Evolution of recombinant (t-PA) Trichoderma reesei in chemostats selects mutants down-regulated in cellulase production

Karin Lanthaler, Jaana Uusitalo, Tiina Pakula, Merja Penttilä, Geoff D. Robson

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

Abstract

In order to study the stability of a recombinant strain of T.reesei expressing tissue plasminogen activator (t-PA), the recombinant strain 306/36 (with t-PA under the control of the native cbhl promoter) and its parent strain (Rut-C30) were grown in chemostat cultures at a dilution rate of 0.05 h--1 in lactose limited chemostats and the secreted levels of recombinant t-PA and native cellulases determined. It was found, that t-PA and native cellulase production by the recombinant strain 306/36 was not stable with morphological mutants displacing the parental strain. This displacement was correlated with a loss in native cellulase and t-PA secretion. Strain Rut-C30 yielded similar morphological mutants, but their appearance was not correlated with a loss of native cellulase production and some mutants at the end of the cultivation even showed enhanced cellulase secretion. Isolated mutants were grown in batch cultivation and the cellulase and t-PA levels compared to parental strain 306/36 and wild type Rut-C30. All mutant isolates derived from the recombinant strain 306/36 showed severe reduction in their ability to secrete native cellulases and t-PA production was virtually undetectable, although Southern blotting revealed no apparent loss in gene copy numbers of the t-PA::cbh1 construct. The reduction may be well due to the fact, that recombinant protein production challenges the fungal secretion machinery to an extent that favours mutants in which recombinant protein production is suppressed. As both t-PA and native cellulase are down-regulated, we are investigating the potential role of the transcriptional regulators of the cellulase genes, ACEI and ACE II.
Original languageEnglish
Title of host publicationPoster Abstracts
Pages189
Publication statusPublished - 2004
Event7th European Conference on Fungal Genetics - Copenhagen, Denmark
Duration: 17 Apr 200420 Apr 2004

Conference

Conference7th European Conference on Fungal Genetics
CountryDenmark
CityCopenhagen
Period17/04/0420/04/04

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Trichoderma
Cellulase
Tissue Plasminogen Activator
Cellulases
Recombinant Proteins
Gene Dosage
Lactose
Regulator Genes
Southern Blotting

Cite this

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title = "Evolution of recombinant (t-PA) Trichoderma reesei in chemostats selects mutants down-regulated in cellulase production",
abstract = "In order to study the stability of a recombinant strain of T.reesei expressing tissue plasminogen activator (t-PA), the recombinant strain 306/36 (with t-PA under the control of the native cbhl promoter) and its parent strain (Rut-C30) were grown in chemostat cultures at a dilution rate of 0.05 h--1 in lactose limited chemostats and the secreted levels of recombinant t-PA and native cellulases determined. It was found, that t-PA and native cellulase production by the recombinant strain 306/36 was not stable with morphological mutants displacing the parental strain. This displacement was correlated with a loss in native cellulase and t-PA secretion. Strain Rut-C30 yielded similar morphological mutants, but their appearance was not correlated with a loss of native cellulase production and some mutants at the end of the cultivation even showed enhanced cellulase secretion. Isolated mutants were grown in batch cultivation and the cellulase and t-PA levels compared to parental strain 306/36 and wild type Rut-C30. All mutant isolates derived from the recombinant strain 306/36 showed severe reduction in their ability to secrete native cellulases and t-PA production was virtually undetectable, although Southern blotting revealed no apparent loss in gene copy numbers of the t-PA::cbh1 construct. The reduction may be well due to the fact, that recombinant protein production challenges the fungal secretion machinery to an extent that favours mutants in which recombinant protein production is suppressed. As both t-PA and native cellulase are down-regulated, we are investigating the potential role of the transcriptional regulators of the cellulase genes, ACEI and ACE II.",
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Lanthaler, K, Uusitalo, J, Pakula, T, Penttilä, M & Robson, GD 2004, Evolution of recombinant (t-PA) Trichoderma reesei in chemostats selects mutants down-regulated in cellulase production. in Poster Abstracts., VIIIp-5, pp. 189, 7th European Conference on Fungal Genetics, Copenhagen, Denmark, 17/04/04.

Evolution of recombinant (t-PA) Trichoderma reesei in chemostats selects mutants down-regulated in cellulase production. / Lanthaler, Karin; Uusitalo, Jaana; Pakula, Tiina; Penttilä, Merja; Robson, Geoff D.

Poster Abstracts. 2004. p. 189 VIIIp-5.

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

TY - CHAP

T1 - Evolution of recombinant (t-PA) Trichoderma reesei in chemostats selects mutants down-regulated in cellulase production

AU - Lanthaler, Karin

AU - Uusitalo, Jaana

AU - Pakula, Tiina

AU - Penttilä, Merja

AU - Robson, Geoff D.

PY - 2004

Y1 - 2004

N2 - In order to study the stability of a recombinant strain of T.reesei expressing tissue plasminogen activator (t-PA), the recombinant strain 306/36 (with t-PA under the control of the native cbhl promoter) and its parent strain (Rut-C30) were grown in chemostat cultures at a dilution rate of 0.05 h--1 in lactose limited chemostats and the secreted levels of recombinant t-PA and native cellulases determined. It was found, that t-PA and native cellulase production by the recombinant strain 306/36 was not stable with morphological mutants displacing the parental strain. This displacement was correlated with a loss in native cellulase and t-PA secretion. Strain Rut-C30 yielded similar morphological mutants, but their appearance was not correlated with a loss of native cellulase production and some mutants at the end of the cultivation even showed enhanced cellulase secretion. Isolated mutants were grown in batch cultivation and the cellulase and t-PA levels compared to parental strain 306/36 and wild type Rut-C30. All mutant isolates derived from the recombinant strain 306/36 showed severe reduction in their ability to secrete native cellulases and t-PA production was virtually undetectable, although Southern blotting revealed no apparent loss in gene copy numbers of the t-PA::cbh1 construct. The reduction may be well due to the fact, that recombinant protein production challenges the fungal secretion machinery to an extent that favours mutants in which recombinant protein production is suppressed. As both t-PA and native cellulase are down-regulated, we are investigating the potential role of the transcriptional regulators of the cellulase genes, ACEI and ACE II.

AB - In order to study the stability of a recombinant strain of T.reesei expressing tissue plasminogen activator (t-PA), the recombinant strain 306/36 (with t-PA under the control of the native cbhl promoter) and its parent strain (Rut-C30) were grown in chemostat cultures at a dilution rate of 0.05 h--1 in lactose limited chemostats and the secreted levels of recombinant t-PA and native cellulases determined. It was found, that t-PA and native cellulase production by the recombinant strain 306/36 was not stable with morphological mutants displacing the parental strain. This displacement was correlated with a loss in native cellulase and t-PA secretion. Strain Rut-C30 yielded similar morphological mutants, but their appearance was not correlated with a loss of native cellulase production and some mutants at the end of the cultivation even showed enhanced cellulase secretion. Isolated mutants were grown in batch cultivation and the cellulase and t-PA levels compared to parental strain 306/36 and wild type Rut-C30. All mutant isolates derived from the recombinant strain 306/36 showed severe reduction in their ability to secrete native cellulases and t-PA production was virtually undetectable, although Southern blotting revealed no apparent loss in gene copy numbers of the t-PA::cbh1 construct. The reduction may be well due to the fact, that recombinant protein production challenges the fungal secretion machinery to an extent that favours mutants in which recombinant protein production is suppressed. As both t-PA and native cellulase are down-regulated, we are investigating the potential role of the transcriptional regulators of the cellulase genes, ACEI and ACE II.

M3 - Conference abstract in proceedings

SP - 189

BT - Poster Abstracts

ER -