Metabolic fluxes may be regulated "hierarchically," e.g., by changes of gene expression that adjust enzyme capacities (Vmax) and/or "metabolically" by interactions of enzymes with substrates, ...products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the almost equal to10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50-20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75-100% of the regulation of protein levels.
A quantitative analysis of the impact of feedback inhibition on aromatic amino acid biosynthesis was performed in chemostat cultures of
Saccharomyces cerevisiae. Introduction of a ...tyrosine-insensitive allele of
ARO4 (encoding 3-deoxy-
d-
arabino-heptulosonate-7-phosphate synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These amino acids were not detected extracellularly. However, an over 100-fold increase of the extracellular levels of phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux through the aromatic pathway was estimated to be over four-fold. Individual overexpression of either the wild-type or feedback insensitive allele of
ARO7 (encoding chorismate mutase had no significant impact. However when they were combined with the Tyr-insensitive
ARO4 allele in combination with the Tyr-insensitive
ARO4 allele, extracellular concentrations of aromatic compounds were increased by over 200-fold relative to the reference strain, corresponding to a 4.5-fold increase of the flux through the aromatic amino acid biosynthesis pathway. Elimination of allosteric control on these two key reactions in aromatic amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids. This broader impact of amino acid biosynthesis presents a challenge in rational optimization of the production of specific amino acids and derived flavour compounds.
Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a ...nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.
Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the ...past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.
Analysis of predicted fungal proteomes revealed a large family of sequences that showed similarity to the
Saccharomyces cerevisiae
Class-I dihydroorotate dehydrogenase Ura1, which supports synthesis ...of pyrimidines under aerobic and anaerobic conditions. However, expression of codon-optimised representatives of this gene family, from the ascomycete
Alternaria alternata
and the basidiomycete
Schizophyllum commune
, only supported growth of an
S. cerevisiae ura1
Δ mutant when synthetic media were supplemented with dihydrouracil. A hypothesis that these genes encode NAD(P)
+
-dependent dihydrouracil dehydrogenases (EC 1.3.1.1 or 1.3.1.2) was rejected based on absence of complementation in anaerobic cultures. Uracil- and thymine-dependent oxygen consumption and hydrogen-peroxide production by cell extracts of
S. cerevisiae
strains expressing the
A. alternata
and
S. commune
genes showed that, instead, they encode active dihydrouracil oxidases (DHO, EC1.3.3.7). DHO catalyses the reaction dihydrouracil + O
2
→ uracil + H
2
O
2
and was only reported in the yeast
Rhodotorula glutinis
(Owaki in J Ferment Technol 64:205–210, 1986). No structural gene for DHO was previously identified. DHO-expressing strains were highly sensitive to 5-fluorodihydrouracil (5F-dhu) and plasmids bearing expression cassettes for DHO were readily lost during growth on 5F-dhu-containing media. These results show the potential applicability of fungal DHO genes as counter-selectable marker genes for genetic modification of
S. cerevisiae
and other organisms that lack a native DHO. Further research should explore the physiological significance of this enigmatic and apparently widespread fungal enzyme.
Pathway swapping Kuijpers, Niels G. A.; Solis-Escalante, Daniel; Luttik, Marijke A. H. ...
Proceedings of the National Academy of Sciences - PNAS,
12/2016, Volume:
113, Issue:
52
Journal Article
Peer reviewed
Open access
Recent developments in synthetic biology enable one-step implementation of entire metabolic pathways in industrial microorganisms. A similarly radical remodelling of central metabolism could greatly ...accelerate fundamental and applied research, but is impeded by the mosaic organization of microbial genomes. To eliminate this limitation, we propose and explore the concept of “pathway swapping,” using yeast glycolysis as the experimental model. Construction of a “single-locus glycolysis” Saccharomyces cerevisiae platform enabled quick and easy replacement of this yeast’s entire complement of 26 glycolytic isoenzymes by any alternative, functional glycolytic pathway configuration. The potential of this approach was demonstrated by the construction and characterization of S. cerevisiae strains whose growth depended on two nonnative glycolytic pathways: a complete glycolysis from the related yeast Saccharomyces kudriavzevii and a mosaic glycolysis consisting of yeast and human enzymes. This work demonstrates the feasibility and potential of modular, combinatorial approaches to engineering and analysis of core cellular processes.
The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor ...for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker's yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker's yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker's yeast as a "cell factory" for sustainable production of fuels and chemicals.
D-galacturonate, a key constituent of pectin, is a ubiquitous monomer in plant biomass. Anaerobic, fermentative conversion of D-galacturonate is therefore relevant in natural environments as well as ...in microbial processes for microbial conversion of pectin-containing agricultural residues. In currently known microorganisms that anaerobically ferment D-galacturonate, its catabolism occurs via the galacturonate-isomerase pathway. Redox-cofactor balancing in this pathway strongly constrains the possible range of products generated from anaerobic D-galacturonate fermentation, resulting in acetate as the predominant organic fermentation product. To explore metabolic diversity of microbial D-galacturonate fermentation, anaerobic enrichment cultures were performed at pH 4. Anaerobic batch and chemostat cultures of a dominant
strain isolated from these enrichment cultures produced near-equimolar amounts of lactate and acetate from D-galacturonate. A combination of whole-genome sequence analysis, quantitative proteomics, enzyme activity assays in cell extracts, and
product identification demonstrated that D-galacturonate metabolism in
occurs via a novel pathway. In this pathway, mannonate generated by the initial reactions of the canonical isomerase pathway is converted to 6-phosphogluconate by two novel biochemical reactions, catalyzed by a mannonate kinase and a 6-phosphomannonate 2-epimerase. Further catabolism of 6-phosphogluconate then proceeds via known reactions of the phosphoketolase pathway. In contrast to the classical isomerase pathway for D-galacturonate catabolism, the novel pathway enables redox-cofactor-neutral conversion of D-galacturonate to ribulose-5-phosphate. While further research is required to identify the structural genes encoding the key enzymes for the novel pathway, its redox-cofactor coupling is highly interesting for metabolic engineering of microbial cell factories for conversion of pectin-containing feedstocks into added-value fermentation products such as ethanol or lactate. This study illustrates the potential of microbial enrichment cultivation to identify novel pathways for the conversion of environmentally and industrially relevant compounds.
Abstract
In Saccharomyces cerevisiae, reduction of NAD+ to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, ...with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD+. At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial ‘internal’ NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol–acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate–oxaloacetate shuttle, a malate–aspartate shuttle and a malate–pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate–citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.
Abstract
Background
In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial ...respiration makes their in vivo activity dependent on oxygen availability.
Saccharomyces cerevisiae
and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete
Anaeromyces robustus
and the yeasts
Schizosaccharomyces japonicus
and
Dekkera bruxellensis
) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD.
Results
Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (
Arura9, SjURA9, DbURA9
) in an
S. cerevisiae ura1Δ
strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing
DbURA9
showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing
DbURA9
-expressing strains showed mutations in
FUM1
, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to
S. cerevisiae
mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of
SjURA9
in
S. cerevisiae
reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9.
Conclusions
Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in
S. cerevisiae
, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in
S. cerevisiae
strains expressing an anaerobically active DHOD from
Sch. japonicus
.
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