scholarly journals Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris (Komagataella phaffii): Impact on Cell Physiology and Recombinant Production of Secreted Proteins

2019 ◽  
Vol 86 (6) ◽  
Author(s):  
Màrius Tomàs-Gamisans ◽  
Cristiane Conte Paim Andrade ◽  
Francisco Maresca ◽  
Sergi Monforte ◽  
Pau Ferrer ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Recombinant Protein ◽  
Protein Production ◽  
Metabolic Flux ◽  
Heterologous Protein ◽  
Antibody Fragment ◽  
Content Type ◽  
Protein Overproduction ◽  
Model Protein ◽  
Nadh Kinase

ABSTRACT 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 cerevisiae POS5-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.

scholarly journals Optimizing Recombinant Protein Production in theEscherichia coliPeriplasm Alleviates Stress

2018 ◽  
Vol 84 (12) ◽  
pp. e00270-18 ◽  
Author(s):  
Thomas Baumgarten ◽  
A. Jimmy Ytterberg ◽  
Roman A. Zubarev ◽  
Jan-Willem de Gier
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Target Gene ◽  
Antibody Fragment ◽  
Content Type ◽  
Expression Intensity ◽  
Target Gene Expression ◽  
Sec Translocon ◽  
Gene Expression Intensity

ABSTRACTInEscherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is—besides the produced target—the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins inE. coli.IMPORTANCEThe bacteriumEscherichia coliis widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm ofE. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

scholarly journals Posttranslational Targeting of a Recombinant Protein Promotes Its Efficient Secretion into the Escherichia coli Periplasm

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
A. Jimmy Ytterberg ◽  
Roman A. Zubarev ◽  
Thomas Baumgarten
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Antibody Fragment ◽  
Secretory Proteins ◽  
Content Type ◽  
Biomass Formation ◽  
E Coli ◽  
Sec Translocon

ABSTRACT Many recombinant proteins that are produced in Escherichia coli have to be targeted to the periplasm to be functional. N-terminal signal peptides can be used to direct recombinant proteins to the membrane-embedded Sec translocon, a multiprotein complex that translocates proteins across the membrane into the periplasm. We have recently shown that the cotranslational targeting of the single-chain variable antibody fragment BL1 saturates the capacity of the Sec translocon leading to impaired translocation of secretory proteins and protein misfolding/aggregation in the cytoplasm. In turn, protein production yields and biomass formation were low. Here, we study the consequences of targeting BL1 posttranslationally to the Sec translocon. Notably, the posttranslational targeting of BL1 does not saturate the Sec translocon capacity, and both biomass formation and protein production yields are increased. Analyzing the proteome of cells producing the posttranslationally targeted BL1 indicates that the decreased synthesis of endogenous secretory and membrane proteins prevents a saturation of the Sec translocon capacity. Furthermore, in these cells, highly abundant chaperones and proteases can clear misfolded/aggregated proteins from the cytoplasm, thereby improving the fitness of these cells. Thus, the posttranslational targeting of BL1 enables its efficient production in the periplasm due to a favorable adaptation of the E. coli proteome. We envisage that our observations can be used to engineer E. coli for the improved production of recombinant secretory proteins. IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. To fold properly, many recombinant proteins have to be targeted to the E. coli periplasm, but so far the impact of the targeting pathway of a recombinant protein to the periplasm has not been extensively investigated. Here, we show that the targeting pathway of a recombinant antibody fragment has a tremendous impact on cell physiology, ultimately affecting protein production yields in the periplasm and biomass formation. This indicates that studying the targeting and secretion of proteins into the periplasm could be used to design strategies to improve recombinant protein production yields.

scholarly journals Draft Genome Sequence of Paenibacillus polymyxa DSM 292, a Gram-Positive, Spore-Forming Soil Bacterium with High Biotechnological Potential

2020 ◽  
Vol 9 (11) ◽  
Author(s):  
Simon Heinze ◽  
Ilias Lagkouvardos ◽  
Wolfgang Liebl ◽  
Wolfgang H. Schwarz ◽  
Petra Kornberger ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Protein Production ◽  
Heterologous Protein ◽  
Draft Genome ◽  
Paenibacillus Polymyxa ◽  
Illumina Miseq ◽  
Draft Genome Sequence ◽  
Biotechnological Potential ◽  
Content Type ◽  
Paired End Sequencing

Paenibacillus polymyxa DSM 292 was originally isolated from soil in 1947 due to its ability to produce antibiotics. The low proteolytic properties of strain DSM 292 warrant its examination as a host for heterologous protein production. Here, we report the draft genome sequence of DSM 292 as established by Illumina MiSeq paired-end sequencing.

Recombinant Protein Production and Plasmid Stability in Escherichia coli Biofilms

2020 ◽  
Author(s):  
Luciana C. Gomes ◽  
Gabriel A. Monteiro ◽  
Filipe J. Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Heterologous Protein ◽  
Plasmid Stability ◽  
Gene Dosage ◽  
Heterologous Protein Expression ◽  
E Coli ◽  
The Impact

<p><em>Escherichia coli</em> biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.</p> <p>In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from <em>E. coli</em> biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of <em>E. coli</em> JM109(DE3) transformed with a plasmid containing a T7 promoter.</p> <p>It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].</p> <p>It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.</p> <p> </p> <p> </p> <p>References:</p> <p>[1] Gomes, L.C., & Mergulhão, F.J. (2017) Heterologous protein production in <em>Escherichia coli</em> biofilms: A non-conventional form of high cell density cultivation. <em>Process Biochemistry, 57, 1-8</em>. https://doi.org/10.1016/j.procbio.2017.03.018</p> <p>[2] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by <em>Escherichia coli</em> Biofilms. <em>International Journal of Molecular Sciences, 21(2), 576</em>. https://doi.org/10.3390/ijms21020576</p>

scholarly journals Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

2015 ◽  
Vol 81 (17) ◽  
pp. 5976-5986 ◽  
Author(s):  
Karin M. Münch ◽  
Johannes Müller ◽  
Sarah Wienecke ◽  
Simone Bergmann ◽  
Steffi Heyber ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Division ◽  
Recombinant Protein ◽  
Bacillus Megaterium ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Population Heterogeneity ◽  
Exponential Growth Phase ◽  
Scale Production ◽  
Content Type

ABSTRACTDuring the past 2 decades,Bacillus megateriumhas been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size ofB. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However,in vivo andin vitroexperiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in aBacillusspecies.

scholarly journals Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Javier Garrigós-Martínez ◽  
Kiira Vuoristo ◽  
Miguel Angel Nieto-Taype ◽  
Juha Tähtiharju ◽  
Jaana Uusitalo ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Cell Factory ◽  
The Novel ◽  
Expression Systems ◽  
Fed Batch ◽  
Model Protein

Abstract Background Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (PAOX1), and the constitutive GAP promoter (PGAP). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (PPDF, PUPP) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Results Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. Conclusions As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.

scholarly journals Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0258005
Author(s):  
Worarat Kruasuwan ◽  
Aekkachai Puseenam ◽  
Chitwadee Phithakrotchanakoon ◽  
Sutipa Tanapongpipat ◽  
Niran Roongsawang
Keyword(s):  
Er Stress ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Pathway ◽  
Heterologous Protein ◽  
Methylotrophic Yeast ◽  
Heterologous Proteins ◽  
Loss Of Function ◽  
Heterologous Protein Secretion ◽  
Model Protein

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O. thermomethanolica and used it to activate SOD1, VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O. thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.

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