scholarly journals Optimal protein production by a synthetic microbial consortium: Coexistence, distribution of labor, and syntrophy

2022 ◽  
Author(s):  
Carlos Martinez ◽  
Eugenio Cinquemani ◽  
Hidde de Jong ◽  
Jean-Luc Gouze
Keyword(s):  
Recombinant Proteins ◽  
Protein Production ◽  
Performance Metrics ◽  
Sufficient Conditions ◽  
Optimal Operation ◽  
Microbial Consortia ◽  
Overflow Metabolism ◽  
Chemostat Model ◽  
E Coli ◽  
Trade Offs

The bacterium E. coli is widely used to produce recombinant proteins such as growth hormone and insulin. One inconvenience with E. coli cultures is the secretion of acetate through overflow metabolism. Acetate inhibits cell growth and represents a carbon diversion, which results in several negative effects on protein production. One way to overcome this problem is the use of a synthetic consortium of two different E. coli strains, one producing recombinant proteins and one reducing the acetate concentration. In this paper, we study a chemostat model of such a synthetic community where both strains are allowed to produce recombinant proteins. We give necessary and sufficient conditions for the existence of a coexistence equilibrium and show that it is unique. Based on this equilibrium, we define a multi-objective optimization problem for the maximization of two important bioprocess performance metrics, process yield and productivity. Solving numerically this problem, we find the best available trade-offs between the metrics. Under optimal operation of the mixed community, both strains must produce the protein of interest, and not only one (distribution instead of division of labor). Moreover, in this regime acetate secretion by one strain is necessary for the survival of the other (syntrophy). The results thus illustrate how complex multi-level dynamics shape the optimal production of recombinant proteins by synthetic microbial consortia.

scholarly journals Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

2006 ◽  
Vol 72 (8) ◽  
pp. 5225-5231 ◽  
Author(s):  
Emmanuel Frachon ◽  
Vincent Bondet ◽  
Hélène Munier-Lehmann ◽  
Jacques Bellalou
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Batch Cultures ◽  
E Coli ◽  
Working Volume ◽  
Versatile Tool ◽  
Labview Program ◽  
Medium Formulation ◽  
Conventional Medium

ABSTRACT A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

scholarly journals Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

2000 ◽  
Vol 66 (3) ◽  
pp. 884-889 ◽  
Author(s):  
Kazuyo Nishihara ◽  
Masaaki Kanemori ◽  
Hideki Yanagi ◽  
Takashi Yura
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Trigger Factor ◽  
Human Lysozyme ◽  
E Coli ◽  
Constructed Plasmids ◽  
Expression Plasmids

ABSTRACT To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production inE. coli.

DYNAMICS OF A MODEL FOR VIRULENT PHAGE T4

2008 ◽  
Vol 16 (04) ◽  
pp. 597-611 ◽  
Author(s):  
ZHIPENG QIU
Keyword(s):  
Population Density ◽  
Reproduction Number ◽  
Bacteriophage T4 ◽  
Sufficient Conditions ◽  
Chemostat Model ◽  
Virulent Phage ◽  
E Coli ◽  
Phage T4 ◽  
Theoretical Results ◽  
The Basic Reproduction Number

In this paper, the asymptotical behavior of a chemostat model for E. coli and the virulent phage T4 is analyzed. The basic reproduction number R0 is proved to be a threshold which determines the outcome of the virulent phage T4. If R0 < 1, the virus dies out; if R0 > 1, the virus persists. Sufficient conditions for the Hopf bifurcation are also established. The theoretical results show that increasing the input of nutrient will result in an increase in the equilibrium population density of the virulent bacteriophage T4, but will have no effect on the equilibrium population density of E. coli. The results also show that increasing the input of nutrient or increasing the average lytic time for the infected E. coli can destabilize the interaction between E. coli and T4.

scholarly journals Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

2021 ◽  
Vol 12 ◽  
Author(s):  
Gema Lozano Terol ◽  
Julia Gallego-Jara ◽  
Rosa Alba Sola Martínez ◽  
Adrián Martínez Vivancos ◽  
Manuel Cánovas Díaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Copy Number ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

scholarly journals Bacterial signal peptides: structure, optimization, and applications

Eureka ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Esra Erkut
Keyword(s):  
Recombinant Proteins ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Bacterial Species ◽  
Structure Optimization ◽  
Biochemical Properties ◽  
Signal Peptides ◽  
Signal Sequences ◽  
Transport Pathways ◽  
E Coli

Bacterial signal peptides are N-terminal tags that direct proteins for export through one of various transport pathways. These signal peptides are highly important as they are the key determinants of transport, ensuring that the correct protein ends up at the correct pathway. While these peptides consist of three domains with well conserved biochemical properties, there still remains a large amount of diversity between the signal sequences for different proteins, transport pathways, and bacterial species. Recent advancements have allowed us to predict signal sequences and manipulate them in an attempt to optimize export efficiency. This knowledge can then be exploited in the field of recombinant protein production wherein bacterial species can be used to produce and secrete proteins of interest. By fusing the protein with an optimized signal peptide, the yield or rate of export can be improved. This review focuses on signal peptides for two primary transport pathways (Sec and Tat) in E. coli specifically, with an emphasis on applications and the production of recombinant proteins.

scholarly journals Enhanced production of heterologous proteins by a synthetic microbial community: Conditions and trade-offs

2020 ◽  
Author(s):  
Marco Mauri ◽  
Jean-Luc Gouzé ◽  
Hidde de Jong ◽  
Eugenio Cinquemani
Keyword(s):  
Heterologous Protein ◽  
Coarse Grained ◽  
Microbial Consortia ◽  
Individual Species ◽  
General Interest ◽  
Heterologous Proteins ◽  
E Coli ◽  
Enhanced Production ◽  
Trade Offs ◽  
Depth Analysis

AbstractSynthetic microbial consortia have been increasingly utilized in biotechnology and experimental evidence shows that suitably engineered consortia can outperform individual species in the synthesis of valuable products. Despite significant achievements, though, a quantitative understanding of the conditions that make this possible, and of the trade-offs due to the concurrent growth of multiple species, is still limited. In this work, we contribute to filling this gap by the investigation of a known prototypical synthetic consortium. A first E. coli strain, producing a heterologous protein, is sided by a second E. coli strain engineered to scavenge toxic byproducts, thus favoring the growth of the producer at the expense of diverting part of the resources to the growth of the cleaner. The simplicity of the consortium is ideal to perform an in depth-analysis and draw conclusions of more general interest. We develop a coarse-grained mathematical model that quantitatively accounts for literature data from different key growth phenotypes. Based on this, assuming growth in chemostat, we first investigate the conditions enabling stable coexistence of both strains and the effect of the metabolic load due to heterologous protein production. In these conditions, we establish when and to what extent the consortium outperforms the producer alone in terms of productivity. Finally, we show in chemostat as well as in a fed-batch scenario that gain in productivity comes at the price of a reduced yield, reflecting at the level of the consortium resource allocation trade-offs that are well-known for individual species.

scholarly journals A freeze-thaw method for disintegration of Escherichia coli cells producing T7 lysozyme used in pBAD expression systems.

2007 ◽  
Vol 54 (3) ◽  
pp. 671-672 ◽  
Author(s):  
Marta Wanarska ◽  
Piotr Hildebrandt ◽  
Józef Kur
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Protein Gene ◽  
Expression Systems ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Deinococcus Geothermalis ◽  
Lac Promoter ◽  
Cell Disintegration

The pLysN plasmid containing the T7 lysozyme gene under control of the lac promoter was constructed to facilitate cell disintegration after expression of recombinant proteins in arabinose-induced expression systems. The usefulness of this plasmid was tested in Escherichia coli TOP10 and E. coli LMG194 cells carrying pBADMHADgeSSB plasmid containing Deinococcus geothermalis SSB protein gene under control of the araBAD promoter. The results showed that low-level expression of T7 lysozyme did not interfere with the target SSB protein production, and that the freezing-thawing treatment was sufficient for disruption of the E. coli cells producing low amounts of T7 lysozyme.

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 Regulating the T7 RNA Polymerase Expression in E. coli BL21 (DE3) to Provide More Host Options for Recombinant Protein Production

2021 ◽  
Author(s):  
Ying-Shuang Xu ◽  
Fei Du ◽  
Zi-Jia Li ◽  
Yu-Zhou Wang ◽  
Zi-Xu Zhang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Recombinant Protein ◽  
Rna Polymerase ◽  
Recombinant Proteins ◽  
Protein Production ◽  
T7 Rna Polymerase ◽  
Foreign Protein ◽  
Atpase Subunit ◽  
E Coli ◽  
Prokaryotic Expression System

Abstract Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PLacUV5), which produces more T7 RNAP than wild-type lac promoter (PLacWT) to promote the production of recombinant proteins. However, there is a resource allocated limitation between cell growth and protein production when producing autolytic proteins or membrane proteins. T7 RNAP is the key factor to solve this problem. Hence, we replaced respectively PLacUV5 with other inducible promoters: arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet) to optimize the production of recombinant protein by regulating the transcription level of T7 RNAP. Compared with BL21 (DE3), the constructed engineering strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the glucose dehydrogenase (GDH) production, the engineered strains BL21 (DE3::tet) exhibited great biomass, cell survival rate and foreign protein expression level. In addition, these engineered strains had been successfully applied to the production of other membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and E. coli F-ATPase subunit b (Ecb). The engineering strains constructed in this paper provided more host choices for the production of recombinant proteins.

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

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