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Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control
KTH, School of Biotechnology (BIO), Bioprocess Technology.
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO), Bioprocess Technology.ORCID iD: 0000-0002-6979-0069
2008 (English)In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 31, no 1, 11-20 p.Article in journal (Refereed) Published
Abstract [en]

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h(-1), respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.

Place, publisher, year, edition, pages
2008. Vol. 31, no 1, 11-20 p.
Keyword [en]
fed-batch technique, acetate formation, high cell density, recombinant product formation, phosphotransferase system, PTS mutations
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-8368DOI: 10.1007/s00449-007-0144-xISI: 000251649100003Scopus ID: 2-s2.0-37249015436OAI: oai:DiVA.org:kth-8368DiVA: diva2:13671
Note
QC 20100902. Uppdaterad från Accepted till Published (20100902)Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Methodology for high-throughput production of soluble recombinant proteins in Escherichia coli
Open this publication in new window or tab >>Methodology for high-throughput production of soluble recombinant proteins in Escherichia coli
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The aim of this work was to investigate and determine central parameters that can be used to control and increase the solubility, quality and productivity of recombinant proteins. These central parameters should be applicable under the constraints of high-throughput protein production in Escherichia coli.

The present investigation shows that alternative methods exist to improve solubility, quality and productivity of the recombinant protein. The hypothesis is that by reducing the synthesis rate of the recombinant protein, a higher quality protein should be produced. The feed rate of glucose can be used to decrease the synthesis rate of the recombinant protein.

The influence of feed rate on solubility and proteolysis was investigated using the lacUV5-promoter and two model proteins, Zb-MalE and Zb-MalE31. Zb-MalE31 is a mutated form of Zb-MalE that contains two different amino acids. These altered amino acids greatly affect the solubility of the protein. The soluble fraction is generally twice as high using Zb-MalE compared to Zb-MalE31. Using a low feed rate compared to high benefits the formation of the full-length soluble protein. Furthermore, by using a low feed rate, the proteolysis can be decreased. One other factor that influences the solubility is the amount of inducer used. An increase from 100 µM to 300 µM IPTG only results in more inclusion bodies being formed, the fraction of soluble protein is the same.

The quality aspect of protein production was investigated for a secreted version of Zb-MalE using two different feed rates of glucose and the maltose induced promoter PmalK. It was shown that when the protein was secreted to the periplasm, the stringent response as well as the accumulation of acetic acid (even for high feed rates) was reduced. The stringent response and accumulation of acetic acid are factors that are known to affect the quality and quantity of recombinant proteins. Transporting the protein to the periplasm results in this case on a lower burden on the cell, which leads to less degradation products being formed when the protein is secreted to the periplasm.

Seeing the feed rate as a critical parameter, the high-throughput production would benefit from a variation in the feed rate. However, since the fed-batch technique is technically complicated for small volumes another approach is needed. E.coli strains that have been mutated to create an internal growth limitation that simulate fed-batch were cultivated in batch and were compared to the parent strain. It was shown that the growth rate and acetic acid formation was comparable to the parent strain in fed-batch. Furthermore it was shown that a higher cell mass was reached using one of the mutants when the cells were cultivated for as long time as possible. The higher cell mass can be used to reach a higher total productivity.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. 42 p.
Keyword
Escherichia coli, high-throughput methodology, recombinant protein production, solubility, productivity, quality, cultivation technology, parallel reactors
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4270 (URN)978-91-7178-561-9 (ISBN)
Presentation
2007-02-16, Sal FB54, AlbaNova universitetscentrum, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20101112Available from: 2007-02-02 Created: 2007-02-02 Last updated: 2010-11-12Bibliographically approved
2. Growth rate control of periplasmic product retention in Escherichia coli
Open this publication in new window or tab >>Growth rate control of periplasmic product retention in Escherichia coli
2008 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The recombinant product is secreted to the periplasm in many processes where E. coli is used as host. One drawback with secretion is the undesired leakage of the periplasmic products to the medium.

The aim of this work was to find strategies to influence the periplasmic retention of recombinant products. We have focused on the role of the specific growth rate, a parameter that is usually controlled in industrial bioprocesses. The hypothesis was that the stability of the outer membrane in E. coli is gained from a certain combination of specific phospholipids and fatty acids on one side and the amount and specificity of the outer membrane proteins on the other side, and that the specific growth rate influences this structure and therefore can be used to control the periplasmic retention.

We found that is possible to control the periplasmic retention by the growth rate. The leakage of the product increased as the growth rate increased. It was however also found that a higher growth rate resulted in increased productivity. This resulted in equal amounts of product inside the cells regardless of growth rate.

We also showed that the growth rate influenced the outer membrane composition with respect to OmpF and LamB while OmpA was largely unaffected. The total amount of outer membrane proteins decreased as the growth rate increased. There were further reductions in outer membrane protein accumulation when the recombinant product was secreted to the periplasm. The lowered amount of outer membrane proteins may have contributed to the reduced ability for the cell to retain the product in the periplasm.

The traditional way to control the growth rate is through a feed of substrate in a fed-batch process. In this work we used strains with a set of mutations in the phosphotransferase system (PTS) with a reduced uptake rate of glucose to investigate if these strains could be used for growth rate control in batch cultivations without the use of fed-batch control equipment. The hypothesis was that the lowering of the growth rate on cell level would result in the establishment of fed-batch similar conditions.

This study showed that it is possible to control the growth rate in batch cultivations by using mutant strains with a decreased level of substrate uptake rate. The mutants also produced equivalent amounts of acetic acid as the wild type did in fed-batch cultivation with the same growth rate. The oxygen consumption rates were also comparable. A higher cell density was reached with one of the mutants than with the wild type in batch cultivations. It is possible to control the growth rate by the use of the mutants in small-scale batch cultivations without fed-batch control equipment.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2008. 39 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:9
Keyword
Escherichia coli, fed-batch, outer membrane proteins, recombinant proteins, specific growth rate, periplasmic retention, phosphotransferase system, high cell density cultivation, acetate formation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4732 (URN)978-91-7178-953-2 (ISBN)
Presentation
2008-05-21, FB51, AlbaNova, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20101108Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2012-02-16Bibliographically approved
3. Impact of glucose uptake rate on recombinant protein production in Escherichia coli
Open this publication in new window or tab >>Impact of glucose uptake rate on recombinant protein production in Escherichia coli
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli.

E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures.  

The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation.

For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm.

The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:18
Keyword
AIDA-autotransporter, Escherichia coli, fed-batch, glucose uptake rate, integral membrane proteins, outer membrane proteins, periplasmic retention, phosphotransferase system, recombinant proteins, specific growth rate, surface expression
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-34019 (URN)978-91-7415-994-3 (ISBN)
Public defence
2011-06-15, FB52, AlbaNova, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20110608Available from: 2011-06-08 Created: 2011-05-23 Last updated: 2012-02-14Bibliographically approved

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