Physiological conditions which promote the synthesis of L-ascorbic acid (vitamin C) by cultures of metabolically engineered Kluyveromyces lactis

O ácido L-ascórbico (ALA) é produzido naturalmente por plantas a partir de D-glicose. Leveduras sintetizam um metabólito semelhante, o ácido D-eritroascórbico (ADEA). Embora este composto não mostre atividade contra o escorbuto, ele contém uma função antioxidante, mas é produzido pelo micro-organismo em baixas concentrações. Recentemente, com a finalidade de fazer as leveduras serem capazes de converter o componente D-galactose da D-lactose do soro de queijo em ALA, a linhagem selvagem de Kluyveromyces lactis CBS2359 foi transformada com genes, que integram a via de biossíntese de L-galactose, isolados de Arabidopsis thaliana. Na presença do intermediário L-galactose, ALA pode então ser sintetizado pela levedura com as enzimas responsáveis pela produção de ADEA. Foi demonstrado que a linhagem engenheirada JVC 1-56 foi capaz de sintetizar ALA em comparação com a selvagem, mas com baixo rendimento. Neste sentido, estudos que auxiliem o aumento da produção de ALA são relevantes. Uma vez que a via engenheirada da síntese de ALA e a via nativa da formação da parede celular de K. lactis possuem um intermediário comum, GDP-D-manose, faz-se necessário avaliar o efeito do desacoplamento da produção de ALA do crescimento que requer constante síntese da parede. Além disso, considerando a propriedade antioxidante do ALA, a expectativa é de que a produção deste metabólito aumentasse quando JVC 1-56 fosse exposta a uma condição de estresse oxidativo por menadiona. Neste contexto, este trabalho investigou as condições fisiológicas mencionadas utilizando os meios Yeast Galactose Base (YGB) e soro de queijo ultrafiltrado (SQU - subproduto de indústrias de queijo em que predomina lactose como fonte de carbono). Foi averiguado que Kluyveromyces lactis JVC 1-56 produz ALA com rendimentos maiores quando cultivada por 96 horas em batelada no meio YPGal. O cultivo em baixas velocidades de crescimento (0,04 h -1) predispõe as células a sintetizar mais ALA por unidade de massa celular (0,80 mg/mg). Entretanto, o rendimento da cultura contínua operada como quimostato, tendo nitrogênio como substrato limitante, ainda mostrou ser inferior ao previsto na batelada (1,94 mg/mg). Já o estresse oxidativo por 12,5 μM de menadiona sobre K. lactis JVC1- 56, nas condições aplicadas, não foi eficaz no aumento da produção de ALA (1,47 mg/mg). Ainda, o rendimento de ADEA foi superior ao de ALA nas duas estratégias: 1,37 mg/mg a 0,21 h-1 na cultura sob regime permanente, 8,52 mg/mg na batelada sob estresse oxidativo e 10,33 mg/mg na batelada na ausência do agente oxidante, indicando que ele pode estar em uma configuração mais estável do que a vitamina C. Finalmente, o permeado do soro de queijo continua sendo uma perspectiva para a conversão da lactose em um produto biotecnológico de maior valor agregado.The L-ascorbic acid (ALA) is produced naturally by plants from D-glucose. Yeasts synthesize a similar metabolite, D-eritroascorbic acid (ADEA). Although this compound does not show activity against scurvy, it contains an antioxidant function, but it is produced in low concentrations by the microorganism. Recently, in order to make yeasts are capable of converting D-galactose D-lactose from cheese whey into ALA, the wild strain Kluyveromyces lactis CBS2359 has been transformed with genes that integrate the L-galactose biosynthesis pathway, isolated from Arabidopsis thaliana. In the presence of intermediate L-galactose, ALA can be synthesized by yeast with enzymes responsible for ADEA production. It has been demonstrated that the engineered strain JVC 1-56 was able to synthesize ALA compared to the wild type, but the yield was still low. In this regard, studies to assist the increased of ALA production are relevant. Once the engineered ALA pathway and native cell wall formation pathway of K. lactis have a common intermediate, GDP-D-mannose, we need to evaluate the effect of uncoupling of vitamin C synthesis route of the growth that requires constant wall synthesis. Moreover, considering the antioxidant properties of ALA, the expectation is that the production of this metabolite increase when JVC 1-56 is exposed to a oxidative stress condition by menadione. In this context, this study investigated the both physiological conditions, using the media Yeast Galactose Base (YGB) and ultrafiltered cheese whey (SQU - byproduct of cheese industry that predominates lactose as carbon source). It was analyzed that Kluyveromyces lactis JVC 1-56 produces higher yields of ALA when it is grown for 96 hours in batch culture in the middle YPGal. Cultivation at low growth rates (0.04 h-1) predisposes the cells to synthesize more ALA per unit cell mass (0.80 mg/mg). However, the yield of continuous culture operated as quimostato, limiting the substrate nitrogen, still proved to be lower than expected in the batch (1.94 mg/mg). Oxidative stress by 12.5 mM menadione on K. lactis JVC1 -56, in the conditions applied, was not effective in the increasing the ALA production (1.47 mg/mg). Still, ADEA yield was higher than ALA in two strategies: 1.37 mg/mg at 0.21 h-1 in culture under steady state, 8.52 mg/mg in batch under oxidative stress and 10.33 mg/mg in the batch in the absence of oxidizing agent, indicating that ADEA may be in a more stable configuration than vitamin C. Finally, the permeate cheese whey remains a prospect for the conversion of lactose into a biotechnological product of higher value aggregate.Fundação de Amparo a Pesquisa do Estado de Minas Gerai

Respostas de Kluyveromyces marxianus CCT 7735 ao estresse por etanol reveladas pelas análises proteômica e metabolômica

Non-Saccharomyces yeasts, such as Kluyveromyces marxianus, have called attention as promising strains for bioethanol production. K. marxianus displays desirable physiological characteristics for bioethanol production, such as capacity to assimilate pentoses and disaccharides beyond sucrose – present in the agro- industrial and forest residues – and thermotolerance. However, K. marxianus, contrary to Saccharomyces cerevisiae, is not tolerant to high ethanol concentrations. Moreover, its physiological responses to ethanol are not well elucidated; therefore, characterizing its physiological responses under ethanol stress is pivotal to apply this knowledge into metabolic engineering approaches to construct strains tolerant to ethanol. Thus, the aim of this work was to determine the ethanol responses of K. marxianus CCT 7735 subjected to 1 and 4 h of ethanol stress through both protein and metabolic profiles. It was observed that at 1 h of stress, although glycolytic enzymes and alcohol dehydrogenases were more abundant lactose and ethanol are not consumed by the yeast. On the other hand, at 4 h the abundance of proteins involved in carbon pathways and protein translation was reduced. In addition, there was an increase in the abundance of proteins and metabolites related to the response to oxidative and osmotic stresses, as well as to heat shock proteins. Interestingly, at 1 h of exposure to ethanol, plasma membrane ATPase was highly abundant, likely to counteract the proton motive force dissipation, which is related to the influx of protons due to increased permeability caused by ethanol. However, at 1 and 4 h of stress, respectively, the level of the metabolites trehalose, ergosterol and some amino acids reduced. Therefore, the results obtained in this study provide important information to improve the understanding of the ethanol responses displayed by K. marxianus.No contexto de produção de bioetanol, leveduras não-Saccharomyces, como Kluyveromyces marxianus, vêm recebendo atenção especial. K. marxianus apresenta características fisiológicas desejáveis para a produção desse biocombustível, tais como capacidade de assimilar pentoses e dissacarídeos além da sacarose – presentes nos resíduos agroindustriais e florestais – e termotolerância. Entretanto, K. marxianus não tolera altas concentrações deste álcool, ao contrário da levedura convencional Saccharomyces cerevisiae. Além disso, as respostas fisiológicas dessas leveduras ao etanol não são bem elucidadas; portanto, faz-se necessário caracterizá-las, tendo como perspectiva a aplicação deste conhecimento em estratégias de engenharia metabólica para a obtenção de linhagens tolerantes a este álcool. Assim, este trabalho teve como objetivo determinar, via perfis proteico e metabólico, as respostas apresentadas por K. marxianus CCT 7735 ao etanol por 1 e 4 horas. Foi observado em 1 h de estresse que, embora as enzimas da via glicolítica e álcool desidrogenases sejam mais abundantes, lactose e etanol não são consumidos pela levedura. Por outro lado, em 4 h a abundância de proteínas envolvidas nas vias de consumo de carbono e no processo de tradução reduziu. Além disso, houve aumento na abundância de proteínas e metabólitos relacionados com respostas aos estresses oxidativo e osmótico, bem como de proteínas de choque térmico. Curiosamente, em 1 h de exposição ao etanol, a abundância da ATPase de membrana plasmática aumentou, o que possivelmente ocorreu como uma resposta adaptativa a fim de evitar a dissipação da força próton-motiva que está associada ao aumento da permeabilidade causada pelo etanol. No entanto, em 1 e 4 h de estresse, respectivamente, o nível dos metabólitos trealose, ergosterol e de alguns aminoácidos diminuiu. Portanto, os resultados obtidos neste estudo forneceram informações relevantes para auxiliar o entendimento das respostas apresentadas por K. marxianus, ao estresse por etanol.Fundação de Amparo à Pesquisa do Estado de Minas Gerai

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

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 β-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 β-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

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