Dataset Information

Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection.

ABSTRACT: BACKGROUND:Pichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein. RESULTS:The recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the ?-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to?~?46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb. CONCLUSIONS:rPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.

SUBMITTER: Bando-Campos G

PROVIDER: S-EPMC6339365 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection.

Bando-Campos Giroshi G Juárez-López Daniel D Román-González Sergio A SA Castillo-Rodal Antonia I AI Olvera Clarita C López-Vidal Yolanda Y Arreguín-Espinosa Roberto R Espitia Clara C Trujillo-Roldán Mauricio A MA Valdez-Cruz Norma A NA

Microbial cell factories 20190119 1

<h4>Background</h4>Pichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein.<h4>Results</h4>The recombina ...[more]

PMID: 30660186

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Project description:High-level expression and secretion of heterologous proteins in yeast cause an increased energy demand, which may result in altered metabolic flux distributions. Moreover, recombinant protein overproduction often results in endoplasmic reticulum (ER) stress and oxidative stress, causing deviations from the optimal NAD(P)H regeneration balance. In this context, overexpression of genes encoding enzymes catalyzing endogenous NADPH-producing reactions, such as the oxidative branch of the pentose phosphate pathway, has been previously shown to improve protein production in Pichia pastoris (syn. Komagataella spp.). In this study, we evaluate the overexpression of the Saccharomyces cerevisiaePOS5-encoded NADH kinase in a recombinant P. pastoris strain as an alternative approach to overcome such redox constraints. Specifically, POS5 was cooverexpressed in a strain secreting an antibody fragment, either by directing Pos5 to the cytosol or to the mitochondria. The physiology of the resulting strains was evaluated in continuous cultivations with glycerol or glucose as the sole carbon source, as well as under hypoxia (on glucose). Cytosolic targeting of Pos5 NADH kinase resulted in lower biomass-substrate yields but allowed for a 2-fold increase in product specific productivity. In contrast, Pos5 NADH kinase targeting to the mitochondria did not affect growth physiology and recombinant protein production significantly. Growth physiological parameters were in silico evaluated using the recent upgraded version (v3.0) of the P. pastoris consensus genome-scale metabolic model iMT1026, providing insights on the impact of POS5 overexpression on metabolic flux distributions.IMPORTANCE Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae, as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris, and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris.

Project description:BackgroundPlants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli-E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported.ResultsHere, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine.ConclusionsThis is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites.

Dataset Information

Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection.

Publications

Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection.

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