Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

In Hypocrea jecorina, Xyr1 (xylanase regulator 1) is the main transcription activator of hydrolase-encoding genes, such as xyn1, xyn2, bxl1, cbh1, cbh2, egl1, and bgl1. Even though Xyr1 mediates the induction signal for all these genes derived from various inducing carbon sources and compounds, xyr1 transcription itself is not inducible by any of these substances. However, cultivation on glucose as the carbon source provokes carbon catabolite repression of xyr1 transcription mediated by Cre1. In addition, xyr1 transcription is repressed by the specific transcription factor Ace1. Moreover, Xyr1 is permanently available in the cell, and no de novo synthesis of this factor is needed for a first induction of xyn1 transcription. The constitutive expression of xyr1 leads to a significant elevation/deregulation of the xyn1, xyn2, and bxl1 transcription compared to what is seen for the parental strain. Overall, the corresponding xylanolytic enzyme activities are clearly elevated in a constitutively xyr1-expressing strain, emphasizing this factor as an auspicious target for genetically engineered strain improvement.

The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO 2 . By addition of eight heterologous genes and deletion of three Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

BackgroundSome 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.ResultsIn 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.ConclusionsTaken 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 materialThe online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users.

Pichia pastoris is the most frequently used yeast system for heterologous protein production today. The last few years have seen several products based on this platform reach approval as biopharmaceutical drugs. Successful glycoengineering to humanize N-glycans is further fuelling this development. However, detailed understanding of the yeast's physiology, genetics and regulation has only developed rapidly in the last few years since published genome sequences have become available. An expanding toolbox of genetic elements and strains for the improvement of protein production is being generated, including promoters, gene copy-number enhancement, gene knockout and high-throughput methods. Protein folding and secretion have been identified as significant bottlenecks in yeast expression systems, pinpointing a major target for strain optimization. At the same time, it has become obvious that P. pastoris, as an evolutionarily more 'ancient' yeast, may in some cases be a better model for human cell biology and disease than Saccharomyces cerevisiae.

Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at μ = 0.015 to 0.15 h–1 in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing μ, while autophagy and other proteolytic processes, carbon source-responsive genes and other targets of the TOR pathway as well as many transcriptional regulators were downregulated at higher μ. Mating and sporulation genes were most active at intermediate μ of 0.05 and 0.075 h–1. At very slow growth (μ = 0.015 h–1) gene regulation differs significantly, affecting many transporters and glucose sensing. Analysis of a subset of genes related to protein folding and secretion reveals that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones are upregulated with increasing growth rate while proteolytic degradation of secretory proteins is downregulated. We conclude that a high μ positively affects specific protein secretion rates by acting on multiple cellular processes.

Besides its use for efficient production of recombinant proteins the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) has been increasingly employed as a platform to produce metabolites of varying origin. We summarize here the impressive methodological developments of the last years to model and analyze the metabolism of P. pastoris, and to engineer its genome and metabolic pathways. Efficient methods to insert, modify or delete genes via homologous recombination and CRISPR/Cas9, supported by modular cloning techniques, have been reported. An outstanding early example of metabolic engineering in P. pastoris was the humanization of protein glycosylation. More recently the cell metabolism was engineered also to enhance the productivity of heterologous proteins. The last few years have seen an increased number of metabolic pathway design and engineering in P. pastoris, mainly towards the production of complex (secondary) metabolites. In this review, we discuss the potential role of P. pastoris as a platform for metabolic engineering, its strengths, and major requirements for future developments of chassis strains based on synthetic biology principles.

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.