Protein constraints in genome-scale metabolic models: Data integration, parameter estimation, and prediction of metabolic phenotypes.

Genome-scale metabolic models provide a valuable resource to study metabolism and cell physiology. These models are employed with approaches from the constraint-based modeling framework to predict metabolic and physiological phenotypes. The prediction performance of genome-scale metabolic models can be improved by including protein constraints. The resulting protein-constrained models consider data on turnover numbers (kcat ) and facilitate the integration of protein abundances. In this systematic review, we present and discuss the current state-of-the-art regarding the estimation of kinetic parameters used in protein-constrained models. We also highlight how data-driven and constraint-based approaches can aid the estimation of turnover numbers and their usage in improving predictions of cellular phenotypes. Finally, we identify standing challenges in protein-constrained metabolic models and provide a perspective regarding future approaches to improve the predictive performance.

PARROT: Prediction of enzyme abundances using protein-constrained metabolic models.

Protein allocation determines the activity of cellular pathways and affects growth across all organisms. Therefore, different experimental and machine learning approaches have been developed to quantify and predict protein abundance and how they are allocated to different cellular functions, respectively. Yet, despite advances in protein quantification, it remains challenging to predict condition-specific allocation of enzymes in metabolic networks. Here, using protein-constrained metabolic models, we propose a family of constrained-based approaches, termed PARROT, to predict how much of each enzyme is used based on the principle of minimizing the difference between a reference and an alternative growth condition. To this end, PARROT variants model the minimization of enzyme reallocation using four different (combinations of) distance functions. We demonstrate that the PARROT variant that minimizes the Manhattan distance between the enzyme allocation of a reference and an alternative condition outperforms existing approaches based on the parsimonious distribution of fluxes or enzymes for both Escherichia coli and Saccharomyces cerevisiae. Further, we show that the combined minimization of flux and enzyme allocation adjustment leads to inconsistent predictions. Together, our findings indicate that minimization of protein allocation rather than flux redistribution is a governing principle determining steady-state pathway activity for microorganism grown in alternative growth conditions.

Effect of carbon and nitrogen concentrations on lipid accumulation and regulation of acetyl-CoA carboxylase in Papiliotrema laurentii.

Biodiesel is an interesting alternative to petroleum diesel as it is renewable, biodegradable, and has a low pollutant content. Yeast oils can be used for biodiesel production instead of edible oils, mitigating the use of arable land and water for biodiesel production. Maximum lipid accumulation is reached at 48 h of cultivation by the oleaginous yeast Papiliotrema laurentii UFV-1. Nevertheless, the effects of carbon and nitrogen concentrations on lipid accumulation, as well as the regulation of lipid metabolism in this yeast are still not well-characterised. Therefore, this work evaluated the effects of carbon and nitrogen concentrations on the lipid accumulation in P. laurentti, the expression of the ACC gene, and the activity of the enzyme acetyl-CoA carboxylase (ACCase) in different carbon:nitrogen ratios (C:N) and glucose concentrations. The variation of ammonium sulfate concentration did not affect the growth and lipid accumulation in P. laurentii UFV-1. On the other hand, glucose concentration remarkably influenced biomass and lipid production by this yeast. Therefore, the carbon concentration is more important than the nitrogen concentration for lipid production by P. laurentii UFV-1. Importantly, the levels of both ACC gene expression and ACCase activity were maximum during the late-exponential growth phase and decreased after reaching the highest lipid contents, which was easier evidenced during the accumulation and maximum lipid levels. As such, the reduction of ACCase enzyme activity seems to be related to the decrease in the expression level of the ACC gene.

Identification of the main proteins secreted by Kluyveromyces marxianus and their possible roles in antagonistic activity against fungi.

Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous cocultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.

Papiliotrema laurentii: general features and biotechnological applications.

Papiliotrema laurentii, previously classified as Cryptococcus laurentii, is an oleaginous yeast that has been isolated from soil, plants, and agricultural and industrial residues. This variety of habitats reflects the diversity of carbon sources that it can metabolize, including monosaccharides, oligosaccharides, glycerol, organic acids, and oils. Compared to other oleaginous yeasts, such as Yarrowia lipolytica and Rhodotorula toruloides, there is little information regarding its genetic and physiological characteristics. From a biotechnological point of view, P. laurentii can produce surfactants, enzymes, and high concentrations of lipids, which can be used as feedstock for fatty acid-derived products. Moreover, it can be applied for the biocontrol of phytopathogenic fungi, contributing to quality maintenance in post- and pre-harvest fruits. It can also improve mycorrhizal colonization, nitrogen nutrition, and plant growth. P. laurentii is also capable of degrading polyester and diesel derivatives and acting in the bioremediation of heavy metals. In this review, we present the current knowledge about the basic and applied aspects of P. laurentii, underscoring its biotechnological potential and future perspectives. KEY POINTS: • The physiological characteristics of P. laurentii confer a wide range of biotechnological applications. • The regulation of the acetyl-CoA carboxylase in P. laurentii is different from most other oleaginous yeasts. • The GEM is a valuable tool to guide the construction of engineered P. laurentii strains with improved features for bio-based products.

Physiological comparisons among Spathaspora passalidarum, Spathaspora arborariae, and Scheffersomyces stipitis reveal the bottlenecks for their use in the production of second-generation ethanol.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44 g/g) and productivity (1.02 g/L.h) were obtained using Sp. passalidarum grown in 100 g/L xylose at 32 °C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.

Ethanol stress responses in Kluyveromyces marxianus: current knowledge and perspectives.

The rising concern with the emission of greenhouse gases has boosted new incentives for biofuels production, which are less polluting than fossil fuels. Special attention has been given to the second-generation ethanol, as it is produced from abundant feedstocks which do not compete with food production, such as lignocellulosic biomass and whey. Kluyveromyces marxianus stands out in second-generation ethanol production due to its capacity of assimilating lactose, the sugar found in whey, and tolerating high temperatures used in simultaneous saccharification processes. Nonetheless, contrary to Saccharomyces cerevisiae, K. marxianus does not tolerate high ethanol concentrations. Ethanol causes a broad range of toxic effects on yeasts, acting on cell membrane and proteins, as well as inducing the generation of reactive oxygen species (ROS). The ethanol stress responses are not fully understood, mainly in non-conventional yeasts such as K. marxianus. Indeed, many molecular responses to ethanol stress are still inferred from S. cerevisiae. As such, a better understanding of the ethanol stress responses in K. marxianus may provide the basis for improving its use in the biofuel industry. Additionally, the selection of ethanol-tolerant strains by metabolic engineering is useful to provide strains with improved capacity to withstand stressful conditions, as well as to obtain new insights about the ethanol stress responses. Key points • It is still not totally clear why K. marxianus is less tolerant to ethanol than S. cerevisiae. • Understanding the ethanol stress response in K. marxianus is pivotal for improving its application in the biofuel industry. • The Metabolic engineering is expected to improve the ethanol tolerance in K. marxianus.

Adaptive responses of Kluyveromyces marxianus CCT 7735 to 2-phenylethanol stress: Alterations in membrane fatty-acid composition, ergosterol content, exopolysaccharide production and reduction in reactive oxygen species.

2-phenylethanol (2-PE) is a higher aromatic alcohol with a rose-like aroma used in the cosmetic and food industries as a flavoring and displays potential for application as an antifungal. Biotechnological production of 2-PE from yeast is an interesting alternative due to the non-use of toxic compounds and the generation of few by-products. Kluyveromyces marxianus CCT 7735 is a thermotolerant strain capable of producing high 2-PE titers from L-Phenylalanine; however, like other yeast species, its growth has been strongly inhibited by this alcohol. Herein, we aimed to evaluate the effect of 2-PE on cell growth, cell viability, membrane permeability, glucose uptake, metabolism, and morphology in K. marxianus CCT 7735, as well as its adaptive responses. The stress condition was imposed after 4 h of cultivation by adding 3.0 g.L-1 of 2-PE in exponential growing cells. 2-PE stress impaired yeast growth, glucose uptake, fermentative metabolism, membrane permeability, and cell viability. Moreover, the stress condition provoked changes in both morphology and surface roughness. The reactive oxygen species (ROS) increased immediately on exposure to 2-PE. Changes in membrane fatty-acid composition, ergosterol content, exopolysaccharides production, and reduction of the ROS levels appear to be the result of adaptive responses in K. marxianus. Our results provided insights into a better understanding of the effects of 2-PE on K. marxianus and its adaptive responses.

Isolation of a new Papiliotrema laurentii strain that displays capacity to achieve high lipid content from xylose.

In this work, we isolated and selected oleaginous yeasts from rock field soils from two National Parks in Brazil (Caparaó and Serra dos Órgãos) with the potential to accumulate oil from xylose, the main pentose sugar found in lignocellulosic biomass. From the 126 isolates, two were selected based on their lipid contents. They were taxonomically identified as Papiliotrema laurentii (UFV-1 and UFV-2). Of the two, P. laurentii UFV-1 was selected as the best lipid producer. Under unoptimized conditions, lipid production by P. laurentii UFV-1 was higher in glucose than in xylose. To improve its lipid production from xylose, we applied response surface methodology (RSM) with a face-centered central composite design (CCF). We evaluated the effects of agitation rate, initial cell biomass (OD600), carbon/nitrogen ratio (C/N ratio) and pH on lipid production. P. laurentii UFV-1 recorded the highest lipid content, 63.5% (w/w) of the cell dry mass, under the following conditions: C/N ratio = 100:1, pH value = 7.0, initial OD600 = 0.8 and agitation = 300 rpm. Under these optimized conditions, biomass, lipid titer and volumetric lipid productivity were 9.31 g/L, 5.90 g/L and 0.082 g/L.h, respectively. Additionally, we determined the fatty acid composition of P. laurentii UFV-1 as follows: C14:0 (0.5%), C16:0 (28.4-29.4%), C16:1 (0.2%), C18:0 (9.5-11%), C18:1 (58.6-60.5%), and C20:0 (0.7-0.8%). Based on this composition, the predicted properties of biodiesel showed that P. laurentii UFV-1 oil is suitable for use as feedstock in biodiesel production.

Assessment of ethanol tolerance of Kluyveromyces marxianus CCT 7735 selected by adaptive laboratory evolution.

Kluyveromyces marxianus CCT 7735 shows potential for producing ethanol from lactose; however, its low ethanol tolerance is a drawback for its industrial application. The first aim of this study was to obtain four ethanol-tolerant K. marxianus CCT 7735 strains (ETS1, ETS2, ETS3, and ETS4) by adaptive laboratory evolution. The second aim was to select among them the strain that stood out and to evaluate metabolic changes associated with the improved ethanol tolerance in this strain. The ETS4 was selected for displaying a specific growth rate higher than the parental strain under ethanol stress (122%) and specific ethanol production rate (0.26 g/g/h) higher than those presented by the ETS1 (0.22 g/g/h), ETS2 (0.17 g/g/h), and ETS3 (0.17 g/g/h) under non-stress condition. Further analyses were performed with the ETS4 in comparison with its parental strain in order to characterize metabolic changes. Accumulation of valine and metabolites of the citric acid cycle (isocitric acid, citric acid, and cis-aconitic acid) was observed only in the ETS4 subjected to ethanol stress. Their accumulation in this strain may have been important to increase ethanol tolerance. Furthermore, the contents of fatty acid methyl esters and ergosterol were higher in the ETS4 than in the parental strain. These differences likely contributed to enhance ethanol tolerance in the ETS4. KEY POINTS: • K. marxianus ethanol-tolerant strains were selected by adaptive laboratory evolution. • Valine and metabolites of the TCA cycle were accumulated in the ETS4. • High contents of fatty acids and ergosterol contributed to enhance ethanol tolerance.

Isolation of xylose-assimilating yeasts and optimization of xylitol production by a new Meyerozyma guilliermondii strain.

Production of xylitol from lignocellulosic biomass is of interest to modern biorefineries, because this biomass should be processed into a spectrum of chemicals (bio-based products) and not only energy. The isolation of new yeast strains capable of efficiently converting xylose into xylitol and withstanding inhibitors released from biomass hydrolysis can contribute to making its production feasible in biorefineries. Forty-three out of 128 yeast strains isolated from the gut of Passalidae beetles were capable of assimilating xylose as the sole carbon source. Meyerozyma guilliermondii UFV-1 was selected due to its ability to grow and ferment D-xylose in a synthetic medium. This yeast assimilated the broad range of sugars present in lignocellulosic biomass hydrolysates, such as xylose, raffinose, cellobiose, rhamnose, arabinose, and glucose. Its optimum growth conditions were pH 8.0 and a temperature of 30 °C. In concentrations of 0.07 mol/L acetic acid, 0.05 mol/L 5-hydroximethylfurfural, and 0.04 mol/L furfural, M. guilliermondii UFV-1 did not grow. Maximum xylitol production in aerobiosis and hypoxia were 51.88 and 27.73 g/L, respectively. Under aerobic condition, xylose concentration and agitation rate were the factors which were statistically significant, while only the agitation rate was significant in hypoxia. We fitted a response surface (RS) that estimated the best agitation rate (113.33 rpm) and xylose concentration (90 g/L) for maximum xylitol production in aerobiosis. Therefore, M. guilliermondii UFV-1 displays potential for being used for xylitol production in biorefineries.

Genomic analysis and lactose transporter expression in Kluyveromyces marxianus CCT 7735.

Kluyveromyces marxianus CCT 7735 has been used to produce ethanol, aromatic compounds, enzymes and heterologous proteins besides assimilates lactose as carbon source. Its genome has 10.7 Mb and encodes 4787 genes distributed in 8 nuclear chromosomes and one mitochondrial. Contrary to Kluyveromyces lactis, which has a unique LAC12 gene (encodes lactose permease), K. marxianus possesses four. The presence of degenerated copies and Solo-LTRs related to retrotransposon TKM close to the LAC12 genes in K. marxianus indicates ectopic recombinations. The Lac12 permeases of K. marxianus and K. lactis are conserved, however the conservation is higher between the copy of the left side of the chromosome three and the unique copy of K. lactis, indicating that this copy is the ancestor. The expression of the four LAC12 genes occurred in aerobiosis and hypoxia. Notably, the high lactose consumption in hypoxia seems to be related to the high expression of the LAC12 genes.

Ethanol stress responses of Kluyveromyces marxianus CCT 7735 revealed by proteomic and metabolomic analyses.

Kluyveromyces marxianus CCT 7735 offers advantages to ethanol production over Saccharomyces cerevisiae, including thermotolerance and the ability to convert lactose to ethanol. However, its growth is impaired at high ethanol concentrations. Herein we report on the protein and intracellular metabolite profiles of K. marxianus at 1 and 4 h under ethanol exposure. The concentration of some amino acids, trehalose and ergosterol were also measured. We observed that proteins and metabolites from carbon pathways and translation were less abundant, mainly at 4 h of ethanol stress. Nevertheless, the concentration of some amino acids and trehalose increased at 8 and 12 h under ethanol stress, indicating an adaptive response. Moreover, our results show that the abundance of proteins and metabolites related to the oxidative stresses responses increased. The results obtained in this study provide insights into understanding the physiological changes in K. marxianus under ethanol stress, indicating possible targets for ethanol tolerant strains construction.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress.

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure.

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes ß-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three ß-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).

The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 µg mL(-1) Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.

Oligonucleotide primers for specific detection of actinobacterial laccases from superfamilies I and K.

Although many putative laccase-like genes have been assigned to members of the phylum Actinobacteria, few of the related enzymes have been characterized so far. It is noteworthy, however, that this small number of enzymes has presented properties with industrial relevance. This observation, combined with the recognized biotechnological potential and the capability of this phylum to degrade recalcitrant soil polymers, has attracted attention for bioprospective approaches. In the present work, we have designed and tested primers that were specific for detection of sub-groups of laccase-like genes within actinomycetes, which corresponded to the superfamilies I and K from the classification presented by the laccase and multicopper oxidase engineering database. The designed primers have amplified laccase-like gene fragments from actinomycete isolates that were undetectable by primers available from the literature. Furthermore, phylogenetic alignments suggest that some of these fragments may belong to new laccases-like proteins, and thus emphasize the benefits of designing subgroup-specific primers.

The influence of presaccharification, fermentation temperature and yeast strain on ethanol production from sugarcane bagasse.

Ethanol can be produced from cellulosic biomass in a process known as simultaneous saccharification and fermentation (SSF). The presence of yeast together with the cellulolytic enzyme complex reduces the accumulation of sugars within the reactor, increasing the ethanol yield and saccharification rate. This paper reports the isolation of Saccharomyces cerevisiae LBM-1, a strain capable of growth at 42 °C. In addition, S. cerevisiae LBM-1 and Kluyveromyces marxianus UFV-3 were able to ferment sugar cane bagasse in SSF processes at 37 and 42 °C. Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50°C than at 37 or 42° C. Furthermore, the volumetric productivity of fermentation increased with presaccharification time, from 0.43 g/L/h at 0 h to 1.79 g/L/h after 72 h of presaccharification. The results suggest that the use of thermotolerant yeasts and a presaccharification stage are key to increasing yields in this process.

Screening of filamentous fungi for production of xylitol from D-xylose

Foram avaliados onze fungos filamentosos para a producão de xilitol em batelada. A producão foi baixa nas condicões de cultivo utilizadas. A máxima, 0,52 g L-1 de xilitol a partir de 11,50 g L-1 de xilose, foi obtida com Penicillium crustosum, com consumo de 76 per center da xilose inicial.