3-hydroxypropionic acid production from crude glycerol with Lactobacillus diolivorans

Biodiesel consists of fatty acid methyl esters and is produced via transesterification of long chain fatty acids, derived from vegetable oil, with methanol. The principle by-product of this process, which makes up to 10% of the biodiesel production, is crude glycerol. Since its purification is rather unsustainable, the microbial upgrading to value-added products opens new opportunities. Lactobacillus diolivorans metabolizes glycerol to balance its electron household by reducing the intermediate 3-hydroxypropione aldehyde to 1,3-propanediol or oxidizing it to 3-hydroxypropionic acid. This species of Lactobacillus is a very effective natural producer of 1,3-propanediol, with titers up to 90 g/L, but as well shows potential for the production of 3-hydroxypropionic acid, which is considered as one of the top twelve value-added platform compounds from biomass according to the US Department of Energy. In this study, it was shown, that process engineering to manipulate the redox household of L. diolivorans is a valuable tool to shift the product pattern. By switching to an aerobic process, the production of 3-hydroxypropionic acid could be improved to titers up to 40 g/L compared to 27 g/L in the anaerobic process. Another target is the feeding strategy, since L. diolivorans is not able to use glycerol as sole energy source. The metabolization of sugars like glucose generates excess electrons which favor the reductive pathway for glycerol utilization. Different molar ratios of glucose to glycerol as well as other carbon sources were tested to study their impact on the product pattern. These process engineering approaches together with the relatively high robustness of L. diolivorans towards this toxic product are promising steps towards the optimization of 3-hydroxypropionic acid production and render L. diolivorans a future host for metabolic modeling target

A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging

The combination of functional genomics with next generation sequencing facilitates new experimental strategies for addressing complex biological phenomena. Here, we report the identification of a gain-of-function allele of peroxiredoxin (thioredoxin peroxidase, Tsa1p) via whole-genome re-sequencing of a dominantSaccharomyces cerevisiae mutant obtained by chemical mutagenesis. Yeast strain K6001, a screening system for lifespan phenotypes, was treated with ethyl methanesulfonate (EMS). We isolated an oxidative stress-resistant mutant (B7) which transmitted this phenotype in a background-independent, monogenic and dominant way. By massive parallel pyrosequencing, we generated an 38.8 fold whole-genome coverage of the strains, which differed in 12,482 positions from the reference (S288c) genome. Via a subtraction strategy, we could narrow this number to 13 total and 4 missense nucleotide variations that were specific for the mutant. Via expression in wild type backgrounds, we show that one of these mutations, exchanging a residue in the peroxiredoxin Tsa1p, was responsible for the mutant phenotype causing background-independent dominant oxidative stress-resistance. These effects were not provoked by altered Tsa1p levels, nor could they be simulated by deletion, haploinsufficiency or over-expression of the wild-type allele. Furthermore, via both a mother-enrichment technique and a micromanipulation assay, we found a robust premature aging phenotype of this oxidant-resistant strain. Thus, TSA1-B7 encodes for a novel dominant form of peroxiredoxin, and establishes a new connection between oxidative stress and aging. In addition, this study shows that the re-sequencing of entire genomes is becoming a promising alternative for the identification of functional alleles in approaches of classic molecular genetics

Systems-level organization of yeast methylotrophic lifestyle

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

Interaction of Werner gene and SNEV

Der Alterungsprozesses ist ein komplexer Vorgang und geht einher mit der Abnahme der Effizienz und Genauigkeit der DNA-Reparaturmechanismen. Die Zelle trifft während des Lebenszyklus auf verschiedenste DNA-Schäden. Zu den gefährlichsten DNA-Schäden gehören die DNA Interstrand Crosslinks (ICLs). Der exakte Reparaturmechanismus für ICLs ist bis zum jetzigen Zeitpunkt nicht bekannt. Es herrscht daher ein großes Interesse diesen Reparaturmechanismus besser zu verstehen. Diese Diplomarbeit beschäftigt sich mit 2 Genen, welche auch für die ICL Reparatur wesentlich sind. Die Kompelixiziät von multizellulären Organismen erschwert das Verständnis komplexer zellulärer Vorgänge. Aus diesem Grund wurde Saccharomyces cerevisiae als eukaryotischer Modelorganismus verwendet. Das erste der oben erwähnten Gene ist Prp19/Pso4, das eine duale Rolle in der Zelle spielt. Einerseits besitzt Prp19/Pso4 eine wichtige Funktion in Bezug auf pre-mRNA splicing, aber es spielt auch eine wichtige Rolle bei der Reparatur von DNA-Schäden. Das zweite Gen ist Sgs1 und wird der Familie der RecQ-Helicasen zugerechnet. RecQ-Helicasen sind hoch konservierte DNA-Helicasen, die in verschiedenste Prozesse , wie DNA-Reparatur oder DNA-Replikation, involviert sind. Das humane Ortholog für Sgs1 ist das Werner Gen. Eine Mutation innerhalb dieses Gens verursacht das Werner Syndrom. Ein anderer Teil dieser Diplomarbeit beschäftigt sich mit der Analyse von Next Generation Sequencing Daten. Das Ziel hier eine Mutation zu finden, die eine spezifischen Phänotyp verursacht. Dieser Teil beinhaltet zwei Projekte, die unterschiedliche Next Generation Sequencing Technologie verwenden, jedoch das gleiche Ziel haben. Die 454 Sequenzierungs Technologie wurde verwendet, um eine dominante Mutation in K6001-B7 zu finden. Die Mutation konnte schließlich im Tsa1 Gen identifiziert werden. Im zweiten Projekt wurden zwei unabhängig entstandenen Revertanten und der parentale pso4-1 Stamm mit der Illumina/Solexa Technologie sequenziert. Das Ergebnis hier ist, dass nach der bioinformatischen Analyse ein SNP für Revertante 1 vorhanden ist. Für die Revertante 2 bleiben 10 SNPs und INDELS übrig. Diese Variationen müssen nun mittels PCR geprüft werden.The accumulation of somatic mutation is a major cause of aging and age-related diseases. In addition to this accumulation the efficiency and fidelity of the DNA repair mechanism decline with progression of aging. During the lifecycle of a cell it faces various types of DNA damages. One of the most harmful DNA damages is DNA interstrand cross links (ICLs). The exact repair process of ICLs is not known yet, but it involves different DNA repair pathways. So there is a big effort to understand the ICL repair pathway. This diploma thesis deals with two specific genes, which have a role in ICL repair. The complexity of multicellular organism often makes it hard to understand cellular processes. Therefore Saccharomyces cerevisiae was used as a single cell eukaryote model organism. The first gene is Prp19/Pso4, which plays a dual role in the cell. It has an essential function in pre-mRNA splicing and a role in processing of DNA lesions especially ICLs The second gene Sgs1 belongs to the RecQ-helicase family. RecQ-helicases are highly conserved DNA helicases that are involved in various processes like as DNA repair, DNA replication. The human homologue of Sgs1 is the WRN-gene. A mutation within this gene causes the Werner Syndrome. Werner syndrome is an autosomal recessive disorder and belongs to the group of pre-mature aging disorder. Another chapter of this diploma thesis was the analysis of next generation sequencing data. Two different next generation sequencing technologies were used to identify specific mutations in yeast strains. 454 Life Sciences/Roche sequencing technology was used to identify a dominant mutation in K6001-B7, which causes oxidants resistance. The phenotype causing mutation was identified by subtractive bioinformatic analysis and finally was located in the Tsa1 gene. The same approach was used to identify mutations in two independent developed reverant yeast strains. The Illumina/Solexa sequencing technology was used to sequence the two revertants and the parental pso4-1 strain. The final outcome was that 1 putative SNP was found in the revertant 1 and 10 SNPs and INDELS were found in revertant 2. This variations have to be checked by PCR and test for restoring the revertant phenotype in the pso4-1 strain.von Hannes RußmayerWien, Univ. für Bodenkultur, Dipl.-Arb., 2010(VLID)112768

Customizing amino acid metabolism of <i>Pichia pastoris</i> for recombinant protein production

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems-level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6-fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures

Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production

The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial β-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy

Additional file 3: of Systems-level organization of yeast methylotrophic lifestyle

Enrichment of the peroxisomal marker protein Pex3p in the peroxisomal fraction. (PDF 271 kb

Additional file 4: of Systems-level organization of yeast methylotrophic lifestyle

Proteomic identification and quantification of methanol metabolic enzymes and control proteins in peroxisomal fractions and homogenates of P. pastoris cells grown on methanol. Containing the following three sheets: Protein hits: contains all identified proteins that met the threshold in at least one sample, with their respective MASCOT scores, number of peptides, and percent sequence coverage. Peptide hits: list of all identified peptides, their MASCOT scores, mass and charge values, and intensities. Peptides used for quant + areas: lists all peptides of the proteins in Table 3 that were used for quantification, and their respective peak areas in the different samples. (XLSX 879 kb