferm. Principal component analysis of the VOC data demonstrated that Uvaferm correlated with ethyl acetate and ethyl hexanoate production, R2 negatively correlated with the acetate esters, and Opale positively correlated with fusel alcohols. Biomass formation was similar for all strains, pointing to metabolic differences in the utilization of nutrients to form VOCs. Partial least-squares linear regression showed that total aroma production is a function of nitrogen utilization (R2 = 0.87). We found that glycine, tyrosine, leucine, and lysine utilization were positively correlated with fusel alcohols and acetate esters. Mechanistic modeling of the yeast metabolic network via parsimonious flux balance analysis and flux enrichment analysis revealed enzymes with crucial roles, such as transaminases and decarboxylases. Our work provides insights in VOC production in wine yeasts.

The foodborne pathogen Listeria monocytogenes can form proteinaceous organelles called bacterial microcompartments (BMCs) that optimize the utilization of substrates, such as 1,2-propanediol, and confer an anaerobic growth advantage. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol, next to acetate and lactate. Rhamnose-derived 1,2-propanediol was found to link with BMCs in some human pathogens such as Salmonella enterica, but the involvement of BMCs in rhamnose metabolism and potential physiological effects on L. monocytogenes are still unknown. In this study, we first test the effect of rhamnose uptake and utilization on anaerobic growth of L. monocytogenes EGDe without or with added vitamin B12, followed by metabolic analysis. We show that vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via 1,2-propanediol degradation into 1-propanol and propionate. Transmission electron microscopy of pdu-induced cells shows that BMCs are formed, and additional proteomics experiments confirm expression of pdu BMC shell proteins and enzymes. Finally, we discuss the physiological effects and energy efficiency of L. monocytogenes pdu BMC-driven anaerobic rhamnose metabolism and the impact on competitive fitness in environments such as the human intestine. IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness and, as such, it is crucial to understand the molecular mechanisms contributing to its survival strategy and pathogenicity. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol. In our previous study, the utilization of 1,2-propanediol (pdu) in L. monocytogenes was proved to be metabolized in bacterial microcompartments (BMCs), which are self-assembling subc

The fitness impact of loss-of-function mutations is generally assumed to reflect the loss of specific molecular functions associated with the perturbed gene. Here, we propose that rewiring of the transcriptome upon deleterious gene inactivation is frequently nonspecific and mimics stereotypic responses to external environmental change. Consequently, transcriptional response to gene deletion could be suboptimal and incur an extra fitness cost. Analysis of the transcriptomes of ∼1,500 single-gene deletion Saccharomyces cerevisiae strains supported this scenario. First, most transcriptomic changes are not specific to the deleted gene but are rather triggered by perturbations in functionally diverse genes. Second, gene deletions that alter the expression of dosage-sensitive genes are especially harmful. Third, by elevating the expression level of downregulated genes, we could experimentally mitigate the fitness defect of gene deletions. Our work shows that rewiring of genomic expression upon gene inactivation shapes the harmful effects of mutations.

Bacterial microcompartments (BMCs) are proteinaceous prokaryotic organelles that enable the utilization of substrates such as 1,2-propanediol and ethanolamine. BMCs are mostly linked to the survival of particular pathogenic bacteria by providing a growth advantage through utilization of 1,2-propanediol and ethanolamine which are abundantly present in the human gut. Although a 1,2-propanediol utilization cluster was found in the probiotic bacterium Propionibacterium freudenreichii, BMC-mediated metabolism of 1,2-propanediol has not been demonstrated experimentally in P. freudenreichii. In this study we show that P. freudenreichii DSM 20271 metabolizes 1,2-propanediol in anaerobic conditions to propionate and 1-propanol. Furthermore, 1,2-propanediol induced the formation of BMCs, which were visualized by transmission electron microscopy and resembled BMCs found in other bacteria. Proteomic analysis of 1,2-propanediol grown cells compared to L-lactate grown cells showed significant upregulation of proteins involved in propanediol-utilization (pdu-cluster), DNA repair mechanisms and BMC shell proteins while proteins involved in oxidative phosphorylation were down-regulated. 1,2-Propanediol utilizing cells actively produced vitamin B12 (cobalamin) in similar amounts as cells growing on L-lactate. The ability to metabolize 1,2-propanediol may have implications for human gut colonization and modulation, and can potentially aid in delivering propionate and vitamin B12 in situ.

Ethanolamine (EA) is a valuable microbial carbon and nitrogen source derived from cell membranes. EA catabolism is suggested to occur in a cellular metabolic subsystem called a bacterial microcompartment (BMC), and the activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that the activation of eut is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent in Listeria monocytogenes. Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics, and electron microscopy. First, we show vitamin B12-induced activation of the eut operon in L. monocytogenes coupled to the utilization of EA, thereby enabling growth. Next, we demonstrate BMC formation connected with EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP-generating acetate branch causes an apparent redox imbalance due to the reduced regeneration of NAD1 in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMCs to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild-type, BMC mutant, and EET mutant strains, we demonstrate an interaction between BMCs and EET and provide evidence for a role of Fe31 as an electron acceptor. Taken together, our results suggest an important role of BMC-dependent EA catabolism in L. monocytogenes growth in anaerobic environments like the human gastrointestinal tract, with a crucial role for the flavin-based EET system in redox balancing.

One approach for elucidating strain-to-strain metabolic differences is the use of genome-scale metabolic models (GSMMs). To date GSMMs have not focused on the industrially important area of flavor production and, as such; do not cover all the pathways relevant to flavor formation in yeast. Moreover, current models for Saccharomyces cerevisiae generally focus on carbon-limited and/or aerobic systems, which is not pertinent to enological conditions. Here, we curate a GSMM (iWS902) to expand on the existing Ehrlich pathway and ester formation pathways central to aroma formation in industrial winemaking, in addition to the existing sulfur metabolism and medium-chain fatty acid (MCFA) pathways that also contribute to production of sensory impact molecules. After validating the model using experimental data, we predict key differences in metabolism for a strain (EC 1118) in two distinct growth conditions, including differences for aroma impact molecules such as acetic acid, tryptophol, and hydrogen sulfide. Additionally, we propose novel targets for metabolic engineering for aroma profile modifications employing flux variability analysis with the expanded GSMM. The model provides mechanistic insights into the key metabolic pathways underlying aroma formation during alcoholic fermentation and provides a potential framework to contribute to new strategies to optimize the aroma of wines.

Here we propose to use bacterial cells as factories that generate EVs harboring both the RNP complex (or any other CRISPR‐mediated tool) and a specific ligand molecule. The ligand would allow specific binding of EVs to cells with the matching receptors, adding a level of specificity to the delivery, hence stimulating precise genome editing.

Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations. Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promiscuity in the acquisition and optimization of growth on predicted non-native substrates in Escherichia coli K-12 MG1655. After as few as approximately 20 generations, evolved populations repeatedly acquired the capacity to grow on five predicted non-native substrates-D-lyxose, D-2-deoxyribose, D-arabinose, m-tartrate, and monomethyl succinate. Altered promiscuous activities were shown to be directly involved in establishing high-efficiency pathways. Structural mutations shifted enzyme substrate turnover rates toward the new substrate while retaining a preference for the primary substrate. Finally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity.