-
Synthetic and Systems Biotechnology Dec 2021Pretreatment of lignocellulosic biomass is crucial for the release of biofermentable sugars for biofuels production, which could greatly alleviate the burgeoning...
Inhibitor tolerance and bioethanol fermentability of levoglucosan-utilizing were enhanced by overexpression of stress-responsive gene : The proteomics-guided metabolic engineering.
Pretreatment of lignocellulosic biomass is crucial for the release of biofermentable sugars for biofuels production, which could greatly alleviate the burgeoning environment and energy crisis caused by the massive usage of traditional fossil fuels. Pyrolysis is a cost-saving pretreatment process that can readily decompose biomass into levoglucosan, a promising anhydrosugar; however, many undesired toxic compounds inhibitory to downstream microbial fermentation are also generated during the pyrolysis, immensely impeding the bioconversion of levoglucosan-containing pyrolysate. Here, we took the first insight into the proteomic responses of a levoglucosan-utilizing and ethanol-producing to three representative biomass-derived inhibitors, identifying large amounts of differentially expressed proteins (DEPs) that could guide the downstream metabolic engineering for the development of inhibitor-resistant strains. Fifteen up- and eight down-regulated DEPs were further identified as the biomarker stress-responsive proteins candidate for cellular tolerance to multiple inhibitors. Among these biomarker proteins, YcfR exhibiting the highest expression fold-change level was chosen as the target of overexpression to validate proteomics results and develop robust strains with enhanced inhibitor tolerance and fermentation performance. Finally, based on four plasmid-borne genes encoding the levoglucosan kinase, pyruvate decarboxylase, alcohol dehydrogenase, and protein YcfR, a new recombinant strain LGE- was successfully created, showing much higher acetic acid-, furfural-, and phenol-tolerance levels compared to the control without overexpression of . The specific growth rate, final cell density, ethanol concentration, ethanol productivity, and levoglucosan consumption rate of the recombinant were also remarkably improved. From the proteomics-guided metabolic engineering and phenotypic observations, we for the first time corroborated that YcfR is a stress-induced protein responsive to multiple biomass-derived inhibitors, and also developed an inhibitors-resistant strain that could produce bioethanol from levoglucosan in the presence of inhibitors of relatively high concentration. The newly developed LGE- strain that could eliminate the commonly-used costly detoxicification processes, is of great potential for the cost-effective bioethanol production from the biomass-derived pyrolytic substrates.
PubMed: 34853817
DOI: 10.1016/j.synbio.2021.11.003 -
Microorganisms Nov 2021Sourdough is a fermentation culture which is formed following metabolic activities of a multiple bacterial and fungal species on raw dough. However, little is known...
Sourdough is a fermentation culture which is formed following metabolic activities of a multiple bacterial and fungal species on raw dough. However, little is known about the mechanism of interaction among different species involved in fermentation. In this study, Sx3 and Sq7 were selected. Protein changes in sourdough, fermented with single culture (either Sx3 or Sq7) and mixed culture (both Sx3 and Sq7), were evaluated by proteomics. The results show that carbohydrate metabolism in mixed-culture-based sourdough is the most important metabolic pathway. A greater abundance of L-lactate dehydrogenase and UDP-glucose 4-epimerase that contribute to the quality of sourdough were observed in mixed-culture-based sourdough than those produced by a single culture. Calreticulin, enolase, seryl-tRNA synthetase, ribosomal protein L23, ribosomal protein L16, and ribosomal protein L5 that are needed for the stability of proteins were increased in mixed-culture-based sourdough. The abundance of some compounds which play an important role in enhancing the nutritional characteristics and flavour of sourdough (citrate synthase, aldehyde dehydrogenase, pyruvate decarboxylase, pyruvate dehydrogenase E1 and acetyl-CoA) was decreased. In summary, this approach provided new insights into the interaction between and in sourdough, which may serve as a base for further research into the detailed mechanism.
PubMed: 34835478
DOI: 10.3390/microorganisms9112353 -
Proceedings of the National Academy of... Nov 2021α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of...
α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high-molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.
Topics: Actinobacteria; Bacteria; Biochemical Phenomena; Computational Biology; Crystallography, X-Ray; Ketoglutarate Dehydrogenase Complex; Kinetics; Molecular Conformation; Mycobacterium tuberculosis; Plasmids; Pyruvate Dehydrogenase Complex; Pyruvic Acid
PubMed: 34819376
DOI: 10.1073/pnas.2112107118 -
Cellular and Molecular Life Sciences :... Dec 2021In human metabolism, pyruvate dehydrogenase complex (PDC) is one of the most intricate and large multimeric protein systems representing a central hub for cellular... (Review)
Review
In human metabolism, pyruvate dehydrogenase complex (PDC) is one of the most intricate and large multimeric protein systems representing a central hub for cellular homeostasis. The worldwide used antiepileptic drug valproic acid (VPA) may potentially induce teratogenicity or a mild to severe hepatic toxicity, where the underlying mechanisms are not completely understood. This work aims to clarify the mechanisms that intersect VPA-related iatrogenic effects to PDC-associated dihydrolipoamide dehydrogenase (DLD; E3) activity. DLD is also a key enzyme of α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, α-ketoadipate dehydrogenase, and the glycine decarboxylase complexes. The molecular effects of VPA will be reviewed underlining the data that sustain a potential interaction with DLD. The drug-associated effects on lipoic acid-related complexes activity may induce alterations on the flux of metabolites through tricarboxylic acid cycle, branched-chain amino acid oxidation, glycine metabolism and other cellular acetyl-CoA-connected reactions. The biotransformation of VPA involves its complete β-oxidation in mitochondria causing an imbalance on energy homeostasis. The drug consequences as histone deacetylase inhibitor and thus gene expression modulator have also been recognized. The mitochondrial localization of PDC is unequivocal, but its presence and function in the nucleus were also demonstrated, generating acetyl-CoA, crucial for histone acetylation. Bridging metabolism and epigenetics, this review gathers the evidence of VPA-induced interference with DLD or PDC functions, mainly in animal and cellular models, and highlights the uncharted in human. The consequences of this interaction may have significant impact either in mitochondrial or in nuclear acetyl-CoA-dependent processes.
Topics: 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); Acetyl Coenzyme A; Acetylation; Animals; Dihydrolipoamide Dehydrogenase; Glycine Dehydrogenase (Decarboxylating); Histone Deacetylase Inhibitors; Humans; Iatrogenic Disease; Ketoglutarate Dehydrogenase Complex; Ketone Oxidoreductases; Liver; Mitochondria; Oxidation-Reduction; Pyruvate Dehydrogenase Complex; Teratogens; Valproic Acid
PubMed: 34718827
DOI: 10.1007/s00018-021-03996-3 -
Plant Physiology Feb 2022Plants have evolutionarily conserved NifU (NFU)-domain proteins that are targeted to plastids or mitochondria. "Plastid-type" NFU1, NFU2, and NFU3 in Arabidopsis... (Comparative Study)
Comparative Study
Plants have evolutionarily conserved NifU (NFU)-domain proteins that are targeted to plastids or mitochondria. "Plastid-type" NFU1, NFU2, and NFU3 in Arabidopsis (Arabidopsis thaliana) play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here, we confirmed that NFU4 and NFU5 are targeted to the mitochondria. The proteins were constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of NFU4 and NFU5 proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes or aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth improved with elevated CO2 treatment. In addition, pyruvate, 2-oxoglutarate, and branched-chain amino acids accumulated in nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, most likely providing Fe-S clusters to lipoyl synthase.
Topics: Arabidopsis Proteins; Gene Expression Regulation, Plant; Genes, Plant; Genetic Variation; Genotype; Iron-Sulfur Proteins; Lipoylation; Mitochondria; Mutation
PubMed: 34718778
DOI: 10.1093/plphys/kiab501 -
Plants (Basel, Switzerland) Sep 2021This study investigated the complex phenotypic and genetic response of Monterey pine () seedlings to co-infections by , the causal agent of pine pitch canker disease,...
This study investigated the complex phenotypic and genetic response of Monterey pine () seedlings to co-infections by , the causal agent of pine pitch canker disease, and the oomycetes and . Monterey pine seedlings were wound-inoculated with each single pathogen and with the combinations / and /. Initially, seedlings inoculated only with showed less severe symptoms than seedlings co-inoculated or inoculated only with or . However, 30 days post-inoculation (dpi), all inoculated seedlings, including those inoculated only with , showed severe symptoms with no significant differences among treatments. The transcriptomic profiles of three genes encoding pathogenesis-related proteins, i.e., chitinase (PR3), thaumatin-like protein (PR5), phenylalanine ammonia-lyase (PAL), and the pyruvate decarboxylase (PDC)-encoding gene were analyzed at various time intervals after inoculation. In seedlings inoculated with single pathogens, stimulated the up-regulation of all genes, while between the two oomycetes, only induced significant up-regulations. In seedlings co-inoculated with and or none of the genes showed a significant over-expression 4 dpi. In contrast, at 11 dpi, significant up-regulation was observed for PR5 in the combination / and PDC in the combination , thus suggesting a possible synergism of multiple infections in triggering this plant defense mechanism.
PubMed: 34685785
DOI: 10.3390/plants10101976 -
Microbial Cell Factories Oct 2021Metabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms,...
BACKGROUND
Metabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms, including Saccharomyces cerevisiae. Although yeast physiology in laboratory strains is well-studied, the metabolic states under industrially relevant scenarios such as winemaking are still not sufficiently understood, especially as there is considerable variation in metabolism between commercial strains. To study the potential causes of strain-dependent variation in the production of volatile compounds during enological conditions, random flux sampling and statistical methods were used, along with experimental extracellular metabolite flux data to characterize the differences in predicted intracellular metabolic states between strains.
RESULTS
It was observed that four selected commercial wine yeast strains (Elixir, Opale, R2, and Uvaferm) produced variable amounts of key volatile organic compounds (VOCs). Principal component analysis was performed on extracellular metabolite data from the strains at three time points of cell cultivation (24, 58, and 144 h). Separation of the strains was observed at all three time points. Furthermore, Uvaferm at 24 h, for instance, was most associated with propanol and ethyl hexanoate. R2 was found to be associated with ethyl acetate and Opale could be associated with isobutanol while Elixir was most associated with phenylethanol and phenylethyl acetate. Constraint-based modeling (CBM) was employed using the latest genome-scale metabolic model of yeast (Yeast8) and random flux sampling was performed with experimentally derived fluxes at various stages of growth as constraints for the model. The flux sampling simulations allowed us to characterize intracellular metabolic flux states and illustrate the key parts of metabolism that likely determine the observed strain differences. Flux sampling determined that Uvaferm and Elixir are similar while R2 and Opale exhibited the highest degree of differences in the Ehrlich pathway and carbon metabolism, thereby causing strain-specific variation in VOC production. The model predictions also established the top 20 fluxes that relate to phenotypic strain variation (e.g. at 24 h). These fluxes indicated that Opale had a higher median flux for pyruvate decarboxylase reactions compared with the other strains. Conversely, R2 which was lower in all VOCs, had higher median fluxes going toward central metabolism. For Elixir and Uvaferm, the differences in metabolism were most evident in fluxes pertaining to transaminase and hexokinase associated reactions. The applied analysis of metabolic divergence unveiled strain-specific differences in yeast metabolism linked to fusel alcohol and ester production.
CONCLUSIONS
Overall, this approach proved useful in elucidating key reactions in amino acid, carbon, and glycerophospholipid metabolism which suggest genetic divergence in activity in metabolic subsystems among these wine strains related to the observed differences in VOC formation. The findings in this study could steer more focused research endeavors in developing or selecting optimal aroma-producing yeast stains for winemaking and other types of alcoholic fermentations.
Topics: Fermentation; Food Microbiology; Metabolic Flux Analysis; Metabolome; Metabolomics; Models, Biological; Odorants; Saccharomyces cerevisiae; Volatile Organic Compounds; Wine
PubMed: 34674718
DOI: 10.1186/s12934-021-01694-0 -
PloS One 20212-Phenylethanol (2-PE) is a valuable aromatic compound with favorable flavors and good properties, resulting in its widespread application in the cosmetic, food and...
2-Phenylethanol (2-PE) is a valuable aromatic compound with favorable flavors and good properties, resulting in its widespread application in the cosmetic, food and medical industries. In this study, a mutant strain, AD032, was first obtained by adaptive evolution under 2-PE stress. Then, a fusion protein from the Ehrlich pathway, composed of tyrB from Escherichia coli, kdcA from Lactococcus lactis and ADH2 from Saccharomyces cerevisiae, was constructed and expressed. As a result, 3.14 g/L 2-PE was achieved using L-phenylalanine as a precursor. To further increase 2-PE production, L-glutamate oxidase from Streptomyces overexpression was applied for the first time in our research to improve the supply of α-ketoglutarate in the transamination of 2-PE synthesis. Furthermore, we found that the disruption of the pyruvate decarboxylase encoding gene PDC5 caused an increase in 2-PE production, which has not yet been reported. Finally, assembly of the efficient metabolic modules and process optimization resulted in the strain RM27, which reached 4.02 g/L 2-PE production from 6.7 g/L L-phenylalanine without in situ product recovery. The strain RM27 produced 2-PE (0.8 mol/mol) with L-phenylalanine as a precursor, which was considerably high, and displayed manufacturing potential regarding food safety and process simplification aspects. This study suggests that innovative strategies regarding metabolic modularization provide improved prospects for 2-PE production in food exploitation.
Topics: Adaptation, Physiological; Biological Evolution; Carboxy-Lyases; Fermentation; Ketoglutaric Acids; Metabolic Engineering; Mutation; Phenylethyl Alcohol; Recombinant Fusion Proteins; Saccharomyces cerevisiae
PubMed: 34665833
DOI: 10.1371/journal.pone.0258180 -
Frontiers in Plant Science 2021Sugarcane is an economically important crop contributing to the sugar and ethanol production of the world with 80 and 40%, respectively. Despite its importance as the...
Sugarcane is an economically important crop contributing to the sugar and ethanol production of the world with 80 and 40%, respectively. Despite its importance as the main crop for sugar production, the mechanisms involved in the regulation of sucrose accumulation in sugarcane culms are still poorly understood. The aim of this work was to compare the quantitative changes of proteins in juvenile and maturing internodes at three stages of plant development. Label-free shotgun proteomics was used for protein profiling and quantification in internodes 5 (I) and 9 (I) of 4-, 7-, and 10-month-old-plants (4M, 7M, and 10M, respectively). The I/I ratio was used to assess the differences in the abundance of common proteins at each stage of internode development. I of 4M plants showed statistically significant increases in the abundance of several enzymes of the glycolytic pathway and proteoforms of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC). The changes in content of the enzymes were followed by major increases of proteins related to O transport like hemoglobin 2, ROS scavenging enzymes, and enzymes involved in the ascorbate/glutatione system. Besides, intermediates from tricarboxylic acid cycle (TCA) were reduced in I-4M, indicating that the increase in abundance of several enzymes involved in glycolysis, pentose phosphate cycle, and TCA, might be responsible for higher metabolic flux, reducing its metabolites content. The results observed in I-4M indicate that hypoxia might be the main cause of the increased flux of glycolysis and ethanolic fermentation to supply ATP and reducing power for plant growth, mitigating the reduction in mitochondrial respiration due to the low oxygen availability inside the culm. As the plant matured and sucrose accumulated to high levels in the culms, the proteins involved in glycolysis, ethanolic fermentation, and primary carbon metabolism were significantly reduced.
PubMed: 34659289
DOI: 10.3389/fpls.2021.716964 -
Metabolic Engineering Communications Dec 2021Engineered strains of the yeast are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids....
Engineered strains of the yeast are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use l-phenylalanine and/or l-tyrosine, generated by the yeast shikimate pathway, as aromatic precursors. The Ehrlich pathway converts these precursors to aromatic fusel alcohols and acids, which are undesirable by-products of yeast strains engineered for production of high-value aromatic compounds. Activity of the Ehrlich pathway requires any of four 2-oxo-acid decarboxylases (2-OADCs): Aro10 or the pyruvate-decarboxylase isoenzymes Pdc1, Pdc5, and Pdc6. Elimination of pyruvate-decarboxylase activity from is not straightforward as it plays a key role in cytosolic acetyl-CoA biosynthesis during growth on glucose. In a search for pyruvate decarboxylases that do not decarboxylate aromatic 2-oxo acids, eleven yeast and bacterial 2-OADC-encoding genes were investigated. Homologs from (), (), (), () and ( and ) complemented a Pdc strain of for growth on glucose. Enzyme-activity assays in cell extracts showed that these genes encoded active pyruvate decarboxylases with different substrate specificities. In these assays, Pdc1, Pdc1.2 or Pdc1.3 had no substrate specificity towards phenylpyruvate. Replacing Aro10 and Pdc1,5,6 by these bacterial decarboxylases completely eliminated aromatic fusel-alcohol production in glucose-grown batch cultures of an engineered coumaric acid-producing strain. These results outline a strategy to prevent formation of an important class of by-products in 'chassis' yeast strains for production of non-native aromatic compounds.
PubMed: 34584841
DOI: 10.1016/j.mec.2021.e00183