Strangers in a foreign land: 'Yeastizing' plant enzymes.

Expressing plant metabolic pathways in microbial platforms is an efficient, cost-effective solution for producing many desired plant compounds. As eukaryotic organisms, yeasts are often the preferred platform. However, expression of plant enzymes in a yeast frequently leads to failure because the enzymes are poorly adapted to the foreign yeast cellular environment. Here, we first summarize the current engineering approaches for optimizing performance of plant enzymes in yeast. A critical limitation of these approaches is that they are labour-intensive and must be customized for each individual enzyme, which significantly hinders the establishment of plant pathways in cellular factories. In response to this challenge, we propose the development of a cost-effective computational pipeline to redesign plant enzymes for better adaptation to the yeast cellular milieu. This proposition is underpinned by compelling evidence that plant and yeast enzymes exhibit distinct sequence features that are generalizable across enzyme families. Consequently, we introduce a data-driven machine learning framework designed to extract 'yeastizing' rules from natural protein sequence variations, which can be broadly applied to all enzymes. Additionally, we discuss the potential to integrate the machine learning model into a full design-build-test cycle.

Cryo-EM reveals a phosphorylated R-domain envelops the NBD1 catalytic domain in an ABC transporter.

Many ATP-binding cassette transporters are regulated by phosphorylation on long and disordered loops which presents a challenge to visualize with structural methods. We have trapped an activated state of the regulatory domain (R-domain) of yeast cadmium factor 1 (Ycf1) by enzymatically enriching the phosphorylated state. A 3.23 Å cryo-EM structure reveals an R-domain structure with four phosphorylated residues and the position for the entire R-domain. The structure reveals key R-domain interactions including a bridging interaction between NBD1 and NBD2 and an interaction with the R-insertion, another regulatory region. We scanned these interactions by systematically replacing segments along the entire R-domain with scrambled combinations of alanine, glycine, and glutamine and probing function under cellular conditions that require the Ycf1 function. We find a close match with these interactions and interacting regions on our R-domain structure that points to the importance of most well-structured segments for function. We propose a model where the R-domain stabilizes a transport-competent state upon phosphorylation by enveloping NBD1 entirely.

The role of hexokinases in epigenetic regulation: altered hexokinase expression and chromatin stability in yeast.

BACKGROUND: Human hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication-coupled epigenetic inheritance process, and chromatin stability. RESULTS: By comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ, hxk2Δ, or double-loss of hxk1Δ hxk2Δ to wild-type cells, we firstly confirmed that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a clinical glycolysis inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability. CONCLUSION: Our findings suggest that in yeast cells lacking HXK2, alternative HXKs such as HXK1 or glucokinase 1 (GLK1) play a role in supporting glycolysis at a level that adequately maintains epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur dependent on non-glycolytic function of Hxk2. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.

Putting It All Together: The Roles of Ribosomal Proteins in Nucleolar Stages of 60S Ribosomal Assembly in the Yeast Saccharomyces cerevisiae.

Here we review the functions of ribosomal proteins (RPs) in the nucleolar stages of large ribosomal subunit assembly in the yeast Saccharomyces cerevisiae. We summarize the effects of depleting RPs on pre-rRNA processing and turnover, on the assembly of other RPs, and on the entry and exit of assembly factors (AFs). These results are interpreted in light of recent near-atomic-resolution cryo-EM structures of multiple assembly intermediates. Results are discussed with respect to each neighborhood of RPs and rRNA. We identify several key mechanisms related to RP behavior. Neighborhoods of RPs can assemble in one or more than one step. Entry of RPs can be triggered by molecular switches, in which an AF is replaced by an RP binding to the same site. To drive assembly forward, rRNA structure can be stabilized by RPs, including clamping rRNA structures or forming bridges between rRNA domains.

TFIIB-Termination Factor Interaction Affects Termination of Transcription on Genome-Wide Scale.

Apart from its well-established role in the initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remain largely unexplored. Using GRO-seq analyses, we compared the terminator readthrough phenotype in the sua7-1 mutant (TFIIBsua7-1) and the isogenic wild type (TFIIBWT) strains. Approximately 74% of genes analyzed exhibited a 2-3-fold increase in readthrough of the poly(A)-termination signal in the TFIIBsua7-1 mutant compared to TFIIBWT cells. To understand the mechanistic basis of TFIIB's role in termination, we performed the mass spectrometry of TFIIB-affinity purified from chromatin and soluble cellular fractions-from TFIIBsua7-1 and TFIIBWT cells. TFIIB purified from the chromatin fraction of TFIIBWT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with CF1A and Rat1 complexes in the TFIIBsua7-1 mutant. ChIP assays revealed about a 90% decline in the recruitment of termination factors in the TFIIBsua7-1 mutant compared to wild type cells. The overall conclusion of these results is that TFIIB affects the termination of transcription on a genome-wide scale, and the TFIIB-termination factor interaction plays a crucial role in the process.

High-efficiency conversion of corn bran to ethanol at 150 L scale.

Fractionated corn bran was processed to maximize ethanol production from starch, cellulose, and xylan. After various bench-scale experiments, an optimized process with dilute acid pretreatment (1.5 % w/w H2SO4) at 90 °C for 60 min was utilized followed by enzymatic hydrolysis using cellulase and hemicellulase for 48 hr. After simultaneous saccharification (regarding starch) and fermentation at 150 L using an engineered yeast, which consumes both glucose and xylose to make ethanol, the 86 % total sugar conversion yield was achieved, including conversions of 95 % for starch, 77 % for cellulose and 77 % for xylan. Also, an accurate mass balance was formulated for ethanol-producing carbohydrates including starch, cellulose, and xylan from feedstock to final ethanol. A highly efficient process of converting corn fiber to ethanol was successfully scaled up to 150 L.

DNA polymerase δ subunit Pol32 binds histone H3-H4 and couples nucleosome assembly with Okazaki fragment processing.

During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3exo-) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1Δ and mcm2-3A, do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.

Amelioration of Smoke Taint in Wine via Addition of Molecularly Imprinted Polymers during or after Fermentation.

The adsorbents used to remove taint compounds from wine can also remove constituents that impart desirable color, aroma, and flavor attributes, whereas molecularly imprinted polymers (MIPs) are tailor-made to selectively bind one or more target compounds. This study evaluated the potential for MIPs to ameliorate smoke taint in wine via removal of volatile phenols during or after fermentation. The addition of MIPs to smoke-tainted Pinot Noir wine (for 24 h with stirring) achieved 35-57% removal of guaiacol, 4-methylguaiacol, cresols, and phenol, but <10% of volatile phenol glycoconjugates were removed and some wine color loss occurred. Of the MIP treatments that were subsequently applied to Semillon and Merlot fermentations or wine, MIP addition post-inoculation of yeast yielded the best outcomes, both in terms of volatile phenol removal and wine sensory profiles. Despite some impact on other aroma volatiles and red wine color, the findings demonstrate that MIPs can ameliorate smoke-tainted wine.

Development of a model for the ethanol concentration limit as a function of temperature and initial substrate concentration using the yeast Saccharomyces cerevisiae.

In this study, a model was developed to simulate the effect of temperature ( T $T$ ) and initial substrate concentration ( S 0 ${S}_{0}$ ) on the ethanol concentration limit ( P max ${P}_{\max }$ ) using the yeast Saccharomyces cerevisiae. To achieve this, regressions were performed using data provided by other authors for P max ${P}_{\max }$ to establish a model dependent on T $T$ and S 0 ${S}_{0}$ capable of predicting results with statistical significance. After constructing the model, a response surface was generated to determine the conditions where P max ${P}_{\max }$ reaches higher values: temperatures between 28°C and 32°C and an initial substrate concentration around 200 g/L. Thus, the proposed model is consistent with the observations that increasing temperatures decrease the ethanol concentration obtained, and substrate concentrations above 200 g/L lead to a reduction in ethanol concentration even at low temperatures such as 28°C.

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Mitochondrial-derived compartments remove surplus proteins from the outer mitochondrial membrane.

The outer mitochondrial membrane (OMM) creates a boundary that imports most of the mitochondrial proteome while removing extraneous or damaged proteins. How the OMM senses aberrant proteins and remodels to maintain OMM integrity remains unresolved. Previously, we identified a mitochondrial remodeling mechanism called the mitochondrial-derived compartment (MDC) that removes a subset of the mitochondrial proteome. Here, we show that MDCs specifically sequester proteins localized only at the OMM, providing an explanation for how select mitochondrial proteins are incorporated into MDCs. Remarkably, selective sorting into MDCs also occurs within the OMM, as subunits of the translocase of the outer membrane (TOM) complex are excluded from MDCs unless assembly of the TOM complex is impaired. Considering that overloading the OMM with mitochondrial membrane proteins or mistargeted tail-anchored membrane proteins induces MDCs to form and sequester these proteins, we propose that one functional role of MDCs is to create an OMM-enriched trap that segregates and sequesters excess proteins from the mitochondrial surface.

De novo biosynthesis of anthocyanins in Saccharomyces cerevisiae using metabolic pathway synthases from blueberry.

BACKGROUND: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency. RESULTS: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique. CONCLUSIONS: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.

Irc20 modulates LOH frequency and distribution in S. cerevisiae.

Loss of Heterozygosity (LOH) due to mitotic recombination is frequently associated with the development of various cancers (e.g. retinoblastoma). LOH is also an important source of genetic diversity, especially in organisms where meiosis is infrequent. Irc20 is a putative helicase, and E3 ubiquitin ligase involved in DNA double-strand break repair pathway. We analyzed genome-wide LOH events, gross chromosomal changes, small insertion-deletions and single nucleotide mutations in eleven S. cerevisiae mutation accumulation lines of irc20∆, which underwent 50 mitotic bottlenecks. LOH enhancement in irc20∆ was small (1.6 fold), but statistically significant as compared to the wild type. Short (≤ 1 kb) and long (> 10 kb) LOH tracts were significantly enhanced in irc20∆. Both interstitial and terminal LOH events were also significantly enhanced in irc20∆ compared to the wild type. LOH events in irc20∆ were more telomere proximal and away from centromeres compared to the wild type. Gross chromosomal changes, single nucleotide mutations and in-dels were comparable between irc20∆ and wild type. Locus based and genome-wide analysis of meiotic recombination showed that meiotic crossover frequencies are not altered in irc20∆. These results suggest Irc20 primarily regulates mitotic recombination and does not affect meiotic crossovers. Our results suggest that the IRC20 gene is important for regulating LOH frequency and distribution.

Investigating biological mechanisms of colour changes in sustainable food systems: The role of Starmerella bacillaris in white wine colouration using a combination of genomic and biostatistics strategies.

This study explores the biological mechanisms behind colour changes in white wine fermentation using different strains of Starmerella bacillaris. We combined food engineering, genomics, machine learning, and physicochemical analyses to examine interactions between S. bacillaris and Saccharomyces cerevisiae. Significant differences in total polyphenol content were observed, with S. bacillaris fermentation yielding 6 % higher polyphenol content compared to S. cerevisiae EC1118. Genomic analysis identified 12 genes in S. bacillaris with high variant counts that could impact phenotypic properties related to wine color. Notably, SNP analysis revealed numerous missense and synonymous variants, as well as stop-gained and start-lost variants between PAS13 and FRI751, suggesting changes in metabolic pathways affecting pigment production. Besides that, high upstream gene variants in SSK1 and HIP1R indicated potential regulatory changes influencing gene expression. Fermentation trials revealed FRI751 consistently showed high antioxidant activity and polyphenol content (Total Polyphenol: 299.33 ± 3.51 mg GAE/L, DPPH: 1.09 ± 0.01 mmol TE/L, FRAP: 0.95 ± 0.02 mmol TE/L). PAS13 exhibited a balanced profile, while EC1118 had lower values, indicating moderate antioxidant activity. The Weibull model effectively captured nitrogen consumption dynamics, with EC1118 serving as a reliable benchmark. The scale parameter delta for EC1118 was 23.04 ± 2.63, indicating moderate variability in event times. These findings highlight S. bacillaris as a valuable component in sustainable winemaking, offering an alternative to chemical additives for maintaining wine quality and enhancing colours profiles. This study provides insights into the biotechnological and fermented food systems applications of yeast strains in improving food sustainability and supply chain, opening new avenues in food engineering and microbiology.

Unraveling the Structure-Spectrum Relationship of Yeast Phenylalanine Transfer RNA: Insights from Theoretical Modeling of Infrared Spectroscopy.

Yeast phenylalanine tRNA (tRNAphe) is a paradigmatic model in structural biology. In this work, we combine molecular dynamics simulations and spectroscopy modeling to establish a direct link between its structure, conformational dynamics, and infrared (IR) spectra. Employing recently developed vibrational frequency maps and coupling models, we apply a mixed quantum/classical treatment of the line shape theory to simulate the IR spectra of tRNAphe in the 1600-1800 cm-1 region across its folded and unfolded conformations and under varying concentrations of Mg2+ ions. The predicted IR spectra of folded and unfolded tRNAphe are in good agreement with experimental measurements, validating our theoretical framework. We then elucidate how the characteristic L-shaped tertiary structure of the tRNA and its modulation in response to diverse chemical environments give rise to distinct IR absorption peaks and line shapes. These calculations effectively bridge IR spectroscopy experiments and atomistic molecular simulations, unraveling the molecular origins of the observed IR spectra of tRNAphe. This work presents a robust theoretical protocol for modeling the IR spectroscopy of nucleic acids, which will facilitate its application as a sensitive probe for detecting the fluctuating secondary and tertiary structures of these essential biological macromolecules.

Optimization of enzymatic hydrolysis of cellulose extracted from bamboo culm for bioethanol production by Saccharomyces cerevisiae modified via CRISPR/Cas9.

In the context of biorefinery, researchers have been looking for lignocellulosic biomasses and ideal treatments to produce economically viable biofuels. In this scenario, the bamboo culm appears as a plant matrix of great potential, given the high cellulose content of low crystallinity. Thus, the objective and differential of this work was to determine the best conditions for enzymatic hydrolysis of cellulose extracted from bamboo culm and to evaluate its potential application in the production of bioethanol through Separate Hydrolysis and Fermentation (SHF) and Saccharification and Simultaneous Fermentation (SSF) by Saccharomyces cerevisiae modified via CRISPR/Cas9. The average cellulose extraction yield was 41.87 % with an extraction efficiency of 86.76 %. In general, as the hydrolysis time increased, an increase in glucose production was observed in almost all assays, with higher hydrolysis efficiency values at 72 h. The results ranged from 2.09 to 19.8 g/L of glucose obtained with efficiency values of 10.47 to 99 %. The best conditions were found in test 5 (temperature of 36 °C and pH 5.0, with only 10 FPU/g of substrate Cellic Ctec2 Novozymes ® cocktail). It is observed that for all hydrolysis times the independent variables pH and temperature were significant under the hydrolysis efficiency, showing a negative effect, indicating that higher values of the same promote lower values of the response variable. For bioethanol production, a maximum concentration of 7.84 g/L was observed for the SSH process after 4 h of fermentation, while for the SSF process it was 12.6 g/L after 24 h of fermentation, indicating the large potential of the simultaneous process together with the application of bamboo culm biomass for high production of biofuel.

Deep-learning-based design of synthetic orthologs of SH3 signaling domains.

Evolution-based deep generative models represent an exciting direction in understanding and designing proteins. An open question is whether such models can learn specialized functional constraints that control fitness in specific biological contexts. Here, we examine the ability of generative models to produce synthetic versions of Src-homology 3 (SH3) domains that mediate signaling in the Sho1 osmotic stress response pathway of yeast. We show that a variational autoencoder (VAE) model produces artificial sequences that experimentally recapitulate the function of natural SH3 domains. More generally, the model organizes all fungal SH3 domains such that locality in the model latent space (but not simply locality in sequence space) enriches the design of synthetic orthologs and exposes non-obvious amino acid constraints distributed near and far from the SH3 ligand-binding site. The ability of generative models to design ortholog-like functions in vivo opens new avenues for engineering protein function in specific cellular contexts and environments.

Nucleophagy in Aspergillus oryzae is Mediated by Autophagosome Formation and Vacuole-Mediated Degradation.

We previously reported autophagy-mediated degradation of nuclei, nucleophagy, in the filamentous fungus Aspergillus oryzae. In this study, we examined whether nuclei are degraded as a whole. We generated A. oryzae mutants deleted for orthologs of Saccharomyces cerevisiae YPT7 and ATG15 which are required, respectively, for autophagosome-vacuole fusion and vacuolar degradation of autophagic bodies. Degradation of histone H2B-EGFP under starvation conditions was greatly decreased in the ΔAoypt7 and ΔAoatg15 mutants. Fluorescence and electron microscopic observations showed that autophagosomes and autophagic bodies surrounding the entire nuclei were accumulated in the cytoplasm of ΔAoypt7 and the vacuole of ΔAoatg15, respectively. These results indicate that nuclei are engulfed in the autophagosomes as a whole and transported/released into the vacuolar lumen where they are degraded.

Activation of the yeast Retrograde Response pathway by adaptive laboratory evolution with S-(2-aminoethyl)-L-cysteine reduces ethanol and increases glycerol during winemaking.

BACKGROUND: Global warming causes an increase in the levels of sugars in grapes and hence in ethanol after wine fermentation. Therefore, alcohol reduction is a major target in modern oenology. Deletion of the MKS1 gene, a negative regulator of the Retrograde Response pathway, in Saccharomyces cerevisiae was reported to increase glycerol and reduce ethanol and acetic acid in wine. This study aimed to obtain mutants with a phenotype similar to that of the MKS1 deletion strain by subjecting commercial S. cerevisiae wine strains to an adaptive laboratory evolution (ALE) experiment with the lysine toxic analogue S-(2-aminoethyl)-L-cysteine (AEC). RESULTS: In laboratory-scale wine fermentation, isolated AEC-resistant mutants overproduced glycerol and reduced acetic acid. In some cases, ethanol was also reduced. Whole-genome sequencing revealed point mutations in the Retrograde Response activator Rtg2 and in the homocitrate synthases Lys20 and Lys21. However, only mutations in Rtg2 were responsible for the overactivation of the Retrograde Response pathway and ethanol reduction during vinification. Finally, wine fermentation was scaled up in an experimental cellar for one evolved mutant to confirm laboratory-scale results, and any potential negative sensory impact was ruled out. CONCLUSIONS: Overall, we have shown that hyperactivation of the Retrograde Response pathway by ALE with AEC is a valid approach for generating ready-to-use mutants with a desirable phenotype in winemaking.

Deuteration of non-labile protium in starch: Biosynthesis and characterisation from yeast-derived starch granules.

Deuterium labelling of the non-labile protium atoms in starch granules has been achieved for the first time, by growing genetically modified yeast on deuterated media. Mass spectrometry of the glucose monomers from digested starch showed 44 % average deuteration of the non-labile protium when grown on partially deuterated raffinose (with average deuteration 48 %); yielding starch with 26 % average overall deuteration. Non-labile deuteration was also demonstrated using D2O solvent in the culture medium. Solid-state NMR revealed that deuteration was not evenly distributed across the monomer, being highest at the C6 carbon and lowest at the C1 carbon. SANS revealed two structural features at q = 0.05 Å-1 and 0.4 Å-1, the first corresponding to a lamellar repeat of approximately 12-13 nm while the latter is consistent with B-type crystalline polymer packing. Furthermore, solvent contrast variation SANS analysis yielded a contrast match point of 66 mol% D2O indicative of approximately 30-35 % average deuteration of the bulk granules, consistent with mass spectroscopy. When coupled with the more traditional process of exchange of labile protium in the hydroxyl groups by D2O solvent exchange, the biosynthesis of highly deuterated starch opens new opportunities for neutron scattering experiments involving multicomponent starch-based systems.