Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization.
ABSTRACT: The classic AOX1 replacement approach is still one of the most often used techniques for expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Although this approach is largely successful, it frequently delivers clones with unpredicted production characteristics and a work-intense screening process is required to find the strain with desired productivity.In this project 845 P. pastoris clones, transformed with a GFP expression cassette, were analyzed for their methanol-utilization (Mut)-phenotypes, GFP gene expression levels and gene copy numbers. Several groups of strains with irregular features were identified. Such features include GFP expression that is markedly higher or lower than expected based on gene copy number as well as strains that grew under selective conditions but where the GFP gene cassette and its expression could not be detected. From these classes of strains 31 characteristic clones were selected and their genomes sequenced. By correlating the assembled genome data with the experimental phenotypes novel insights were obtained. These comprise a clear connection between productivity and cassette-to-cassette orientation in the genome, the occurrence of false-positive clones due to a secondary recombination event, and lower total productivity due to the presence of untransformed cells within the isolates were discovered. To cope with some of these problems, the original vector was optimized by replacing the AOX1 terminator, preventing the occurrence of false-positive clones due to the secondary recombination event.Standard methods for transformation of P. pastoris led to a multitude of unintended and sometimes detrimental integration events, lowering total productivity. By documenting the connections between productivity and integration event we obtained a deeper understanding of the genetics of mutation in P. pastoris. These findings and the derived improved mutagenesis and transformation procedures and tools will help other scientists working on recombinant protein production in P. pastoris and similar non-conventional yeasts.
Project description:Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an autonomously replicating sequence (ARS). Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named "pMito" consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector transformation in Saccharomyces cerevisiae, widening the potential for biotechnological application. pMito displayed strong potential to reduce clonal variability in experiments targeting recombinant protein production. These findings also showcase the as of yet largely untapped potential of mitochondrial ARS from different yeasts for biotechnological applications.
Project description:BACKGROUND: Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. RESULTS: In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3-5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. CONCLUSIONS: We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.
Project description:Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes. Here, we demonstrate that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human beta2-adrenergic receptor. Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gene. In three expression plasmids, the receptors were fused to the Saccharomyces cerevisiae alpha-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors. After transformation into P. pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT5A receptor and the beta2-adrenergic receptor by radioligand binding using [N-methyl-3H]lysergic acid diethylamide and [5,7-3H](-)CGP-12177 respectively. The production level of the 5HT5A receptor was improved by a factor of three by fusion with the alpha-factor prepropeptide. Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors. The addition of the adrenergic antagonist alprenolol to the culture medium had a positive effect on the number of specific binding sites detectable in clones producing the beta2-adrenergic receptor. For the 5HT5A receptor the addition of yohimbine resulted in a similar but smaller effect. Binding assays revealed that approx. 25 pmol of beta2-adrenergic receptor and approx. 40 pmol of 5HT5A receptor per mg of membrane protein in crude membrane preparations were produced. The pharmacological profiles for the heterologously produced receptors, estimated by ligand-displacement analysis using certain adrenergic and serotoninergic agonists and antagonists, were comparable with those reported for the receptors expressed in mammalian systems. Immunoblot analysis of the 5HT5A receptor revealed an apparent molecular mass about 20 kDa higher than expected from the amino acid sequence. Here, the Kex2 endopeptidase failed to process the alpha-factor leader correctly. Blocking glycosylation in vivo by tunicamycin or in vitro deglycosylation of membranes by endoglycosidase H resulted in correct processing. In contrast, the beta2-adrenergic receptor fusion to the alpha-factor leader was correctly processed by the internal Kex2 endopeptidase. The Kex2-processed beta2-adrenergic receptor was not glycosylated. In conclusion, the high-level production of the two receptors in P. pastoris will allow their purification in quantities sufficient for subsequent biophysical and structural studies.
Project description:BACKGROUND:The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. RESULTS:The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. CONCLUSIONS:PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression-heterologous genes included-, thus directly influencing the production kinetics of recombinant protein.
Project description:Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P(AOX1)) sequence. This first library initially spanned an activity range between approximately 6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a 'real case', i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in P(AOX1)-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new P(AOX1) synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.
Project description:Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV-G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV-G in P. pastoris.
Project description:The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability.
Project description:BACKGROUND:The methanol-regulated AOX1 promoter (PAOX1) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of PAOX1 restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of PAOX1. The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal. RESULTS:The characteristics of PAOX1 are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (PAOX2). This novel approach enhanced PAOX1 efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow PAOX1 to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on PAOX1 was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction. CONCLUSIONS:We successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol.
Project description:In this study, we targeted Olig2, a basic helix-loop-helix transcription factor that plays an important role in motoneuron and oligodendrocyte development, in human embryonic stem cell (hESC) line BG01 by homologous recombination. One allele of Olig2 locus was replaced by a green fluorescent protein (GFP) cassette with a targeting efficiency of 5.7%. Targeted clone R-Olig2 (like the other clones) retained pluripotency, typical hESC morphology, and a normal parental karyotype 46,XY. Most importantly, GFP expression recapitulated endogenous Olig2 expression when R-Olig2 was induced by sonic hedgehog and retinoic acid, and GFP-positive cells could be purified by fluorescence-activated cell sorting. Consistent with previous reports on rodents, early GFP-expressing cells appeared biased to a neuronal fate, whereas late GFP-expressing cells appeared biased to an oligodendrocytic fate. This was corroborated by myoblast coculture, transplantation into the rat spinal cords, and whole genome expression profiling. The present work reports an hESC reporter line generated by homologous recombination targeting a neural lineage-specific gene, which can be differentiated and sorted to obtain pure neural progenitor populations.
Project description:Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non-homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ?20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the ?ku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25-fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR-Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.