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An investigation into the hex1 gene and gene promoter for the enhancement of protein production in Trichoderma reesei

thesis
posted on 28.03.2022, 09:19 by Natalie Claire Curach
For Trichoderma reesei to be developed as an effiecient producer of a large variety of proteins, the expression system requires diversification. In particular, the choice of promoters available needs to be broadened to include promoters which are active in conditions other than those conducive to induction of cellulase expression. Using proteomics, the HEX1 protein was identified as an abundant protein of the cell envelope of T. reesei when grown on a range of carbon sources, suggesting that a strong constitutive promoter drives the expression of this physiologically important protein. This thesis is an exploration into the hex1 gene promoter and the role of hex1 in the maintenance of mycelium integrity in T. reesei with consideration for the application of this gene in the further development of filamentous fungi as protein expression systems. -- The single copy hex1 gene and flanking regions were isolated from T. reesei and another biotechnologically important fungus, Ophiostoma floccosum. The fluorescent reporter protein DsRed1-E5 was expressed under the T. reesei hex1 promoter and promoter activity was monitored by fluorescence CLSM and RNA analysis. During the rapid growth phase of a culture, the hex1 promoter was active in a range of carbon sources and three transcipt types with alternative tsp and splicing sites were discovered for the hex1 gene. The distribution of fluorescence throughout the mycelium suggested spatial regulation of the hex1 promoter as well as temporal regulation. The promoter was continually active in the absence of a functional hex1 gene product suggesting that the hex1 promoter is regulated in part, by negative feedback from the endogenous gene product. Interruption of the hex1 gene produced hyphae that leaked excessive volumes of cytoplasm when physically damaged which may be advantageous for the externalisation of selected protein products. The results indicate that the regulation of the hex1 hene promoter is complex and that the hex1 gene is integral to the maintenance of the integrity of the fungal mycelium.

History

Alternative Title

Investigation into the hex1 gene and gene promoter in Trichoderma reesei

Table of Contents

Introduction -- Materials and methods -- Isolation of the hex1 gene from Trichoderma reesei and Ophiostoma floccosum -- Expression of DsRed under the cbh1 promoter and the hex1 promoter with random integration -- Modified expression vectors containing a fusion to a portion of hex1 gene sequence -- Expression of DsRed from the hex1 locus and the phenotypic characteristics of a hex1 deletion mutant -- Summary and concluding discussion.

Notes

Supplementary material to figures contained on DVD only available with manuscript. Bibliography: p. 221-244

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

Thesis (PhD), Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences

Department, Centre or School

Dept. of Biological Sciences

Year of Award

2005

Principal Supervisor

Helena Nevalainen

Additional Supervisor 1

Peter Bergquist

Additional Supervisor 2

Junior T'eo

Rights

Copyright disclaimer: http://www.copyright.mq.edu.au Copyright Natalie Claire Curach 2005.

Language

English

Extent

xv, 244 p. ill

Former Identifiers

mq:7032 http://hdl.handle.net/1959.14/71199 1346811