RESUMEN
Cat8 is a C6 zinc cluster transcription activator in yeast. It is generally recognized that the transcription of CAT8 is inhibited and that Cat8 is inactive in the presence of high concentrations of glucose. However, our recent study found that constitutively overexpressed Cat8 played a regulatory role in Saccharomyces cerevisiae in the presence of 20 g/L glucose. To explore the regulatory network of Cat8 at high glucose concentrations, CAT8 was both overexpressed and deleted in this study. Cell growth and glucose consumption in different media were significantly accelerated by the deletion of CAT8, while the lag period was greatly shortened. RNA-seq and genetic modification showed that the deletion of CAT8 changed the type of energy metabolism in yeast cells. Many genes related to the mitochondrial respiratory chain were downregulated, resulting in a reduction in aerobic respiration and the tricarboxylic acid cycle. Meanwhile, both the energy supply of anaerobic ethanol fermentation and the Crabtree effect of S. cerevisiae were enhanced by the deletion of CAT8. CAT8 knockout cells show a higher sugar uptake rate, a higher cell growth rate, and higher tolerance to glucose than the wild-type strain YS58. This study expands the understanding of the regulatory network of Cat8 and provides guidance for modulating yeast cell growth. KEY POINTS: ⢠The deletion of CAT8 promoted cell growth of S. cerevisiae. ⢠Transcriptome analysis revealed the regulation network of Cat8 under 1% glucose condition. ⢠CAT8 deletion increases the glucose tolerance of cells by enhancing the Crabtree effect.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Metabolismo Energético , Fermentación , Glucosa/metabolismo , Transactivadores/genéticaRESUMEN
ß-Caryophyllene is a plant-derived bicyclic sesquiterpene with multiple biological functions. ß-Caryophyllene production by engineered Saccharomyces cerevisiae represents a promising technological route. However, the low catalytic activity of ß-caryophyllene synthase (CPS) is one of the main restrictive factors for ß-caryophyllene production. Here, directed evolution of the Artemisia annua CPS was performed, and variants of CPS enhancing the ß-caryophyllene biosynthesis in S. cerevisiae were obtained, in which an E353D mutant enzyme presented large improvements in Vmax and Kcat. The Kcat/Km of the E353D mutant was 35.5% higher than that of wild-type CPS. Moreover, the E353D variant exhibited higher catalytic activity in much wider pH and temperature ranges. Thus, both the higher catalytic activity and the robustness of the E353D variant contribute to the 73.3% increase in ß-caryophyllene production. Furthermore, the S. cerevisiae chassis was engineered by overexpressing genes related to ß-alanine metabolism and MVA pathway to enhance the synthesis of the precursor, and ATP-binding cassette transporter gene variant STE6T1025N to improve the transmembrane transport of ß-caryophyllene. The combined engineering of CPS and chassis resulted in 70.45 mg/L of ß-caryophyllene after 48 h of cultivation in a test tube, which was 2.93-fold of that of the original strain. Finally, a ß-caryophyllene yield of 594.05 mg/L was obtained by fed-batch fermentation, indicating the potential of ß-caryophyllene production by yeast.
Asunto(s)
Artemisia annua , Sesquiterpenos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos Policíclicos/metabolismo , Sesquiterpenos/metabolismo , Artemisia annua/genética , Óxido Nítrico Sintasa/metabolismo , Ingeniería Metabólica/métodosRESUMEN
Mevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds. Saccharomyces cerevisiae is an attractive chassis for the native active MVA pathway. Here, the truncated form of Enterococcus faecalis MvaE with only 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431-fold and 9-fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and ß-alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3-4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl-CoA supply and ß-alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3-4-CPS generated from expressing ß-caryophyllene synthase in SQ3-4 produced 11.86 ± 0.09 mg l-1 ß-caryophyllene, while strain SQ3-5 resulted from down-regulation of ERG1 in SQ3-4 produced 408.88 ± 0.09 mg l-1 squalene in shake flask cultivations. Strain SQ3-5 produced 4.94 g l-1 squalene in fed-batch fermentation in cane molasses medium, indicating the promising potential for cost-effective production of squalene.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas , Ácido Mevalónico , Saccharomyces cerevisiae , beta-Alanina , Hidroximetilglutaril-CoA Reductasas/metabolismo , Ingeniería Metabólica , Ácido Mevalónico/metabolismo , Ingeniería de Proteínas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Escualeno/metabolismo , Terpenos/metabolismo , beta-Alanina/metabolismoRESUMEN
Cat8 is an important transcription factor regulating the utilization of non-fermentative carbon sources in Saccharomyces cerevisiae. However, our previous studies found that Cat8 may play a critical role in nitrogen metabolism, but the regulatory mechanism has not been elucidated. In this study, the nuclear localization and analysis of regulatory activity showed that the Cat8 function relies on Snf1 kinase. In the fermentation with glucose or glycerol as carbon sources under phenylalanine (Phe) induction, by comparing the changes of cellular gene expression and Cat8 target gene binding profiles after Cat8 overexpression, enhanced transcription was shown among key genes involved in the Ehrlich pathway (e.g., ARO9, ARO10, and ADH2) and its upstream and downstream related factors (e.g., GAP1, AGP1, GAT1, PDR12, and ESPB6), indicating that Cat8 participated in the regulation of nitrogen metabolism. Moreover, highly active Cat8 interacts with transcriptional activator Aro80 and GATA activator Gat1 coordinately to regulate the transcription of ARO10. Altogether, our results showed that Cat8 may act as a global transcription factor in response to nutritional changes, regulating both carbon and nitrogen utilization. This provides a new insight for us to explore the regulation of cell nutrient metabolism networks in yeast.
RESUMEN
Copper is an essential micronutrient for life, whose homeostasis is rigorously regulated to meet the demands of normal biological processes and to minimize the potential toxicity. Copper enriched by yeast is regarded as a safe and bioavailable form of copper supplements. Here, a Saccharomyces cerevisiae mutant strain H247 with expanded storage capability of copper was obtained through atmospheric and room-temperature plasma treatment. Transcriptomic analyses found that transcriptional upregulation of DGA1 might be the major contributor to the enhancement of intracellular copper accumulation in strain H247. The positive correlation between biogenesis of lipid droplets and intracellular accumulation of copper was confirmed by overexpression of the diacylglycerol acyltransferase encoding genes DGA1 and LRO1 or knockout of DGA1. Lipid droplets are not only the storage pool of copper but might prompt the copper trafficking to mitochondria, vacuoles, and Golgi apparatus. These results provide new insights into the sophisticated copper homeostatic mechanisms and the biological functions of lipid droplets.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cobre/farmacología , Gotas Lipídicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , TranscriptomaRESUMEN
BACKGROUND: Saccharomyces cerevisiae is well-known as an ideal model system for basic research and important industrial microorganism for biotechnological applications. Acetic acid is an important growth inhibitor that has deleterious effects on both the growth and fermentation performance of yeast cells. Comprehensive understanding of the mechanisms underlying S. cerevisiae adaptive response to acetic acid is always a focus and indispensable for development of robust industrial strains. eIF5A is a specific translation factor that is especially required for the formation of peptide bond between certain residues including proline regarded as poor substrates for slow peptide bond formation. Decrease of eIF5A activity resulted in temperature-sensitive phenotype of yeast, while up-regulation of eIF5A protected transgenic Arabidopsis against high temperature, oxidative or osmotic stress. However, the exact roles and functional mechanisms of eIF5A in stress response are as yet largely unknown. RESULTS: In this research, we compared cell growth between the eIF5A overexpressing and the control S. cerevisiae strains under various stressed conditions. Improvement of acetic acid tolerance by enhanced eIF5A activity was observed all in spot assay, growth profiles and survival assay. eIF5A prompts the synthesis of Ume6p, a pleiotropic transcriptional factor containing polyproline motifs, mainly in a translational related way. As a consequence, BEM4, BUD21 and IME4, the direct targets of Ume6p, were up-regulated in eIF5A overexpressing strain, especially under acetic acid stress. Overexpression of UME6 results in similar profiles of cell growth and target genes transcription to eIF5A overexpression, confirming the role of Ume6p and its association between eIF5A and acetic acid tolerance. CONCLUSION: Translation factor eIF5A protects yeast cells against acetic acid challenge by the eIF5A-Ume6p-Bud21p/Ime4p/Bem4p axles, which provides new insights into the molecular mechanisms underlying the adaptive response and tolerance to acetic acid in S. cerevisiae and novel targets for construction of robust industrial strains.