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Impact of glucose uptake rate on recombinant protein production in Escherichia coli
KTH, School of Biotechnology (BIO), Bioprocess Technology.
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 [en]
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: urn:nbn:se:kth:diva-34019ISBN: 978-91-7415-994-3OAI: oai:DiVA.org:kth-34019DiVA: diva2:418588
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
List of papers
1. 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 (ScopusID)
Note
QC 20100902. Uppdaterad från Accepted till Published (20100902)Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2011-06-08Bibliographically approved
2. 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 (ScopusID)
Note
QC 20100914. Uppdaterad från Submitted till Published (20100914)Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2011-06-08Bibliographically approved
3. Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli.
Open this publication in new window or tab >>Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli.
2011 (English)In: Microbial Cell Factories, ISSN 1475-2859, Vol. 10, no 1, 35- p.Article in journal (Refereed) Published
Abstract [en]

The production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes. RESULTS: A set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities. CONCLUSIONS: The use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.

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-34433 (URN)10.1186/1475-2859-10-35 (DOI)000291990500001 ()21586123 (PubMedID)2-s2.0-79955953366 (ScopusID)
Note
Updated from submitted to published. QC 20110711Available from: 2011-06-08 Created: 2011-06-08 Last updated: 2011-07-12Bibliographically approved
4. Optimisation of surface expression using the AIDA autotransporter
Open this publication in new window or tab >>Optimisation of surface expression using the AIDA autotransporter
2011 (English)In: Microbial Cell Factories, ISSN 1475-2859, Vol. 10Article in journal (Refereed) Published
Abstract [en]

Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l(-1) could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

Keyword
ESCHERICHIA-COLI, BIOTECHNOLOGICAL APPLICATIONS, DISPLAY, BATCH, PROTEINS, PROTEASE
National Category
Bioprocess Technology
Identifiers
urn:nbn:se:kth:diva-34435 (URN)10.1186/1475-2859-10-72 (DOI)000295830400001 ()2-s2.0-80052833041 (ScopusID)
Note

QC 20111103

Available from: 2011-06-08 Created: 2011-06-08 Last updated: 2013-05-16Bibliographically approved

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