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Impact of glucose feed rate on productivity and recombinant protein quality in Escherichia coli
KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate.

The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, qp, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins.

By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation,

In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied.

In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the PmalK production system.

A novel fed-batch process based on the promoters for the universal stress proteins A and B (PuspA, PuspB) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects.

Place, publisher, year, edition, pages
Stockholm: KTH , 2005. , 66 p.
Keyword [en]
Biochemistry, Escherichia coli, recombinant protein production, fed-batch, feed profile, specific growth rate
Keyword [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-115ISBN: 91-7283-944-9 (print)OAI: oai:DiVA.org:kth-115DiVA: diva2:6936
Public defence
2005-02-04, Kollegiesalen, Valhallavägen 79, Stockholm, 10:00
Opponent
Supervisors
Note

QC 20101008

Available from: 2005-02-01 Created: 2005-02-01 Last updated: 2012-09-27Bibliographically approved
List of papers
1. The influence of time and method of induction on recombinant protein productivity and acetic acid formation in Escherichia coli
Open this publication in new window or tab >>The influence of time and method of induction on recombinant protein productivity and acetic acid formation in Escherichia coli
2004 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290Article in journal (Other academic) Submitted
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-25119 (URN)
Note
QS 20120316Available from: 2010-10-08 Created: 2010-10-08 Last updated: 2017-12-12Bibliographically approved
2. Limiting factors in Escherichia coli fed-batch production of recombinant proteins
Open this publication in new window or tab >>Limiting factors in Escherichia coli fed-batch production of recombinant proteins
Show others...
2003 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 81, no 2, 158-166 p.Article in journal (Refereed) Published
Abstract [en]

Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high (mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.

Keyword
recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation, by-product formation, ppGpp
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-25120 (URN)10.1002/bit.10457 (DOI)000180063000004 ()
Note
QC 20101008Available from: 2010-10-08 Created: 2010-10-08 Last updated: 2017-12-12Bibliographically approved
3. Fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations
Open this publication in new window or tab >>Fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations
Show others...
2003 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 83, no 5, 595-603 p.Article in journal (Refereed) Published
Abstract [en]

A high-level production system using the universal stress promoters uspA and uspB in a fed-batch cultivation based on minimal medium was designed. In development it was shown that a standard industrial fed-batch protocol could not be used for this purpose since it failed to induce the levels of product as compared to the basal level. Instead, a batch protocol followed by a low constant feed of glucose was shown to give full induction. The levels of the product protein, beta-galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the uspA than uspB vectors where uspA showed considerably higher basal level. The data indicate that the sigma(70) regulated promoter, uspA, although affected by the alarmone guanosine tetraphosphate, ppGpp, worked partly in a similar manner to constitutive promoters. An industrial high cell density fedbatch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final beta-galatosidase concentration of 7 g/L and a final cell concentration of 65 g/L. The heterogeneity in production of the individual cell was measured by fluorescence microscopy. The data show that there is a process time independent heterogeneity in production, which is suggested to be caused by heterogeneity in the substrate uptake rate of the individual cell.

Keyword
stress promoters, uspA, uspB, recombinant protein production, fed-batch, high cell density, plasmid and chromosomal construction, escherichia-coli, protein expression, limit growth, genes, strategies, starvation, cloning
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-22690 (URN)10.1002/bit.10716 (DOI)000184310700010 ()
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved
4. Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli
Open this publication in new window or tab >>Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli
Show others...
2005 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 68, no 1, 82-90 p.Article in journal (Refereed) Published
Abstract [en]

The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

Keyword
fed-batch production, cultivations, expression, promoters, fusion, system
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-6752 (URN)10.1007/s00253-004-1855-4 (DOI)000230859900013 ()2-s2.0-23944489637 (Scopus ID)
Note

QC 20101008

Available from: 2007-02-02 Created: 2007-02-02 Last updated: 2017-12-14Bibliographically approved
5. Solubility and proteolysis of the Zb-MaIE and Zb-MaIE31 proteins during overproduction in Escherichia coli
Open this publication in new window or tab >>Solubility and proteolysis of the Zb-MaIE and Zb-MaIE31 proteins during overproduction in Escherichia coli
2005 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 90, no 2, 239-247 p.Article in journal (Refereed) Published
Abstract [en]

From the hypothesis that the rate of expression of a nascent polypeptide controls the accumulation of soluble full-length protein, accumulation of the model fusion proteins Zb-MalE and Zb-MalE31, were studied. MalE and MalE31 are two isoforms of the maltose binding protein, differing only in two consecutive amino acids. Parameters controlling the expression rate were the transcription rate, which was controlled by IPTG induction of the lacUV5 promoter and the substrate addition levels during fed-batch cultivation.

Results show that the two product proteins appear in both soluble and insoluble fractions during cultivation and are both subjected to proteolysis. However, the accumulation of the soluble form of Zb-MalE31 protein is radically lower, at all conditions, due to the small difference in primary structure.

It was shown that both proteolysis and inclusion body formation could be influenced by the selected parameters although a change in feed rate had a considerably higher effect. A high concentration of inducer and a "high" feed rate result in a low accumulation of soluble product, due to a high proteolysis. The concentration of inducer leading to different levels of transcription is, however, an efficient tool to influence inclusion body formation. At low IPTG concentrations (<= 5 mu M), this formation is almost abolished while at a comparatively high concentration (>= 300 mu M) 50% of the total product accumulated was in the form of inclusion bodies.

Keyword
Escherichia coli, Fed-batch, Feed-rate, Inclusion bodies, Proteolysis, Solubility, Zb-MalE, Zb-MalE31
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-6751 (URN)10.1002/bit.20433 (DOI)000227994700009 ()
Note
QC 20100929Available from: 2007-02-02 Created: 2007-02-02 Last updated: 2017-12-14Bibliographically approved

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