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Growth rate control of periplasmic product retention in Escherichia coli
KTH, School of Biotechnology (BIO), Bioprocess Technology.
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 [en]
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: urn:nbn:se:kth:diva-4732ISBN: 978-91-7178-953-2 (print)OAI: oai:DiVA.org:kth-4732DiVA: diva2:13672
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
List of papers
1. Fedbatch design for periplasmic product retention in Escherichia coli
Open this publication in new window or tab >>Fedbatch design for periplasmic product retention in Escherichia coli
2008 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 135, no 4, 358-365 p.Article in journal (Refereed) 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.

Keyword
E. coli, Fedbatch, Feed rate, Outer membrane proteins, Periplasmic retention, Recombinant proteins
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8367 (URN)10.1016/j.jbiotec.2008.05.002 (DOI)000258477200006 ()2-s2.0-47249110176 (Scopus ID)
Note
QC 20100914. Uppdaterad från Submitted till Published (20100914)Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2017-12-14Bibliographically approved
2. Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control
Open this publication in new window or tab >>Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control
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.

Keyword
fed-batch technique, acetate formation, high cell density, recombinant product formation, phosphotransferase system, PTS mutations
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-8368 (URN)10.1007/s00449-007-0144-x (DOI)000251649100003 ()2-s2.0-37249015436 (Scopus ID)
Note
QC 20100902. Uppdaterad från Accepted till Published (20100902)Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2017-12-14Bibliographically approved

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