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Fedbatch design for periplasmic product retention in Escherichia coli
KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.ORCID-id: 0000-0002-6979-0069
2008 (Engelska)Ingår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 135, nr 4, s. 358-365Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h-1 where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.

Ort, förlag, år, upplaga, sidor
2008. Vol. 135, nr 4, s. 358-365
Nyckelord [en]
E. coli, Fedbatch, Feed rate, Outer membrane proteins, Periplasmic retention, Recombinant proteins
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-8367DOI: 10.1016/j.jbiotec.2008.05.002ISI: 000258477200006Scopus ID: 2-s2.0-47249110176OAI: oai:DiVA.org:kth-8367DiVA, id: diva2:13670
Anmärkning
QC 20100914. Uppdaterad från Submitted till Published (20100914)Tillgänglig från: 2008-05-08 Skapad: 2008-05-08 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
Ingår i avhandling
1. Growth rate control of periplasmic product retention in Escherichia coli
Öppna denna publikation i ny flik eller fönster >>Growth rate control of periplasmic product retention in Escherichia coli
2008 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2008. s. 39
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2008:9
Nyckelord
Escherichia coli, fed-batch, outer membrane proteins, recombinant proteins, specific growth rate, periplasmic retention, phosphotransferase system, high cell density cultivation, acetate formation
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-4732 (URN)978-91-7178-953-2 (ISBN)
Presentation
2008-05-21, FB51, AlbaNova, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20101108Tillgänglig från: 2008-05-08 Skapad: 2008-05-08 Senast uppdaterad: 2012-02-16Bibliografiskt granskad
2. Impact of glucose uptake rate on recombinant protein production in Escherichia coli
Öppna denna publikation i ny flik eller fönster >>Impact of glucose uptake rate on recombinant protein production in Escherichia coli
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2011
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2011:18
Nyckelord
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
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
urn:nbn:se:kth:diva-34019 (URN)978-91-7415-994-3 (ISBN)
Disputation
2011-06-15, FB52, AlbaNova, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20110608Tillgänglig från: 2011-06-08 Skapad: 2011-05-23 Senast uppdaterad: 2012-02-14Bibliografiskt granskad

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