University of Oulu

Development of rapid gene expression analysis and its application to bioprocess monitoring

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Author: Rautio, Jari
Organizations: University of Oulu, Faculty of Technology, Department of Process and Environmental Engineering
Format: eBook
Online Access: PDF Full Text (PDF, )
Persistent link: http://urn.fi/urn:isbn:9789513870584
Language: English
Published: 2007
Publish Date: 2007-12-12
Thesis type: Doctoral Dissertation
Defence Note: Academic Dissertation to be presented with the assent of the Faculty of Technology, University of Oulu, for public discussion in Raahensali (Auditorium L10), Linnanmaa, on December 7th, 2007, at 12 noon.
Opponent: Professor Sven-Olof Enfors
Kustos: Professor Peter Neubauer
Description:

Abstract

Cultivation of a microorganism in a bioreactor offers an ideal environment for optimized production of industrial compounds and for studying biological phenomena under reproducible conditions. In order to be able better to understand and control biological systems both for industrial and scientific purposes, development of methods that generate more detailed information about the biosystems is required. The focus in the development of tools for monitoring and control of bioreactor cultivations is on analyses that report on the physiological status of the production organism. Genetic expression is an important and growing aspect of cellular physiology, because the genomic sequences are becoming available for an increasing number of organisms. Technologies enabling studies of whole genome-wide expression analysis have provided large quantities of gene expression data under various conditions. One consequence of this has been the discovery of smaller sets of genes that provide the essential information about the biological system of interest. This has increased the need for technologies enabling rapid and cost-effective detection of specific gene transcripts.

The aim of the present study was to develop methods suitable for expression analysis of defined gene sets in bioprocess conditions, and to apply the methods for monitoring microbial cultures. The environmental conditions in bioreactor cultivations set certain challenges for the methodology. The environmental surroundings are typically in constant change during bioprocesses, requiring frequent analysis. In addition, the conditions are affected by various factors, such as decreasing nutrient and oxygen levels and increasing levels of secreted proteins or ethanol. Thus the number of relevant genes to be monitored in a process is dozens to hundreds rather than a few. For control purposes the response time of the method should be short.

The solution (sandwich) hybridization principle was applied in the development of two mRNA analysis methods: 1. a sandwich hybridization assay with alkaline phosphatase-based signal amplification and 2. a solution hybridization method called TRAC (Transcript analysis with the aid of affinity capture) using a pool of oligonucleotide probes separable and quantifiable by capillary electrophoresis. The basic sandwich hybridization assay detects one target per sample, whereas TRAC was capable of more than 20-plex RNA target detection. Both methods are performed in 96-well format with crude cell lysates as sample material. The developed methods have many advantages that make them suitable for monitoring microbial cultures. The analysis is simple (RNA extraction and cDNA conversions are avoided), the protocol time is short and for large numbers of samples the methods could be semi-automated by using magnetic bead processors. Multiplex target detection by the TRAC method makes it suitable for high-throughput gene expression analysis.

The TRAC method was applied for monitoring protein production processes and chemostat cultures of the filamentous fungus Trichoderma reesei, used widely in industrial enzyme production. In addition conventional beer fermentations by brewer's lager yeast (Saccharomyces pastorianus) were monitored by frequent analysis of gene expression facilitated by TRAC. Altogether about 30 T. reesei and 70 S. pastorianus genes were identified with presumed relevance to the respective processes and were subsequently tested in process conditions. Many of the marker gene expression profiles showed to have value in the prediction of consecutive physiological effects and of process performance both in the filamentous fungus and in yeast. Marker gene expression measured by TRAC could be used e.g. in evaluation of growth and of the production potential of secreted proteins, as well as in evaluation of nutrient and oxygen availability. In addition TRAC was used in the evaluation of gene expression stability during steady state conditions during T. reesei chemostat cultures as well as during transient oxygen deprivations. These data were applicable in the evaluation of steady state quality, which was useful when selecting samples for further systems-level analyses. The data obtained by TRAC confirmed the value of focused and frequent analysis of gene expression in monitoring biotechnical processes, providing a powerful tool for process optimization purposes.


Tiivistelmä

Bioprosesseja käytetään teolliseen biologisten komponenttien tuottamiseen ja biologisten ilmiöiden tutkimiseen toistettavissa olosuhteissa. Jotta biologisia systeemejä voitaisiin sekä ymmärtää että kontrolloida paremmin, tarvitaan sellaisten menetelmien kehitystä, jotka tuottavat yksityiskohtaista informaatiota organismien fysiologisesta tilasta. Geenien ilmentyminen on tärkeä osa solujen fysiologiaa. Genominlaajuiset ilmentymisanalyysit ovat mahdollistaneet pienempien geeniryhmien identifioinnin, jonka avulla voidaan selvittää olennainen informaatio kiinnostuksen kohteena olevasta biologisesta systeemistä. Tämä puolestaan on lisännyt tarvetta tekniikoille, jotka mahdollistavat nopean ja edullisen geenien transkriptien mittauksen.

Tässä tutkimuksessa oli tavoitteena kehittää menetelmiä, jotka soveltuvat tiettyjen geeniryhmien ilmentymisen mittaukseen bioprosesseissa. Liuoshybridisaatioperiaatetta sovellettiin kehitettäessä sandwich-hybridisaatiomenetelmää sekä menetelmää nimeltä TRAC (Transcript analysis with the aid of affinity capture), jossa käytetään sellaisten oligonukleotidikoettimien joukkoja, jotka voidaan erotella ja kvantifioida kapillaarielektroforeesilla. Sandwich-hybridisaatiomenetelmää voidaan käyttää havainnoimaan yksittäisiä kohteita kustakin näytteestä, kun taas TRAC-menetelmä mahdollistaa yli 20 RNA-kohteen yhtäaikaisen mittauksen kustakin näytteestä, mikä tekee siitä tehokkaan ilmentymisanalyysin. Kehitettyjen menetelmien edut bioprosessien seurannassa ovat helppokäyttöisyys ja nopea mittaus suoraan solulysaateista. Näytteenkäsittely on osittain automatisoitu suurien näytemäärien yhtäaikaiseen käsittelyyn.

TRAC-menetelmää sovellettiin rihmasieni Trichoderma reeseillä suoritettujen proteiinituottoprosessien sekä lager-hiivalla (Saccharomyces pastorianus) suoritettujen käymisten seurantaan. Yhteensä identifioitiin 30 T. reesei ja 70 S. pastoriasnus -prosessien kannalta tärkeiksi oletettua merkkigeeniä. Useiden identifioitujen rihmasienen ja hiivan merkkigeenien ilmentymisprofiilien osoitettiin ennustavan sekä fysiologisia vaikutuksia että prosessien etenemistä. Lisäksi TRAC-menetelmää käytettiin geenien ilmentymistasojen stabiilisuuden mittaukseen T. reesei -kemostaattikasvatuksissa. Tätä dataa käytettiin arvioimaan kasvatusten laatua valittaessa näytteitä jatkoanalyyseihin. TRAC-menetelmällä kerätty data osoitti fokusoidun ja tiheään tapahtuvan geenien ilmentymismittauksen hyödyn bioteknisten prosessien seurannassa ja tarjosi täten tehokkaan työkalun prosessien optimointiin.


Series: VTT Publications
ISSN: 1235-0621
ISSN-E: 1455-0849
ISSN-L: 1235-0621
ISBN: 978-951-38-7058-4
ISBN Print: 978-951-38-7057-7
Issue: 661
Subjects:
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