scholarly journals Impact of overproduced heterologous protein characteristics on physiological response in Yarrowia lipolytica steady-state-maintained continuous cultures

2020 ◽  
Vol 104 (22) ◽  
pp. 9785-9800
Author(s):  
Paulina Korpys-Woźniak ◽  
Piotr Kubiak ◽  
Wojciech Białas ◽  
Ewelina Celińska
Keyword(s):  
Steady State ◽  
Yarrowia Lipolytica ◽  
Heterologous Protein ◽  
Target Protein ◽  
Host Cells ◽  
Synthesis Process ◽  
Secretory Proteins ◽  
Heterologous Proteins ◽  
Glycosylation Pattern ◽  
The Impact

Abstract Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27–65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein’s post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed. Key Points • Eight expression systems, producing different reporter proteins were analyzed. • The cells were maintained in steady-state by continuous chemostat culturing. • Protein- and promoter-specific effects were observed.

scholarly journals Contributions of the Pre- and Pro-Regions of a Staphylococcus hyicus Lipase to Secretion of a Heterologous Protein by Bacillus subtilis

2009 ◽  
Vol 76 (3) ◽  
pp. 659-669 ◽  
Author(s):  
Thijs R. H. M. Kouwen ◽  
Allan K. Nielsen ◽  
Emma L. Denham ◽  
Jean-Yves F. Dubois ◽  
Ronald Dorenbos ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Stress Responses ◽  
Heterologous Protein ◽  
Surrounding Medium ◽  
Wall Stress ◽  
Secretory Proteins ◽  
Cell Factory ◽  
Heterologous Proteins ◽  
Established Cell

ABSTRACT Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (prelip) and pro (prolip) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the prolip-peptide, in combination with the lipase signal peptide (prelip), contributes significantly to the efficient secretion of PhoA by B. subtilis and that prelip directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.

scholarly journals Enhanced Recombinant Protein Production Under Special Environmental Stress

2021 ◽  
Vol 12 ◽  
Author(s):  
Xinyi Chen ◽  
Chun Li ◽  
Hu Liu
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Synthesis Process ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Eukaryotic Microorganism ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Environment Stress ◽  
Extracellular Environment

Regardless of bacteria or eukaryotic microorganism hosts, improving their ability to express heterologous proteins is always a goal worthy of elaborate study. In addition to traditional methods including intracellular synthesis process regulation and extracellular environment optimization, some special or extreme conditions can also be employed to create an enhancing effect on heterologous protein production. In this review, we summarize some extreme environmental factors used for the improvement of heterologous protein expression, including low temperature, hypoxia, microgravity and high osmolality. The applications of these strategies are elaborated with examples of well-documented studies. We also demonstrated the confirmed or hypothetical mechanisms of environment stress affecting the host behaviors. In addition, multi-omics techniques driving the stress-responsive research for construction of efficient microbial cell factories are also prospected at the end.

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
Author(s):  
Jörg F. Rippmann ◽  
Michaela Klein ◽  
Christian Hoischen ◽  
Bodo Brocks ◽  
Wolfgang J. Rettig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Binding Activity ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Active Product ◽  
Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

scholarly journals Advanced Constitutive Modeling of the Thixotropic Elasto-Visco-Plastic Behavior of Blood: Steady-State Blood Flow in Microtubes

Materials ◽  
2021 ◽  
Vol 14 (2) ◽  
pp. 367
Author(s):  
Konstantinos Giannokostas ◽  
Yannis Dimakopoulos ◽  
Andreas Anayiotos ◽  
John Tsamopoulos
Keyword(s):  
Blood Flow ◽  
Steady State ◽  
Constitutive Model ◽  
Blood Plasma ◽  
Constitutive Modeling ◽  
Flow Direction ◽  
Momentum Balance ◽  
Plastic Behavior ◽  
Flow Investigation ◽  
The Impact

The present work focuses on the in-silico investigation of the steady-state blood flow in straight microtubes, incorporating advanced constitutive modeling for human blood and blood plasma. The blood constitutive model accounts for the interplay between thixotropy and elasto-visco-plasticity via a scalar variable that describes the level of the local blood structure at any instance. The constitutive model is enhanced by the non-Newtonian modeling of the plasma phase, which features bulk viscoelasticity. Incorporating microcirculation phenomena such as the cell-free layer (CFL) formation or the Fåhraeus and the Fåhraeus-Lindqvist effects is an indispensable part of the blood flow investigation. The coupling between them and the momentum balance is achieved through correlations based on experimental observations. Notably, we propose a new simplified form for the dependence of the apparent viscosity on the hematocrit that predicts the CFL thickness correctly. Our investigation focuses on the impact of the microtube diameter and the pressure-gradient on velocity profiles, normal and shear viscoelastic stresses, and thixotropic properties. We demonstrate the microstructural configuration of blood in steady-state conditions, revealing that blood is highly aggregated in narrow tubes, promoting a flat velocity profile. Additionally, the proper accounting of the CFL thickness shows that for narrow microtubes, the reduction of discharged hematocrit is significant, which in some cases is up to 70%. At high pressure-gradients, the plasmatic proteins in both regions are extended in the flow direction, developing large axial normal stresses, which are more significant in the core region. We also provide normal stress predictions at both the blood/plasma interface (INS) and the tube wall (WNS), which are difficult to measure experimentally. Both decrease with the tube radius; however, they exhibit significant differences in magnitude and type of variation. INS varies linearly from 4.5 to 2 Pa, while WNS exhibits an exponential decrease taking values from 50 mPa to zero.

scholarly journals Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

2021 ◽  
Vol 7 (3) ◽  
pp. 179
Author(s):  
Kai P. Hussnaetter ◽  
Magnus Philipp ◽  
Kira Müntjes ◽  
Michael Feldbrügge ◽  
Kerstin Schipper
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Ustilago Maydis ◽  
Downstream Processing ◽  
Culture Broth ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Regulatory Systems ◽  
Unconventional Secretion

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

scholarly journals Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Neeraja Punde ◽  
Jennifer Kooken ◽  
Dagmar Leary ◽  
Patricia M. Legler ◽  
Evelina Angov
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Dna Sequences ◽  
Structural Integrity ◽  
Host Cells ◽  
Loss Of Function ◽  
Species Specific ◽  
And Function ◽  
The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

scholarly journals Perturbation of host autophagy by the Irish potato famine pathogen

2014 ◽  
Vol 70 (a1) ◽  
pp. C826-C826
Author(s):  
Abbas Maqbool ◽  
Richard Richard ◽  
Tolga Bozkurt ◽  
Yasin Dagdas ◽  
Khaoula Belhai ◽  
...  
Keyword(s):  
Host Cell ◽  
Plant Cells ◽  
Irish Potato ◽  
Effector Proteins ◽  
Host Cells ◽  
Cell Physiology ◽  
Biochemical Basis ◽  
The Impact ◽  
Potato Famine ◽  
Irish Potato Famine

Autophagy is a catabolic process involving degradation of dysfunctional cytoplasmic components to ensure cellular survival under starvation conditions. The process involves formation of double-membrane vesicles called autophagosomes and delivery of the inner constituents to lytic compartments. It can also target invading pathogens, such as intracellular bacteria, for destruction and is thus implicated in innate immune pathways [1]. In response, certain mammalian pathogens deliver effector proteins into host cells that inhibit autophagy and contribute to enabling parasitic infection [2]. Pyhtophthora infestans, the Irish potato famine pathogen, is a causative agent of late blight disease in potato and tomato crops. It delivers a plethora of modular effector proteins into plant cells to promote infection. Once inside the cell, RXLR-type effector proteins engage with host cell proteins, to manipulate host cell physiology for the benefit of the pathogen. As plants lack an adaptive immune system, this provides a robust mechanism for pathogens to circumvent host defense. PexRD54 is an intracellular RXLR-type effector protein produced by P. infestans. PexRD54 interacts with potato homologues of autophagy protein ATG8 in plant cells. We have been investigating the structural and biochemical basis of the PexRD54/ATG8 interaction in vitro. We have purified PexRD54 and ATG8 independently and in complex from E. coli. Using protein/protein interaction studies we have shown that PexRD54 binds ATG8 with sub-micromolar affinity. We have also determined the structure of PexRD54 in the presence of ATG8. This crystal structure provides key insights into how the previously reported WY-fold of oomycete RXLR-type effectors [3] can be organized in multiple repeats. The structural data also provides insights into the interaction between PexRD54 and ATG8, suggesting further experiments to understand the impact of this interaction on host cell physiology and how this benefits the pathogen.

scholarly journals Does footwear type impact the number of steps required to reach gait steady state?: An innovative look at the impact of foot orthoses on gait initiation

2010 ◽  
Vol 32 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Bijan Najafi ◽  
Daniel Miller ◽  
Beth D. Jarrett ◽  
James S. Wrobel
Keyword(s):  
Steady State ◽  
Gait Initiation ◽  
Foot Orthoses ◽  
The Impact

scholarly journals Research on Cooperative Innovation Behavior of Industrial Cluster Based on Subject Adaptability

2016 ◽  
Vol 2016 ◽  
pp. 1-19 ◽  
Author(s):  
Xiaohui Jia ◽  
Minghui Jiang ◽  
Lei Shi
Keyword(s):  
Steady State ◽  
Evolutionary Game ◽  
Industrial Cluster ◽  
Industrial Clusters ◽  
Government Support ◽  
Correlated Equilibrium ◽  
Game Model ◽  
Cooperative Innovation ◽  
The Cost ◽  
The Impact

From the perspective of the interactive cooperation among subjects, this paper portrays the process of cooperative innovation in industrial cluster, in order to capture the correlated equilibrium relationship among them. Through the utilization of two key tools, evolutionary stable strategy and replicator dynamics equations, this paper considers the cost and gains of cooperative innovation and the amount of government support as well as other factors to build and analyze a classic evolutionary game model. On this basis, the subject’s own adaptability is introduced, which is regarded as the system noise in the stochastic evolutionary game model so as to analyze the impact of adaptability on the game strategy selection. The results show that, in the first place, without considering subjects’ adaptability, their cooperation in industrial clusters depends on the cost and gains of innovative cooperation, the amount of government support, and some conditions that can promote cooperation, namely, game steady state. In the second place after the introduction of subjects’ adaptability, it will affect both game theory selection process and time, which means that the process becomes more complex, presents the nonlinear characteristics, and helps them to make faster decisions in their favor, but the final steady state remains unchanged.

Life Prediction of Power Turbine Components for High Exhaust Back Pressure Applications: Part I — Disks

2015 ◽  
Author(s):  
Dipankar Dua ◽  
Brahmaji Vasantharao
Keyword(s):  
Steady State ◽  
Secondary Flow ◽  
Gas Turbines ◽  
Life Prediction ◽  
Creep Damage ◽  
Back Pressure ◽  
System Level ◽  
Cyclic Life ◽  
Power Turbine ◽  
The Impact

Industrial and aeroderivative gas turbines when used in CHP and CCPP applications typically experience an increased exhaust back pressure due to pressure losses from the downstream balance-of-plant systems. This increased back pressure on the power turbine results not only in decreased thermodynamic performance but also changes power turbine secondary flow characteristics thus impacting lives of rotating and stationary components of the power turbine. This Paper discusses the Impact to Fatigue and Creep life of free power turbine disks subjected to high back pressure applications using Siemens Energy approach. Steady State and Transient stress fields have been calculated using finite element method. New Lifing Correlation [1] Criteria has been used to estimate Predicted Safe Cyclic Life (PSCL) of the disks. Walker Strain Initiation model [1] is utilized to predict cycles to crack initiation and a fracture mechanics based approach is used to estimate propagation life. Hyperbolic Tangent Model [2] has been used to estimate creep damage of the disks. Steady state and transient temperature fields in the disks are highly dependent on the secondary air flows and cavity dynamics thus directly impacting the Predicted Safe Cyclic Life and Overall Creep Damage. A System-level power turbine secondary flow analyses was carried out with and without high back pressure. In addition, numerical simulations were performed to understand the cavity flow dynamics. These results have been used to perform a sensitivity study on disk temperature distribution and understand the impact of various back pressure levels on turbine disk lives. The Steady Sate and Transient Thermal predictions were validated using full-scale engine test and have been found to correlate well with the test results. The Life Prediction Study shows that the impact on PSCL and Overall Creep damage for high back pressure applications meets the product design standards.

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