Customizing amino acid metabolism of Pichia pastoris 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.

Process engineering towards an oxidative cellular state improves 3-hydroxypropionic acid production with Lentilactobacillus diolivorans.

3-hydroxypropionic acid (3-HP) is among the top platform chemicals proposed for bio based production by microbial fermentation from renewable resources. A promising renewable substrate for 3-HP production is crude glycerol. Only a few microorganisms can efficiently convert glycerol to 3-HP. Among the most promising organisms is Lentilactobacillus diolivorans. In this study, an already established fed-batch process, accumulating 28 g/L 3-HP, was used as a starting point for process engineering. The engineering approaches focused on modulating the cellular redox household towards a more oxidized state, as these conditions favour 3-HP production. Variations of oxygen and glucose availability (controlled by the glucose/glycerol ratio in the feed medium) individually already improved 3-HP production. However, the combination of both optimal parameters (30% O2, 0.025 mol/mol glu/gly) led to the production of 67.7 g/L 3-HP after 180 h of cultivation, which is so far the highest titer reported for 3-HP production using Lactobacillus spp.

Production, Storage Stability, and Susceptibility Testing of Reuterin and Its Impact on the Murine Fecal Microbiome and Volatile Organic Compound Profile.

Background: Probiotics are generally considered as safe, but infections may rarely occur in vulnerable patients. Alternatives to live microorganisms to manage dysbiosis may be of interest in these patients. Reuterin is a complex component system exhibiting broad spectrum antimicrobial activity and a possible candidate substance in these cases. Methods: Reuterin supernatant was cultured from Lentilactobacillus diolivorans in a bioreactor in a two-step process. Storage stability at -20°C and effect of repeated freeze-thaw cycles were assessed by high performance liquid chromatography (HPLC). Antimicrobial activity was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus (S.) aureus, Staphylococcus epidermidis, Streptococcus (S.) agalactiae, Propionibacterium acnes, and Pseudomonas aeruginosae. Male BALBc mice were gavage fed with reuterin supernatant (n = 10) or culture medium (n = 10). Fecal volatile organic compounds (VOC) were assessed by gas chromatography mass spectroscopy; the microbiome was examined by 16S rRNA gene sequencing. Results: The supernatant contained 13.4 g/L reuterin (3-hydroxypropionaldehyde; 3-HPA). 3-HPA content remained stable at -20°C for 35 days followed by a slow decrease of its concentration. Repeated freezing/thawing caused a slow 3-HPA decrease. Antimicrobial activity was encountered against S. aureus, S. epidermidis, and S. agalactiae. Microbiome analysis showed no differences in alpha and beta diversity markers. Linear discriminant effect size (LEfSe) analysis identified Lachnospiraceae_bacterium_COE1 and Ruminoclostridium_5_uncultured_Clostridiales_ bacterium (in the reuterin medium group) and Desulfovibrio_uncultured_ bacterium, Candidatus Arthromitus, Ruminococcae_NK4A214_group, and Eubacterium_xylanophilum_group (in the reuterin group) as markers for group differentiation. VOC analysis showed a significant decrease of heptane and increase of 3-methylbutanal in the reuterin group. Conclusion: The supernatant produced in this study contained acceptable amounts of 3-HPA remaining stable for 35 days at -20°C and exhibiting an antimicrobial effect against S. aureus, S. agalactiae, and S. epidermidis. Under in vivo conditions, the reuterin supernatant caused alterations of the fecal microbiome. In the fecal, VOC analysis decreased heptane and increased 3-methylbutanal were encountered. These findings suggest the high potential of the reuterin system to influence the intestinal microbiome in health and disease, which needs to be examined in detail in future projects.

Microbial 2-butanol production with Lactobacillus diolivorans.

BACKGROUND: Biobutanol has great potential as biofuel of the future. However, only a few organisms have the natural ability to produce butanol. Amongst them, Clostridium spp. are the most efficient producers. The high toxicity of biobutanol constitutes one of the bottlenecks within the biobutanol production process which often suffers from low final butanol concentrations and yields. Butanol tolerance is a key driver for process optimisation and, therefore, in the search for alternative butanol production hosts. Many Lactobacillus species show a remarkable tolerance to solvents and some Lactobacillus spp. are known to naturally produce 2-butanol from meso-2,3-butanediol (meso-2,3-BTD) during anaerobic sugar fermentations. Lactobacillus diolivorans showed already to be highly efficient in the production of other bulk chemicals using a simple two-step metabolic pathway. Exactly, the same pathway enables this cell factory for 2-butanol production. RESULTS: Due to the inability of L. diolivorans to produce meso-2,3-BTD, a two-step cultivation processes with Serratia marcescens has been developed. S. marcescens is a very efficient producer of meso-2,3-BTD from glucose. The process yielded a butanol concentration of 10 g/L relying on wild-type bacterial strains. A further improvement of the maximum butanol titer was achieved using an engineered L. diolivorans strain overexpressing the endogenous alcohol dehydrogenase pduQ. The two-step cultivation process based on the engineered strain led to a maximum 2-butanol titer of 13.4 g/L, which is an increase of 34%. CONCLUSION: In this study, L. diolivorans is for the first time described as a good natural producer for 2-butanol from meso-2,3-butanediol. Through the application of a two-step cultivation process with S. marcescens, 2-butanol can be produced from glucose in a one-vessel, two-step microbial process.

Spotlight on biodiversity of microbial cell factories for glycerol conversion.

Plant oil based industrial oleochemistry leads to a large side stream of crude glycerol, which offers itself as a low price carbon source for microbial chemical production. Compared to sugar, glycerol is more reduced and less microorganisms are able to use it as carbon source. An interesting feature of glycerol conversion is that many organisms cannot use it as carbon source at all, but they readily use it as electron sink under anaerobic conditions. In any case the number of pathways by which glycerol enters the microbial metabolism is quite limited. Having said this, an interesting variety of products of industrial relevance is accumulated by naturally occurring microorganisms which can use glycerol. These chemicals range from fuels and solvents to polymer precursors up to food additives. The limited number of metabolic pathways and the manageable amount of products allow to highlight the importance of tapping the outstanding resource of biodiversity for industrial purposes. The interplay of microbial biodiversity, metabolic engineering and bioprocess engineering is key for economic success in industrial microbiology. In this article we shed light on the biodiversity of naturally glycerol consuming microorganisms and their impact and importance on microbial chemical production.

Microbial production of high value molecules using rayon waste material as carbon-source.

Rayon filaments composed of regenerated cellulose are used as reinforcement materials in tires and to a lower extent in the clothing industry as personal protective equipment e.g. flame retardant cellulosic based materials. After use, these materials are currently transferred to landfills while chemical degradation does not allow the recovery of the cellulose (as glucose) nor the separation of the high valuable flame-retardant pigment. In this study, rayon fibers were enzymatically hydrolyzed to allow recovery of glucose and valuable additives. The glucose was successfully used as carbon source for the production of high value compounds such as itaconic acid, lactic acid and chitosan. 14.2 g/L of itaconic acid, 36.5 g/L of lactic acid and 39.2 g/L of chitosan containing biomass were produced from Escherichia coli, Lactobacillus paracasei and Aspergillus niger, respectively, comparable to yields obtained when using commercial glucose as carbon source.

The Efficient Clade: Lactic Acid Bacteria for Industrial Chemical Production.

Lactic acid bacteria are well known to be beneficial for food production and, as probiotics, they are relevant for many aspects of health. However, their potential as cell factories for the chemical industry is only emerging. Many physiological traits of these microorganisms, evolved for optimal growth in their niche, are also valuable in an industrial context. Here, we illuminate these features and describe why the distinctive adaptation of lactic acid bacteria is particularly useful when developing a microbial process for chemical production from renewable resources. High carbon uptake rates with low biomass formation combined with strictly regulated simple metabolic pathways, leading to a limited number of metabolites, are among the key factors defining their success in both nature and industry.

Metabolomics of Pichia pastoris: impact of buffering conditions on the kinetics and nature of metabolite loss during quenching.

Mass spectrometry-based metabolomic profiling is a powerful strategy to quantify the concentrations of numerous primary metabolites in parallel. To avoid distortion of metabolite concentrations, quenching is applied to stop the cellular metabolism instantly. For yeasts, cold methanol quenching is accepted to be the most suitable method to stop metabolism, while keeping the cells intact for separation from the supernatant. During this treatment, metabolite loss may occur while the cells are suspended in the quenching solution. An experiment for measuring the time-dependent loss of selected primary metabolites in differently buffered quenching solutions was conducted to study pH and salt concentration-dependent effects. Molecular properties of the observed metabolites were correlated with the kinetics of loss to gain insight into the mechanisms of metabolite leakage. Size and charge-related properties play a major role in controlling metabolite loss. We found evidence that interaction with the cell wall is the main determinant to retain a molecule inside the cell. Besides suggesting an improved quenching protocol to keep loss at a minimum, we could establish a more general understanding of the process of metabolite loss during quenching, which will allow to predict optimal conditions for hitherto not analysed metabolites.

Metabolic Flexibility of Yarrowia lipolytica Growing on Glycerol.

The yeast Yarrowia lipolytica is a fascinating microorganism with an amazing metabolic flexibility. This yeast grows very well on a wide variety of carbon sources from alkanes over lipids, to sugars and glycerol. Y. lipolytica accumulates a wide array of industrially relevant metabolites. It is very tolerant to many environmental factors, above all the pH value. It grows perfectly well over a wide pH range, but it has been described, that the pH has a decisive influence on the metabolite pattern accumulated by this yeast. Here, we set out to characterize the metabolism of different Y. lipolytica strains, isolated from various environments, growing on glycerol at different pH values. The conditions applied for strain characterization are of utmost importance. Shake flask cultures lead to very different results, when compared to controlled conditions in bioreactors regarding pH and aeration. Only one of the tested strains was able to accumulate high amounts of citric acid in shake flask experiments, whereas a group of six strains turned out to accumulate citric acid efficiently under controlled conditions. The present study shows that strains isolated from dairy products predominantly accumulate sugar alcohols at any given pH, when grown on glycerol under nitrogen-limitation.

Increasing pentose phosphate pathway flux enhances recombinant protein production in Pichia pastoris.

Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive influence on recombinant production of cytosolic human superoxide dismutase (hSOD). In this study, different combinations of these genes belonging to the oxidative PPP were generated and analyzed. Thereby, a 3.8-fold increase of hSOD production was detected when glucose-6-phosphate dehydrogenase (ZWF1) and 6-gluconolactonase (SOL3) were simultaneously overexpressed, while the combinations of other genes from PPP had no positive effect on protein production. By measuring isotopologue patterns of (13)C-labelled metabolites, we could detect an upshift in the flux ratio of PPP to glycolysis upon ZWF1 and SOL3 co-overexpression, as well as increased levels of 6-phosphogluconate. The substantial improvement of hSOD production by ZWF1 and SOL3 co-overexpression appeared to be connected to an increase in PPP flux. In conclusion, we show that overexpression of SOL3 together with ZWF1 enhanced both the PPP flux ratio and hSOD accumulation, providing evidence that in P. pastoris Sol3 limits the flux through PPP and recombinant protein production.

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.

Metabolomics sampling of Pichia pastoris revisited: rapid filtration prevents metabolite loss during quenching.

Metabolomics can be defined as the quantitative assessment of a large number of metabolites of a biological system. A prerequisite for the accurate determination of intracellular metabolite concentrations is a reliable and reproducible sample preparation method, which needs to be optimized for each organism individually. Here, we compare the performance of rapid filtration and centrifugation after quenching of Pichia pastoris cells in cold methanol. During incubation in the quenching solution, metabolites are lost from the cells with a half-life of 70-180 min. Metabolites with lower molecular weights showed lower half-lifes compared to metabolites with higher molecular weight. Rapid filtration within 2 min after quenching leads to only minor losses below 2%, and is thus the preferred method for cell separation.

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 ß-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.

Interlaboratory comparison for quantitative primary metabolite profiling in Pichia pastoris.

For the first time, an interlaboratory comparison was performed in the field of quantitative metabolite profiling in Pichia pastoris. The study was designed for the evaluation of different measurement platforms integrating different quantification strategies using internal standardization. Nineteen primary metabolites including amino acids and organic acids were selected for the study. Homogenous samples were obtained from chemostat fermentations after rapid sampling, quenching and filtration, and hot ethanol extraction. Laboratory 1 (BOKU) employed an in vivo-synthesized fully labeled U(13)C cell extracts of P. pastoris for immediate internal standardization upon cell extraction. Quantification was carried out using orthogonal reversed-phase (RP-LC) and hydrophilic interaction chromatography (HILIC) in combination with tandem mass spectrometry. Laboratory 2 (Biocrates) applied a metabolomics kit allowing fully automated, rapid derivatization, solid phase extraction and internal standardization in 96-well plates with immobilized isotopically enriched internal standards in combination with HILIC-MS-MS and RP-LC-MS-MS for organic acids and derivatized amino acids, respectively. In this study, the obtained intracellular concentrations ranged from 0.2 to 108 µmol g(-1) cell dry weight. The total combined uncertainty was estimated including uncertainty contributions from the corresponding MS-based measurement and sample preparation for each metabolite. Evidently, the uncertainty contribution of sample preparation was lower for the values obtained by laboratory 1, implementing isotope dilution upon extraction. Total combined uncertainties (K = 2) ranging from 21 to 48% and from 30 to 57% were assessed for the quantitative results obtained in laboratories 1 and 2, respectively. The major contribution arose from sample preparation, hence from repeatability precision of the extraction procedure. Finally, the laboratory intercomparison was successful as most of the investigated metabolites showed concentration levels agreeing within their total combined uncertainty, implying that accurate quantification was given. The application of isotope dilution upon extraction was an absolute prerequisite for the quantification of the redox-sensitive amino acid methionine, where no agreement between the two laboratories could be achieved.

U13C cell extract of Pichia pastoris--a powerful tool for evaluation of sample preparation in metabolomics.

Quantitative metabolic profiling is preceded by dedicated sample preparation protocols. These multistep procedures require detailed optimization and thorough validation. In this work, a uniformly (13)C-labeled (U(13)C) cell extract was used as a tool to evaluate the recoveries and repeatability precisions of the cell extraction and the extract treatment. A homogenous set of biological replicates (n = 15 samples of Pichia pastoris) was prepared for these fundamental experiments. A range of less than 30 intracellular metabolites, comprising amino acids, nucleotides, and organic acids were measured both in monoisotopic (12)C and U(13)C form by LC-MS/MS employing triple quadrupole MS, reversed phase chromatography, and HILIC. Recoveries of the sample preparation procedure ranging from 60 to 100% and repeatability precisions below 10% were obtained for most of the investigated metabolites using internal standardization approaches. Uncertainty budget calculations revealed that for this complex quantification task, in the optimum case, total combined uncertainty of 12% could be achieved. The optimum case would be represented by metabolites, easy to extract from yeast with high and precise recovery. In other cases the total combined uncertainty was significantly higher.

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.