David Resina

Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics

Both conventional and innovative biomedical approaches require cost-effective protein drugs with high therapeutic potency, improved bioavailability, biocompatibility, stability and pharmacokinetics. The growing longevity of the human population, the increasing incidence and prevalence of age-related diseases and the better comprehension of genetic-linked disorders prompt to develop natural and engineered drugs addressed to fulfill emerging therapeutic demands. Conventional microbial systems have been for long time exploited to produce biotherapeutics, competing with animal cells due to easier operation and lower process costs. However, both biological platforms exhibit important drawbacks (mainly associated to intracellular retention of the product, lack of post-translational modifications and conformational stresses), that cannot be overcome through further strain optimization merely due to physiological constraints. The metabolic diversity among microorganisms offers a spectrum of unconventional hosts, that, being able to bypass some of these weaknesses, are under progressive incorporation into production pipelines. In this review we describe the main biological traits and potentials of emerging bacterial, yeast, fungal and microalgae systems, by comparing selected leading species with well established conventional organisms with a long run in protein drug production.

Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction

BackgroundThe analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organisms responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR) and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1), namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL.ResultsThe transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected.ConclusionThe bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of specific mRNA species in P. pastoris cells grown in fed-batch cultures. As a proof-of-principle, the influence of the carbon and nitrogen sources, the specific growth rate, as well as the ROL overexpression on the transcriptional levels of a reduced set of bioprocess-relevant genes has been quantitatively studied, revealing that ROL overexpression and secretion seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process.

Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter

The yeast Pichia pastoris has been previously used for extracellular expression of a Rhizopus oryzae lipase (Rol). However, limitations in Rol folding and secretion through the cell wall became apparent when producing it in fed-batch cultivations. In this study, we have investigated the effect of combining two cell engineering strategies to alleviate putative bottlenecks in Rol secretion, namely the constitutive expression of the induced form of the Saccharomyces cerevisiae unfolded protein response transcriptional factor Hac1 and the deletion of the GAS1 gene encoding beta-1,3-glucanosyltransglycosylase, GPI-anchored to the outer leaflet of the plasma membrane, playing a key role in yeast cell wall assembly. The performance of these engineered Rol-producing strains has been compared in fed-batch cultivations set at a low specific growth rate of about 0.005 h-(1). It was found that Rol overexpression in a P. pastoris strain expressing constitutively the induced form of S. cerevisiae Hac1 and the deletion of GAS1 resulted in about a 3-fold and 4-fold increase in the overall process specific productivity, respectively, whereas the double mutant HAC1/deltagas1 strain yielded about a 7-fold increase. Overall, these results reflect the multiplicity of physiological bottlenecks at different levels/steps throughout the Rol synthesis, secretion and excretion processes in P. pastoris.

Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris

BackgroundTogether with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP) has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a Rhizopus oryzae lipase (ROL) in Pichia pastoris by means of 2D-fluorimetric techniquesResultsIn this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in P. pastoris batch cultivations. Furthermore, both the fusion enzyme and the fluorescent protein (GFP S65T mutant) retained their biological activity. However, when multiwavelength spectrofluorometry was used for extracellular fusion protein monitoring, riboflavin appeared as a major interfering component with GFP signal. Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activityConclusionP. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in P. pastoris using GFP fusions combined with multiwavelength spectrofluorimetric techniques, riboflavin may interfere with GFP fluorescence signal, thus limiting the application of some GFP variants for on-line extracellular recombinant protein quantification and monitoring purposes.

Limitations using GFP as a protein expression reporter in Pichia pastoris

Background The development of fluorimetric sensors during the last decade and the advantages of fluorimetry as a non invasive, highly specific and sensitive technique have favoured the utilization of this signal not only in biology but also in bioprocesses. In this context, the Aequoria victoria green fluorescent protein (GFP) has appeared as a popular reporter protein to study both prokaryotic and eukaryotic systems. It does not demand any cofactors to fluoresce, is a small molecule and requires no fixation techniques. Among different applications, GFP has been used as a reporter in gene delivery, as a tracer in subcellular trafficking and as a fusion partner to monitor protein location. For bioprocess development, GFP has been used as a protein fusion partner to monitor and optimise recombinant protein production [1]. However, production of soluble, secreted GFP or protein-GFP fusions in P. pastoris has proved to be a difficult task. Also, decrease of the target protein production levels when fused to GFP is case dependent.

Disruption of the GAS1 gene of Pichia pastoris confers a supersecretory phenotype for Rhizopus oryzae lipase, but not for human trypsinogen

on host physiology The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting. Meeting

Transcriptional analysis of protein production and induction of unfolded protein response in Pichia pastoris expressing a Rhizopus oryzae lipase under the FLD1 promoter

Background Methanol-free high cell density fed-batch cultivation strategies for the Pichia pastoris expression system have been recently developed by expressing a Rhizopus oryzae lipase (ROL) under the transcriptional control of FLD1 promoter (P FLD1)[1]. These cultivation strategies were based on the use of sorbitol and methylamine as carbon and nitrogen source, respectively, during the induction phase of the cultivation process. The specific growth rate proved to be an important parameter in the productivity of secreted ROL. Moreover, intracellular active product accumulation and a decrease in the specific product secretion rate were observed along the induction phase of the fermentation process. These results suggested the presence of a bottleneck(s) throughout the synthesis and secretion process of the heterologous lipase. Recently [2], flow cytometry analyses of intracellular ROL levels confirmed that a fraction of the product was retained within the cell. Further, this intracellular product accumulation was concomitant with an increase on the BiP protein, a chaperone of the HSP70 class that plays an important role in the unfolded protein response (UPR). Notably, the increase of BiP and ROL content in the cell was detected soon after the beginning of the induction phase. Interestingly, the intracellular BiP and ROL profiles were different depending on the specific growth rate of the cells. In this study, we report the application of a sandwich hybridization assay-based technique [3] for quantification of specific mRNAs levels during the extracellular production of ROL in P. pastoris under the transcriptional control of PFLD1. These studies have been carried out in fed-batch cultures at two different specific growth rates.

Analysis of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated formaldehyde dehydrogenase promoter (PFLD1)

Background Methanol-free high cell density fed-batch cultivation strategies for the P. pastoris expression system have been recently developed by expressing a Rhizopus oryzae lipase (ROL) under the transcriptional control of the PFLD1 [1]. These cultivation strategies were based on the use of sorbitol and methylamine as carbon and nitrogen source, respectively, during the induction phase of the cultivation process. Fed-batch fermentations were performed at three different specific growth rates and showed that productivities were strongly correlated with this parameter (i.e. with the cells physiological state). Moreover, intracellular active product accumulation and a decrease in the specific product secretion rate were observed along the induction phase of the fermentation process. These results suggested the presence of a bottleneck(s) throughout the synthesis and secretion process of the heterologous lipase.