Heterologous production of the epoxycarotenoid violaxanthin in Saccharomyces cerevisiae.

Microbial production of carotenoids has mainly focused towards a few products, such as ß-carotene, lycopene and astaxanthin. However, other less explored carotenoids, like violaxanthin, have also shown unique properties and promissory applications. Violaxanthin is a plant-derived epoxidated carotenoid with strong antioxidant activity and a key precursor of valuable compounds, such as fucoxanthin and ß-damascenone. In this study, we report for the first time the heterologous production of epoxycarotenoids in yeast. We engineered the yeast Saccharomyces cerevisiae following multi-level strategies for the efficient accumulation of violaxanthin. Starting from a ß-carotenogenic yeast strain, we first evaluated the performance of several ß-carotene hydroxylases (CrtZ), and zeaxanthin epoxidases (ZEP) from different species, together with their respective N-terminal truncated variants. The combined expression of CrtZ from Pantoea ananatis and truncated ZEP of Haematococcus lacustris showed the best performance and led to a yield of 1.6 mg/gDCW of violaxanthin. Further improvement of the epoxidase activity was achieved by promoting the transfer of reducing equivalents to ZEP by expressing several redox partner systems. The co-expression of the plant truncated ferredoxin-3, and truncated root ferredoxin oxidoreductase-1 resulted in a 2.2-fold increase in violaxanthin yield (3.2 mg/gDCW). Finally, increasing gene copy number of carotenogenic genes enabled reaching a final production of 7.3 mg/gDCW in shake flask cultures and batch bioreactors, which is the highest yield of microbially produced violaxanthin reported to date.

l-Malate (-2) Protonation State is Required for Efficient Decarboxylation to l-Lactate by the Malolactic Enzyme of Oenococcus oeni.

Malolactic fermentation (MLF) is responsible for the decarboxylation of l-malic into lactic acid in most red wines and some white wines. It reduces the acidity of wine, improves flavor complexity and microbiological stability. Despite its industrial interest, the MLF mechanism is not fully understood. The objective of this study was to provide new insights into the role of pH on the binding of malic acid to the malolactic enzyme (MLE) of Oenococcus oeni. To this end, sequence similarity networks and phylogenetic analysis were used to generate an MLE homology model, which was further refined by molecular dynamics simulations. The resulting model, together with quantum polarized ligand docking (QPLD), was used to describe the MLE binding pocket and pose of l-malic acid (MAL) and its l-malate (-1) and (-2) protonation states (MAL- and MAL2-, respectively). MAL2- has the lowest ∆Gbinding, followed by MAL- and MAL, with values of -23.8, -19.6, and -14.6 kJ/mol, respectively, consistent with those obtained by isothermal calorimetry thermodynamic (ITC) assays. Furthermore, molecular dynamics and MM/GBSA results suggest that only MAL2- displays an extended open conformation at the binding pocket, satisfying the geometrical requirements for Mn2+ coordination, a critical component of MLE activity. These results are consistent with the intracellular pH conditions of O. oeni cells-ranging from pH 5.8 to 6.1-where the enzymatic decarboxylation of malate occurs.

Development of an International Odor Identification Test for Children: The Universal Sniff Test.

OBJECTIVE: To assess olfactory function in children and to create and validate an odor identification test to diagnose olfactory dysfunction in children, which we called the Universal Sniff (U-Sniff) test. STUDY DESIGN: This is a multicenter study involving 19 countries. The U-Sniff test was developed in 3 phases including 1760 children age 5-7 years. Phase 1: identification of potentially recognizable odors; phase 2: selection of odorants for the odor identification test; and phase 3: evaluation of the test and acquisition of normative data. Test-retest reliability was evaluated in a subgroup of children (n = 27), and the test was validated using children with congenital anosmia (n = 14). RESULTS: Twelve odors were familiar to children and, therefore, included in the U-Sniff test. Children scored a mean ± SD of 9.88 ± 1.80 points out of 12. Normative data was obtained and reported for each country. The U-Sniff test demonstrated a high test-retest reliability (r27 = 0.83, P < .001) and enabled discrimination between normosmia and children with congenital anosmia with a sensitivity of 100% and specificity of 86%. CONCLUSIONS: The U-Sniff is a valid and reliable method of testing olfaction in children and can be used internationally.

Increasing the intracellular isoprenoid pool in Saccharomyces cerevisiae by structural fine-tuning of a bifunctional farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPPS) is a key enzyme responsible for the supply of isoprenoid precursors for several essential metabolites, including sterols, dolichols and ubiquinone. In Saccharomyces cerevisiae, FPPS catalyzes the sequential condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), producing geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Critical amino acid residues that determine product chain length were determined by a comparative study of strict GPP synthases versus strict FPPS. In silico ΔΔG, i.e. differential binding energy between a protein and two different ligands-of yeast FPPS mutants was evaluated, and F96, A99 and E165 residues were identified as key determinants for product selectivity. A99X variants were evaluated in vivo, S. cerevisiae strains carrying A99R and A99H variants showed significant differences on GPP concentrations and specific growth rates. The FPPS A99T variant produced unquantifiable amounts of FPP and no effect on GPP production was observed. Strains carrying A99Q, A99Y and A99K FPPS accumulated high amounts of DMAPP-IPP, with a decrease in GPP and FPP. Our results demonstrated the relevance of the first residue before FARM (First Aspartate Rich Motif) over substrate consumption and product specificity of S. cerevisiae FPPS in vivo. The presence of A99H significantly modified product selectivity and appeared to be relevant for GPP synthesis.

Optogenetic switches for light-controlled gene expression in yeast.

Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

Chemical vs. biotechnological synthesis of C13-apocarotenoids: current methods, applications and perspectives.

Apocarotenoids are natural compounds derived from the oxidative cleavage of carotenoids. Particularly, C13-apocarotenoids are volatile compounds that contribute to the aromas of different flowers and fruits and are highly valued by the Flavor and Fragrance industry. So far, the chemical synthesis of these terpenoids has dominated the industry. Nonetheless, the increasing consumer demand for more natural and sustainable processes raises an interesting opportunity for bio-production alternatives. In this regard, enzymatic biocatalysis and metabolically engineered microorganisms emerge as attractive biotechnological options. The present review summarizes promising bioengineering approaches with regard to chemical production methods for the synthesis of two families of C13-apocarotenoids: ionones/dihydroionones and damascones/damascenone. We discuss each method and its applicability, with a thorough comparative analysis for ionones, focusing on the production process, regulatory aspects, and sustainability.

Production of ß-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae.

BACKGROUND: Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants-like raspberries and roses-by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid ß-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP. RESULTS: Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of ß-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased ß-ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of ß-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase. CONCLUSIONS: An efficient ß-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene-the highest ß-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.

Construction of robust dynamic genome-scale metabolic model structures of Saccharomyces cerevisiae through iterative re-parameterization.

Dynamic flux balance analysis (dFBA) has been widely employed in metabolic engineering to predict the effect of genetic modifications and environmental conditions in the cell׳s metabolism during dynamic cultures. However, the importance of the model parameters used in these methodologies has not been properly addressed. Here, we present a novel and simple procedure to identify dFBA parameters that are relevant for model calibration. The procedure uses metaheuristic optimization and pre/post-regression diagnostics, fixing iteratively the model parameters that do not have a significant role. We evaluated this protocol in a Saccharomyces cerevisiae dFBA framework calibrated for aerobic fed-batch and anaerobic batch cultivations. The model structures achieved have only significant, sensitive and uncorrelated parameters and are able to calibrate different experimental data. We show that consumption, suboptimal growth and production rates are more useful for calibrating dynamic S. cerevisiae metabolic models than Boolean gene expression rules, biomass requirements and ATP maintenance.

Metabolic and transcriptomic response of the wine yeast Saccharomyces cerevisiae strain EC1118 after an oxygen impulse under carbon-sufficient, nitrogen-limited fermentative conditions.

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to continuously changing environmental conditions, such as decreasing sugar and increasing ethanol concentrations. Oxygen, a critical nutrient to avoid stuck and sluggish fermentations, is only discretely available throughout the process after pump-over operation. In this work, we studied the physiological response of the wine yeast S. cerevisiae strain EC1118 to a sudden increase in dissolved oxygen, simulating pump-over operation. With this aim, an impulse of dissolved oxygen was added to carbon-sufficient, nitrogen-limited anaerobic continuous cultures. Results showed that genes related to mitochondrial respiration, ergosterol biosynthesis, and oxidative stress, among other metabolic pathways, were induced after the oxygen impulse. On the other hand, mannoprotein coding genes were repressed. The changes in the expression of these genes are coordinated responses that share common elements at the level of transcriptional regulation. Beneficial and detrimental effects of these physiological processes on wine quality highlight the dual role of oxygen in 'making or breaking wines'. These findings will facilitate the development of oxygen addition strategies to optimize yeast performance in industrial fermentations.

Prevalence of lactose intolerance in Chile: a double-blind placebo study.

BACKGROUND AND STUDY AIMS: Lactase non-persistence (LNP), or primary hypolactasia, is a genetic condition that mediates lactose malabsorption and can cause lactose intolerance. Here we report the prevalence of lactose intolerance in a double-blind placebo study. METHODS: The LCT C>T-13910 variant was genotyped by RT-PCR in 121 volunteers and lactose malabsorption was assessed using the hydrogen breath test (HBT) after consuming 25 g of lactose. Lactose intolerance was assessed by scoring symptoms (SS) using a standardized questionnaire following challenge with a lactose solution or saccharose placebo. RESULTS: The LNP genotype was observed in 57% of the volunteers, among whom 87% were HBT⁺. In the HBT⁺ group the median SS was 9 and in the HBT⁻ group the median SS was 3 (p < 0.001). No difference was observed in the SS when both groups were challenged with the placebo. The most common symptoms included audible bowel sounds, abdominal pain and meteorism. In the ROC curve analysis, an SS ≥ 6 demonstrated 72% sensitivity and 81% specificity for predicting a positive HBT. To estimate prevalence, lactose intolerance was defined as the presence of an SS ≥ 6 points after subtracting the placebo effect and 34% of the study population met this definition. CONCLUSIONS: The LNP genotype was present in more than half of subjects evaluated and the observed prevalence of lactose intolerance was 34%.

Identification of volatile compounds associated with the aroma of white strawberries (Fragaria chiloensis).

BACKGROUND: Fragaria chiloensis (L.) Mill spp. chiloensis form chiloensis, is a strawberry that produces white fruits with unique aromas. This species, endemic to Chile, is one of the progenitors of Fragaria x ananassa Duch. In order to identify the volatile compounds that might be responsible for aroma, these were extracted, and analyzed by gas chromatography-mass spectrometry (GC-MS), gas chromatography-olfactometry (GC-O) and compared with sensory analyses. RESULTS: Three methods of extraction were used: solvent-assisted evaporation (SAFE), headspace solid phase micro-extraction (HS-SPME) and liquid-liquid extraction (LLE). Ninety-nine volatile compounds were identified by GC-MS, of which 75 showed odor activity using GC-O. Based on the highest dilution factor (FD = 1000) and GC-O intensity ≥2, we determined 20 major compounds in white strawberry fruit that contribute to its aroma. We chose 51 compounds to be tested against their commercial standards. The identities were confirmed by comparison of their linear retention indices against the commercial standards. The aroma of white strawberry fruits was reconstituted with a synthetic mixture of most of these compounds. CONCLUSION: The volatile profile of white strawberry fruit described as fruity, green-fresh, floral, caramel, sweet, nutty and woody will be a useful reference for future strawberry breeding programs.

Metabolic behavior for a mutant Oenococcus oeni strain with high resistance to ethanol to survive under oenological multi-stress conditions.

Malolactic fermentation (MLF) positively influences the quality of the wine, and it occurs as a result of a lactic acid bacteria's metabolism, mainly of the Oenococcus oeni species. However, delays and halting of MLF are frequent problems in the wine industry. This is mainly because O. oeni's development is inhibited by different kinds of stress. Even though the sequencing of the genome of the PSU-1 strain of O. oeni, as well as other strains, has made it possible to identify genes involved in the resistance to some types of stress, all of the factors that could be involved are still unknown. With the aim of contributing to this knowledge, the random mutagenesis technique was used in this study as a strategy for genetic improvement of strains of the O. oeni species. The technique proved to be capable of generating a different and improved strain when compared to the PSU-1 strain (the parent from which it descends). Then, we evaluated the metabolic behavior of both strains in three different wines. We used synthetic MaxOeno wine (pH 3.5; 15% v/v ethanol), red wine (Cabernet Sauvignon), and white wine (Chardonnay). Furthermore, we compared the transcriptome of both strains, grown in MaxOeno synthetic wine. The specific growth rate of the E1 strain was on average 39% higher in comparison to the PSU-1 strain. Interestingly, E1 strain showed an overexpression of the OEOE_1794 gene, which encodes a UspA-like protein, which has been described as promoting growth. We observed that the E1 strain was able to convert, on average, 34% more malic acid into lactate than the PSU-1 strain, regardless of the wine being used. On the other hand, the E1 strain showed a flux rate of fructose-6-phosphate production that was 86% higher than the mannitol production rate, and the internal flux rates increase in the direction of pyruvate production. This coincides with the higher number of OEOE_1708 gene transcripts observed in the E1 strain grown in MaxOeno. This gene encodes for an enzyme fructokinase (EC 2.7.1.4) involved in the transformation of fructose to fructose-6-phosphate.

Oxygen response of the wine yeast Saccharomyces cerevisiae EC1118 grown under carbon-sufficient, nitrogen-limited enological conditions.

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 µM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations.

Modeling oxygen dissolution and biological uptake during pulse oxygen additions in oenological fermentations.

Discrete oxygen additions during oenological fermentations can have beneficial effects both on yeast performance and on the resulting wine quality. However, the amount and time of the additions must be carefully chosen to avoid detrimental effects. So far, most oxygen additions are carried out empirically, since the oxygen dynamics in the fermenting must are not completely understood. To efficiently manage oxygen dosage, we developed a mass balance model of the kinetics of oxygen dissolution and biological uptake during wine fermentation on a laboratory scale. Model calibration was carried out employing a novel dynamic desorption-absorption cycle based on two optical sensors able to generate enough experimental data for the precise determination of oxygen uptake and volumetric mass transfer coefficients. A useful system for estimating the oxygen solubility in defined medium and musts was also developed and incorporated into the mass balance model. Results indicated that several factors, such as the fermentation phase, wine composition, mixing and carbon dioxide concentration, must be considered when performing oxygen addition during oenological fermentations. The present model will help develop better oxygen addition policies in wine fermentations on an industrial scale.

Metabolic Modeling of Wine Fermentation at Genome Scale.

Wine fermentation is an ancient biotechnological process mediated by different microorganisms such as yeast and bacteria. Understanding of the metabolic and physiological phenomena taking place during this process can be now attained at a genome scale with the help of metabolic models. In this chapter, we present a detailed protocol for modeling wine fermentation using genome-scale metabolic models. In particular, we illustrate how metabolic fluxes can be computed, optimized and interpreted, for both yeast and bacteria under winemaking conditions. We also show how nutritional requirements can be determined and simulated using these models in relevant test cases. This chapter introduces fundamental concepts and practical steps for applying flux balance analysis in wine fermentation, and as such, it is intended for a broad microbiology audience as well as for practitioners in the metabolic modeling field.

Screening of COVID-19 cases through a Bayesian network symptoms model and psychophysical olfactory test.

The sudden loss of smell is among the earliest and most prevalent symptoms of COVID-19 when measured with a clinical psychophysical test. Research has shown the potential impact of frequent screening for olfactory dysfunction, but existing tests are expensive and time consuming. We developed a low-cost ($0.50/test) rapid psychophysical olfactory test (KOR) for frequent testing and a model-based COVID-19 screening framework using a Bayes Network symptoms model. We trained and validated the model on two samples: suspected COVID-19 cases in five healthcare centers (n = 926; 33% prevalence, 309 RT-PCR confirmed) and healthy miners (n = 1,365; 1.1% prevalence, 15 RT-PCR confirmed). The model predicted COVID-19 status with 76% and 96% accuracy in the healthcare and miners samples, respectively (healthcare: AUC = 0.79 [0.75-0.82], sensitivity: 59%, specificity: 87%; miners: AUC = 0.71 [0.63-0.79], sensitivity: 40%, specificity: 97%, at 0.50 infection probability threshold). Our results highlight the potential for low-cost, frequent, accessible, routine COVID-19 testing to support society's reopening.

Genomic integration of unclonable gene expression cassettes in Saccharomyces cerevisiae using rapid cloning-free workflows.

Most DNA assembly methods require bacterial amplification steps, which restrict its application to genes that can be cloned in the bacterial host without significant toxic effects. However, genes that cannot be cloned in bacteria do not necessarily exert toxic effects on the final host. In order to tackle this issue, we adapted two DNA assembly workflows for rapid, cloning-free construction and genomic integration of expression cassettes in Saccharomyces cerevisiae. One method is based on a modified Gibson assembly, while the other relies on a direct assembly and integration of linear PCR products by yeast homologous recombination. The methods require few simple experimental steps, and their performance was evaluated for the assembly and integration of unclonable zeaxanthin epoxidase expression cassettes in yeast. Results showed that up to 95% integration efficiency can be reached with minimal experimental effort. The presented workflows can be employed as rapid gene integration tools for yeast, especially tailored for integrating unclonable genes.

Engineering Saccharomyces cerevisiae for the Overproduction of ß-Ionone and Its Precursor ß-Carotene.

ß-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment ß-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeast Saccharomyces cerevisiae. However, current ß-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of ß-carotene and its subsequent conversion to ß-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on ß-carotene accumulation, and later, on ß-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date in S. cerevisiae for a constitutive expression system. For ß-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest ß-carotene-producing strains, reaching up to 33 mg/L of ß-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing ß-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.

Protein engineering of carotenoid cleavage dioxygenases to optimize ß-ionone biosynthesis in yeast cell factories.

Synthesis of ß-ionone in recombinant Saccharomyces cerevisiae is limited by the efficiency of Carotenoid Cleavage Dioxygenases (CCD), membrane-tethered enzymes catalyzing the last step in the pathway. We performed in silico design and membrane affinity analysis, focused on single-point mutations of PhCCD1 to improve membrane anchoring. The resulting constructs were tested in a ß-carotene hyper-producing strain by comparing colony pigmentation against colonies transformed with native PhCCD1 and further analyzed by ß-ionone quantification via RP-HPLC. Two single-point mutants increased ß-ionone yields almost 3-fold when compared to native PhCCD1. We also aimed to improve substrate accessibility of PhCCD1 through the amino-terminal addition of membrane destination peptides directed towards the endoplasmic reticulum or plasma membrane. Yeast strains expressing peptide-PhCCD1 constructs showed ß-ionone yields up to 4-fold higher than the strain carrying the native enzyme. Our results demonstrate that protein engineering of CCDs significantly increases the yield of ß-ionone synthesized by metabolically engineered yeast.

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris.

Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/gDCW per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris.