Dissimilatory Pathway Research Articles

Understanding pathways of dissimilatory and assimilatory dissolved inorganic nitrogen uptake in streams

AbstractDissolved inorganic nitrogen (DIN) in streams is mostly available as two different species: nitrate ( ) and ammonium ( ). These two DIN species undergo specific dissimilatory uptake pathways and show distinct preference during biological assimilation. These differences ultimately dictate how DIN is cycled within streams and its further export to downstream ecosystems. Here, we provide a synthesis analysis of the uptake of and at the reach scale and on the contribution of dissimilatory and assimilatory uptake pathways. We combined 15N‐tracer experiments in a single stream with compiled results of and uptake from the literature. As expected, streams were more efficient in processing than at the reach scale. These results were partially explained by the fact that, on average, dissimilatory uptake (i.e., denitrification and DNRA) had a low incidence on total uptake, whereas dissimilatory uptake (i.e., nitrification) contributed to a high proportion of total uptake thereby increasing in‐stream concentration. Furthermore, assimilatory uptake by in‐stream biotic compartments dominated the total uptake of the two DIN species and was generaly higher for than for . Overall, results from this study indicate that assimilatory uptake by biotic compartments rather than permanent removal dominates total uptake in streams. In contrast, both assimilatory and dissimilatory uptake can contribute similarly to total uptake. Our findings have strong implications for a better understanding of N cycling within the context of widespread increases in DIN concentration and changes in the : ratio driven by human activities.

Microbial community analysis of pH 4 thermal springs in Yellowstone National Park.

The pH of the majority of thermal springs in Yellowstone National Park (YNP) is from 1 to 3 and 6 to 10; relatively few springs (~5%) have a pH range of 4-5. We used 16S rRNA gene pyrosequencing to investigate microbial communities sampled from four pH 4 thermal springs collected from four regions of YNP that differed in their fluid temperature and geochemistry. Our results revealed that the composition of bacterial communities varied among the sites, despite sharing similar pH values. The taxonomic composition and metabolic functional potential of the site with the lowest temperature (55°C), a thermal spring from the Seven Mile Hole (SMH) area, were further investigated using shotgun metagenome sequencing. The taxonomic classification, based on 372Mbp of unassembled metagenomic reads, indicated that this community included a high proportion of Chloroflexi, Bacteroidetes, Proteobacteria, and Firmicutes. Functional comparison with other YNP thermal spring metagenomes indicated that the SMH metagenome was enriched in genes related to energy production and conversion, transcription, and carbohydrate transport. Analysis of genes involved in nitrogen metabolism revealed assimilatory and dissimilatory nitrate reduction pathways, whereas the majority of genes involved in sulfur metabolism were related to the reduction of sulfate to adenylylsulfate, sulfite, and H2S. Given that pH 4 thermal springs are relatively less common in YNP and thermal areas worldwide, they may harbor novel microbiota and the communities that inhabit them deserve further investigation.

Microbial oxidative sulfur metabolism: biochemical evidence of the membrane-bound heterodisulfide reductase-like complex of the bacterium Aquifex aeolicus.

The Hdr (heterodisulfide reductase)-like enzyme is predicted, from gene transcript profiling experiments previously published, to be essential in oxidative sulfur metabolism in a number of bacteria and archaea. Nevertheless, no biochemical and physicochemical data are available so far about this enzyme. Genes coding for it were identified in Aquifex aeolicus, a Gram-negative, hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium that uses inorganic sulfur compounds as electron donor to grow. We provide biochemical evidence that this Hdr-like enzyme is present in this sulfur-oxidizing prokaryote (cultivated with thiosulfate or elemental sulfur). We demonstrate, by immunolocalization and cell fractionation, that Hdr-like enzyme is associated, presumably monotopically, with the membrane fraction. We show by co-immunoprecipitation assay or partial purification, that the Hdr proteins form a stable complex composed of at least five subunits, HdrA, HdrB1, HdrB2, HdrC1 and HdrC2, present in two forms of high molecular mass on native gel (∼240 and 450 kDa). These studies allow us to propose a revised model for dissimilatory sulfur oxidation pathways in A. aeolicus, with Hdr predicted to generate sulfite.

Transcriptome analysis reveals physiological characteristics required for magnetosome formation in Magnetospirillum gryphiswaldense MSR-1.

Magnetosome synthesis ability of Magnetospirillum gryphiswaldense MSR-1 in an autofermentor can be precisely controlled through strict control of dissolved oxygen concentration. In this study, using transcriptome data we discovered gene transcriptional differences and compared physiological characteristics of MSR-1 cells cultured under aerobic (high-oxygen) and micro-aerobic (low-oxygen) conditions. The results showed that 77 genes were up-regulated and 95 genes were down-regulated significantly under micro-aerobic situation. These genes were involved primarily in the categories of cell metabolism, transport, regulation and unknown-function proteins. The nutrient transport and physiological metabolism were slowed down under micro-aerobic condition, whereas dissimilatory denitrification pathways were activated and it may supplemental energy was made available for magnetosome synthesis. The result suggested that the genes of magnetosome membrane proteins (Mam and Mms) are not directly regulated by oxygen level, or are constitutively expressed. A proposed regulatory network of differentially expressed genes reflects the complexity of physiological metabolism in MSR-1, and suggests that some yet-unknown functional proteins play important roles such as ferric iron uptake and transport during magnetosome synthesis. The transcriptome data provides a holistic view of the responses of MSR-1 cells to differing oxygen levels. This approach will give new insights into general principles of magnetosome formation.

Tidal pumping facilitates dissimilatory nitrate reduction in intertidal marshes.

Intertidal marshes are alternately exposed and submerged due to periodic ebb and flood tides. The tidal cycle is important in controlling the biogeochemical processes of these ecosystems. Intertidal sediments are important hotspots of dissimilatory nitrate reduction and interacting nitrogen cycling microorganisms, but the effect of tides on dissimilatory nitrate reduction, including denitrification, anaerobic ammonium oxidation and dissimilatory nitrate reduction to ammonium, remains unexplored in these habitats. Here, we use isotope-tracing and molecular approaches simultaneously to show that both nitrate-reduction activities and associated functional bacterial abundances are enhanced at the sediment-tidal water interface and at the tide-induced groundwater fluctuating layer. This pattern suggests that tidal pumping may sustain dissimilatory nitrate reduction in intertidal zones. The tidal effect is supported further by nutrient profiles, fluctuations in nitrogen components over flood-ebb tidal cycles, and tidal simulation experiments. This study demonstrates the importance of tides in regulating the dynamics of dissimilatory nitrate-reducing pathways and thus provides new insights into the biogeochemical cycles of nitrogen and other elements in intertidal marshes.

Nitrous oxide emission by the non-denitrifying, nitrate ammonifier Bacillus licheniformis.

BackgroundFirmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both. The recent renewed interest in their nitrogen metabolism has revealed many interesting features, the most striking being their wide variety of dissimilatory nitrate reduction pathways. In the present study, nitrous oxide production from Bacillus licheniformis, a ubiquitous Gram-positive, spore-forming species with many industrial applications, is investigated.ResultsB. licheniformis has long been considered a denitrifier but physiological experiments on three different strains demonstrated that nitrous oxide is not produced from nitrate in stoichiometric amounts, rather ammonium is the most important end-product, produced during fermentation. Significant strain dependency in end-product ratios, attributed to nitrite and ammonium, and medium dependency in nitrous oxide production were also observed. Genome analyses confirmed the lack of a nitrite reductase to nitric oxide, the key enzyme of denitrification. Based on the gene inventory and building on knowledge from other non-denitrifying nitrous oxide emitters, hypothetical pathways for nitrous oxide production, involving NarG, NirB, qNor and Hmp, are proposed. In addition, all publically available genomes of B. licheniformis demonstrated similar gene inventories, with specific duplications of the nar operon, narK and hmp genes as well as NarG phylogeny supporting the evolutionary separation of previously described distinct BALI1 and BALI2 lineages.ConclusionsUsing physiological and genomic data we have demonstrated that the common soil bacterium B. licheniformis does not denitrify but is capable of fermentative dissimilatory nitrate/nitrite reduction to ammonium (DNRA) with concomitant production of N2O. Considering its ubiquitous nature and non-fastidious growth in the lab, B. licheniformis is a suitable candidate for further exploration of the actual mechanism of N2O production in DNRA bacteria and its relevance in situ.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2382-2) contains supplementary material, which is available to authorized users.

Nitrite Reduction Assay for Whole Pseudomonas Cells

The second step of the dissimilatory denitrification pathway in which nitrite (NO2-) is converted to nitric oxide (NO) is catalyzed by the enzyme nitrite reductase. Two distinct enzymes are found in nature that catalyze this reaction, and they contain different metal sites, either iron (Fe), in the form of heme, or copper (Cu) (Zumft, 1997). The Pseudomonas stutzeri (P. stutzeri) RCH2 strain used in this assay contains both an Fe and a Cu form of nitrite reductase. In this assay, total nitrite reductase activity can be measured in whole cells using fumarate or some other carbon source as an electron source by measuring the disappearance of nitrite over time (Thorgersen et al., 2015).

Sulfur Isotope Effects of Dissimilatory Sulfite Reductase.

The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major (34S/32S) and minor (33S/32S, 36S/32S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in 34S/32S (hereafter, ) to be 15.3 ± 2‰, 2σ. The accompanying minor isotope effect in 33S, described as , is calculated to be 0.5150 ± 0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3–0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in 34εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of is similar to the median value of experimental observations compiled from all known published work, where 34εr−p = 16.1‰ (r–p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments ( 17.3 ± 1.5‰, 2σ) and in modern marine sediments ( 17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in modern and ancient environments.

Nitrogen Cycling Potential of a Grassland Litter Microbial Community.

Because microorganisms have different abilities to utilize nitrogen (N) through various assimilatory and dissimilatory pathways, microbial composition and diversity likely influence N cycling in an ecosystem. Terrestrial plant litter decomposition is often limited by N availability; however, little is known about the microorganisms involved in litter N cycling. In this study, we used metagenomics to characterize the potential N utilization of microbial communities in grassland plant litter. The frequencies of sequences associated with eight N cycling pathways differed by several orders of magnitude. Within a pathway, the distributions of these sequences among bacterial orders differed greatly. Many orders within the Actinobacteria and Proteobacteria appeared to be N cycling generalists, carrying genes from most (five or six) of the pathways. In contrast, orders from the Bacteroidetes were more specialized and carried genes for fewer (two or three) pathways. We also investigated how the abundance and composition of microbial N cycling genes differed over time and in response to two global change manipulations (drought and N addition). For many pathways, the abundance and composition of N cycling taxa differed over time, apparently reflecting precipitation patterns. In contrast to temporal variability, simulated global change had minor effects on N cycling potential. Overall, this study provides a blueprint for the genetic potential of N cycle processes in plant litter and a baseline for comparisons to other ecosystems.

Hydraulic effects on nitrogen removal in a tidal spring‐fed river

AbstractHydraulic properties such as stage and residence time are important controls on riverine N removal. In most rivers, these hydraulic properties vary with stochastic precipitation forcing, but in tidal rivers, hydraulics variation occurs on a predictable cycle. In Manatee Springs, a highly productive, tidally influenced spring‐fed river in Florida, we observed significant reach‐scale N removal that varied in response to tidally driven variation in hydraulic properties as well as sunlight‐driven variation in assimilatory uptake. After accounting for channel residence time and stage variation, we partitioned the total removal signal into assimilatory (i.e., plant uptake) and dissimilatory (principally denitrification) pathways. Assimilatory uptake was strongly correlated with primary production and ecosystem C:N was concordant with tissue stoichiometry of the dominant autotrophs. The magnitude of N removal was broadly consistent in magnitude with predictions from models (SPARROW and RivR‐N). However, contrary to model predictions, the highest removal occurred at the lowest values of τ/d (residence time divided by depth), which occurred at low tide. Removal efficiency also exhibited significant counterclockwise hysteresis with incoming versus outgoing tides. This behavior is best explained by the sequential filling and draining of transient storage zones such that water that has spent the longest time in the storage zone, and thus had the most time for N removal, drains back into the channel at the end of an outgoing tide, concurrent with shortest channel residence times. Capturing this inversion of the expected relationship between channel residence time and N removal highlights the need for nonsteady state reactive transport models.

The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity

Cysteine residues are absolutely indispensable for the reactions of almost all enzymes involved in the dissimilatory oxidation pathways of reduced inorganic sulfur compounds. Tetrathionate hydrolase from the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate. Af-Tth is a key enzyme in the dissimilatory sulfur oxidation pathway in this bacterium. Only one cysteine residue (Cys301) has been identified in the deduced amino acid sequence of the Af-Tth gene. In order to clarify the role of the sole cysteine residue, a site-specific mutant enzyme (C301A) was generated. No difference was observed in the retention volumes of the wild-type and mutant Af-Tth enzymes by gel-filtration column chromatography, and surprisingly the enzyme activities measured in the cysteine-deficient and wild-type enzymes were the same. These results suggest that the sole cysteine residue (Cys301) in Af-Tth is involved in neither the tetrathionate hydrolysis reaction nor the subunit assembly. Af-Tth may thus have a novel cysteine-independent reaction mechanism.

Draft Genome Sequence of Bacillus sp. Strain BSC154, Isolated from Biological Soil Crust of Moab, Utah.

Bacillus sp. BSC154 was isolated from a biological soil crust near Moab, Utah. The strain appears to be capable of chemotaxis and biofilm production. The BSC154 genome contains iron siderophore production, nitrate reduction, mixed acid-butanediol fermentation, and assimilatory and dissimilatory sulfate metabolism pathways.

Draft Genome Sequence of Massilia sp. Strain BSC265, Isolated from Biological Soil Crust of Moab, Utah.

Massilia sp. BSC265 was isolated from a biological soil crust near Moab, Utah. The strain appears to be capable of chemotaxis and exopolysaccharide synthesis for biofilm adhesion. The BSC265 genome contains a complete dissimilatory nitrate reduction pathway as well as a TCA cycle, making it a facultative anaerobe.

Effect of high electron donor supply on dissimilatory nitrate reduction pathways in a bioreactor for nitrate removal

The possible shift of a bioreactor for NO3− removal from predominantly denitrification (DEN) to dissimilatory nitrate reduction to ammonium (DNRA) by elevated electron donor supply was investigated. By increasing the C/NO3− ratio in one of two initially identical reactors, the production of high sulfide concentrations was induced. The response of the dissimilatory NO3− reduction processes to the increased availability of organic carbon and sulfide was monitored in a batch incubation system. The expected shift from a DEN- towards a DNRA-dominated bioreactor was not observed, also not under conditions where DNRA would be thermodynamically favorable. Remarkably, the microbial community exposed to a high C/NO3− ratio and sulfide concentration did not use the most energy-gaining process.

The nitrate-sensing NasST system regulates nitrous oxide reductase and periplasmic nitrate reductase in Bradyrhizobium japonicum.

The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B. japonicum Nos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3(-))-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3(-) , nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3(-) addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3(-). Thus, although NasST is known to regulate the NO3(-)-mediated response of NO3(-) assimilation genes in bacteria, our results show that NasST regulates the NO3(-) -mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B. japonicum.

Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures

BackgroundPichia pastoris is a popular yeast preferably employed for secretory protein production. Secretion is not always efficient and endoplasmic retention of proteins with aberrant folding properties, or when produced at exaggerated rates, can occur. In these cases production usually leads to an unfolded protein response (UPR) and the induction of the endoplasmic reticulum associated degradation (ERAD). P. pastoris is nowadays also an established host for secretory insulin precursor (IP) production, though little is known about the impact of IP production on the host cell physiology, in particular under industrially relevant production conditions. Here, we evaluate the cellular response to aox1 promoter-controlled, secretory IP production in controlled fed-batch processes using a proteome profiling approach.ResultsCells were first grown in a batch procedure using a defined medium with a high glycerol concentration. After glycerol depletion IP production was initiated by methanol addition which was kept constant through continuous methanol feeding. The most prominent changes of the intracellular proteome after the onset of methanol feeding were related to the enzymes of central carbon metabolism. In particular, the enzymes of the methanol dissimilatory pathway - virtually absent in the glycerol batch phase - dominated the proteome during the methanol fed-batch phase. Unexpectedly, a strong decrease of UPR and ERAD related proteins was also observed during methanol-induced IP production. Compared to non-producing control strains grown under identical conditions the UPR down-regulation was less pronounced indicating that IP production elicits a detectable but non prominent UPR response which is repressed by the general culture condition-dependent UPR down-regulation after the shift from glycerol to methanol.ConclusionsThe passage of IP through the secretory pathway using an optimized IP vector and growing the strain at fed-batch conditions with a high initial glycerol concentration does not impose a significant burden on the secretory machinery even under conditions leading to an extracellular accumulation of ~ 3 g L-1 IP. The glycerol batch pre-induction culture conditions are associated with a high constitutive - recombinant protein production independent - induction of the UPR and ERAD pathways probably preconditioning the cells for effective IP secretion in the methanol fed-batch phase.

Microbiological Reduction of Sb(V) in Anoxic Freshwater Sediments

Microbiological reduction of millimolar concentrations of Sb(V) to Sb(III) was observed in anoxic sediments from two freshwater settings: (1) a Sb- and As-contaminated mine site (Stibnite Mine) in central Idaho and 2) an uncontaminated suburban lake (Searsville Lake) in the San Francisco Bay Area. Rates of Sb(V) reduction in anoxic sediment microcosms and enrichment cultures were enhanced by amendment with lactate or acetate as electron donors but not by H2, and no reduction occurred in sterilized controls. Addition of 2-(14)C-acetate to Stibnite Mine microcosms resulted in the production of (14)CO2 coupled to Sb(V) reduction, suggesting that this process proceeds by a dissimilatory respiratory pathway in those sediments. Antimony(V) reduction in Searsville Lake sediments was not coupled to acetate mineralization and may be associated with Sb-resistance. The microcosms and enrichment cultures also reduced sulfate, and the precipitation of insoluble Sb(III)-sulfide complexes was a major sink for reduced Sb. The reduction of Sb(V) by Stibnite Mine sediments was inhibited by As(V), suggesting that As(V) is a preferred electron acceptor for the indigenous community. These findings indicate a novel pathway for anaerobic microbiological respiration and suggest that communities capable of reducing high concentrations of Sb(V) commonly occur naturally in the environment.

Vertical activity distribution of dissimilatory nitrate reduction in coastal marine sediments

Abstract. The relative importance of two dissimilatory nitrate reduction pathways, denitrification (DEN) and dissimilatory nitrate reduction to ammonium (DNRA), was investigated in intact sediment cores from five different coastal marine field sites (Dorum, Aarhus Bight, Mississippi Delta, Limfjord and Janssand). The vertical distribution of DEN activity was examined using the acetylene inhibition technique combined with N2O microsensor measurements, whereas NH4+ production via DNRA was measured with a recently developed gel probe-stable isotope technique. At all field sites, dissimilatory nitrate reduction was clearly dominated by DEN (59–131% of the total NO3− reduced) rather than by DNRA, irrespective of the sedimentary inventories of electron donors such as organic carbon, sulfide, and iron. Highest ammonium production via DNRA, accounting for up to 8.9% of the total NO3− reduced, was found at a site with very high concentrations of total sulfide and NH4+ within and below the layer in which NO3− reduction occurred. Sediment from two field sites, one with low and one with high DNRA activity in the core incubations, was also used for slurry incubations. Now, in both sediments high DNRA activity was detected accounting for 37–77% of the total NO3− reduced. These contradictory results might be explained by enhanced NO3− availability for DNRA bacteria in the sediment slurries compared to the core-incubated sediments in which diffusion of NO3− from the water column may only reach DEN bacteria, but not DNRA bacteria. The true partitioning of dissimilatory nitrate reduction between DNRA and DEN may thus lie in between the values found in whole core (underestimation of DNRA) and slurry incubations (overestimation of DNRA).

Microbial community in packed bed bioreactor involved in nitrate remediation from low level radioactive waste

Nitrate is the second largest contaminant of agriculture soil after pesticides. It also is a major pollutant from nuclear and metallurgical operations. Conventional methods for nitrate removal suffers from high cost and complexity leaving bioremediation as a viable alternative strategy. A pilot plant of 2.5 m(3)/day capacity has been functioning since 2005 based on microbial consortia treating actual effluent from nuclear power plant having pH of 7-8.5 (optimum) with N:C ratio of 1:1.7. The maximum biodegradable nitrate concentration of 3000 ppm could be reduced to below permissible limit (44.2 ppm) within 24 h in presence of sodium acetate as carbon source. Culture independent analysis (16S rDNA based) revealed clones having closest identity with uncultured bacterium, Pseudomonas stutzeri and Azoarcus sp. The existence of dissimilatory pathway of nitrate reduction in the community DNA is indicated by presence of nirS and nirK gene. Though the microbial mass was developed using municipal sewage, absence of Mycobacterium sp was confirmed using PCR. The understanding of the molecular identification of the consortium would help in developing the preservation strategy of the microbial mass for replication and perpetuation of the system.

Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes

BackgroundPichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process.ResultsThe shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation.ConclusionsIn the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.