Broadly applicable genetic tools for fungi

Anssi Rantasalo

Broadly applicable genetic tools for fungi

Julkaisun otsikon käännös: Laajatoimiset geneettiset työkalut hiiva- ja homeorganismeille

Anssi Rantasalo

Tutkimustuotos: Doctoral Thesis › Collection of Articles

Abstrakti

Mikro-organismien geneettinen muokkaus bioteknisiä sovelluksia varten on selvästi helpottunut ja nopeutunut viime vuosien aikana. Keskeisenä syynä ovat uudet molekyylibiologian menetelmät, jotka mahdollistavat tuottoisäntien genomien helpon ja nopean muokkauksen. Tämän seurauksena tehokkaiden solutehtaiden rakentaminen teollisuuden tarpeisiin on tullut mahdolliseksi. Sovellusten kehittämistä rajoittaa kuitenkin edelleen geenien ilmentämiseen tarvittavien tehokkaiden ja luotettavasti toimivien työkalujen puute. Tämän vuoksi esimerkiksi vähän tutkittujen organismien biotekninen hyödyntäminen on vaikeaa. Tässä väitöskirjatyössä kehitettiin teknologia, joka mahdollistaa geenien tarkan ilmentämisen laajasti eri hiiva- ja homelajeissa. Uudet työkalut tarjoavat runsaasti uusia mahdollisuuksia bioteollisuudelle, koska hiivoilla ja homeilla on monia teollisiin prosesseihin sopivia ominaisuuksia. Työkalut ovat erityisen hyödyllisiä sovelluksissa, joissa edellytetään geenien voimakasta tai tarkasti kontrolloitua ilmentymisestä. Työn ensimmäinen osa (julkaisut I ja II) keskittyy kehittämään toiminnallisesti modulaarisia DNA-osia, kuten ydinpromoottoreita ja transkriptiofaktoreita Saccharomyces cerevisiae hiivassa. Kehitetyt osat mahdollistavat monimutkaisten geneettisten järjestelmien rakentamisen, ja niiden avulla on mahdollista säädellä geenien ilmentymistä laajasti eri voimakkuuksilla. Työn seuraava osa (julkaisu III) keskittyy kehittämään universaalisti toimivia työkaluja, jotka mahdollistavat geenien ilmentämisen laajasti eri hiiva- ja homeorganismeissa. Tekniikan toimintaa havainnollistetaan yhteensä kahdeksassa erityyppisessä organismissa: kuudessa eri hiivassa ja kahdessa rihmasienessä. Työn tuloksena syntynyt teknologia mahdollistaa täysin uusien tai vain vähän tutkittujen organismien valjastamisen bioteknisiin sovelluksiin. Työn viimeisessä osassa työkalujen käyttöä havainnollistetaan proteiinintuottosovelluksessa (julkaisu IV). Esimerkkiproteiiniksi valittiin Candida antarctican Lipaasi B, jota ilmennettiin Trichoderma reesei rihmasienessä. Saavutetut tuottotasot vastasivat tasoa, joka saavutettiin yleisesti käytetyllä selluloosa-indusoituvalla cbh1 promoottorilla. Uusien työkalujen olosuhteista riippumattoman toiminnan vuoksi proteiinintuotto oli mahdollista suorittaa myös sellulaasientsyymien ilmentymistä estävässä kasvatusliuoksessa, mahdollistaen kohdeproteiinin tehokkaan tuotannon siten että solun omia proteiineja ei tuotettu kasvuliuokseen. Tämä helpottaa merkittävästi kohdeproteiinin puhdistusta ja alentaa siihen käytettyjä kustannuksia.

Julkaisun otsikon käännös	Laajatoimiset geneettiset työkalut hiiva- ja homeorganismeille
Alkuperäiskieli	Englanti
Pätevyys	Tohtorintutkinto
Myöntävä instituutio	Aalto-yliopisto
Valvoja/neuvonantaja	Frey, Alexander, Vastuuprofessori Mojzita, Dominik, Ohjaaja, Ulkoinen henkilö Jäntti, Jussi, Ohjaaja, Ulkoinen henkilö
Kustantaja	Aalto University
Painoksen ISBN	978-952-60-8361-2
Sähköinen ISBN	978-952-60-8362-9
Tila	Julkaistu - 2019
OKM-julkaisutyyppi	G5 Artikkeliväitöskirja

Tutkimusalat

ekspressiosysteemi
hiiva
home
synteettinen biologia
transkriptiofaktori
teollinen biotekniikka

Pääsy asiakirjaan

http://urn.fi/URN:ISBN:978-952-60-8362-9

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@phdthesis{25d46ed92fb549d49ca79936e6272837,

title = "Broadly applicable genetic tools for fungi",

abstract = "Engineering of microbial hosts for industrial use has became significantly easier and faster in the recent years. This has been enabled by novel molecular biology tools and genome editing methods. However, the gene expression tools required for optimization of the engineered organisms are often inefficient, or completely lacking especially in less-established production hosts. This hinders the rapid development of novel biotechnological applications. This PhD work focused on developing synthetic biology tools that allow accurate control of gene expression in a broad spectrum of fungal species. The outcomes of this thesis represent an important advance in the field of biological engineering because fungi comprise an industrially important group of organisms. The developed tools provide high utility especially in applications in which precise balancing or strong gene expression is required. In the first part of this work (Publication I and II), a library of modular genetic parts, including a set of core promoters and synthetic transcription factors was established in Saccharomyces cerevisiae. These tools enable construction of complex genetic devices and they provided a broad gene expression range. Next part of this work focuses on development of universal gene expression system for fungi (Publication III). The functionality of the system was demonstrated in eight diverse fungal hosts: in six yeasts and two filamentous fungi. Importantly, due to universal performance of the system, these tools also makes it possible to introduce novel eukaryotic microbes for biotechnological use. In the final part, the developed synthetic tools were applied in a protein production application (Publication IV). The selected example protein product was lipase B of Candida antarctica (calB), and the production was carried out in filamentous fungus Trichoderma reesei. The obtained protein levels were comparable to the levels obtained by using the commonly employed inducible cbh1 promoter. In addition, due to highly constitutive expression provided by synthetic expression system, it was possible to produce highly pure protein in cellulase-repressing glucose medium eliminating the secretion of unwanted background enzymes.",

keywords = "expression system, yeast, fungi, synthetic biology, transcription factor, industrial biotechnology, ekspressiosysteemi, hiiva, home, synteettinen biologia, transkriptiofaktori, teollinen biotekniikka, expression system, yeast, fungi, synthetic biology, transcription factor, industrial biotechnology",

author = "Anssi Rantasalo",

year = "2019",

language = "English",

isbn = "978-952-60-8361-2",

series = "Aalto University publication series DOCTORAL DISSERTATIONS",

publisher = "Aalto University",

number = "2",

address = "Finland",

school = "Aalto University",

}

TY - BOOK

T1 - Broadly applicable genetic tools for fungi

AU - Rantasalo, Anssi

PY - 2019

Y1 - 2019

N2 - Engineering of microbial hosts for industrial use has became significantly easier and faster in the recent years. This has been enabled by novel molecular biology tools and genome editing methods. However, the gene expression tools required for optimization of the engineered organisms are often inefficient, or completely lacking especially in less-established production hosts. This hinders the rapid development of novel biotechnological applications. This PhD work focused on developing synthetic biology tools that allow accurate control of gene expression in a broad spectrum of fungal species. The outcomes of this thesis represent an important advance in the field of biological engineering because fungi comprise an industrially important group of organisms. The developed tools provide high utility especially in applications in which precise balancing or strong gene expression is required. In the first part of this work (Publication I and II), a library of modular genetic parts, including a set of core promoters and synthetic transcription factors was established in Saccharomyces cerevisiae. These tools enable construction of complex genetic devices and they provided a broad gene expression range. Next part of this work focuses on development of universal gene expression system for fungi (Publication III). The functionality of the system was demonstrated in eight diverse fungal hosts: in six yeasts and two filamentous fungi. Importantly, due to universal performance of the system, these tools also makes it possible to introduce novel eukaryotic microbes for biotechnological use. In the final part, the developed synthetic tools were applied in a protein production application (Publication IV). The selected example protein product was lipase B of Candida antarctica (calB), and the production was carried out in filamentous fungus Trichoderma reesei. The obtained protein levels were comparable to the levels obtained by using the commonly employed inducible cbh1 promoter. In addition, due to highly constitutive expression provided by synthetic expression system, it was possible to produce highly pure protein in cellulase-repressing glucose medium eliminating the secretion of unwanted background enzymes.

AB - Engineering of microbial hosts for industrial use has became significantly easier and faster in the recent years. This has been enabled by novel molecular biology tools and genome editing methods. However, the gene expression tools required for optimization of the engineered organisms are often inefficient, or completely lacking especially in less-established production hosts. This hinders the rapid development of novel biotechnological applications. This PhD work focused on developing synthetic biology tools that allow accurate control of gene expression in a broad spectrum of fungal species. The outcomes of this thesis represent an important advance in the field of biological engineering because fungi comprise an industrially important group of organisms. The developed tools provide high utility especially in applications in which precise balancing or strong gene expression is required. In the first part of this work (Publication I and II), a library of modular genetic parts, including a set of core promoters and synthetic transcription factors was established in Saccharomyces cerevisiae. These tools enable construction of complex genetic devices and they provided a broad gene expression range. Next part of this work focuses on development of universal gene expression system for fungi (Publication III). The functionality of the system was demonstrated in eight diverse fungal hosts: in six yeasts and two filamentous fungi. Importantly, due to universal performance of the system, these tools also makes it possible to introduce novel eukaryotic microbes for biotechnological use. In the final part, the developed synthetic tools were applied in a protein production application (Publication IV). The selected example protein product was lipase B of Candida antarctica (calB), and the production was carried out in filamentous fungus Trichoderma reesei. The obtained protein levels were comparable to the levels obtained by using the commonly employed inducible cbh1 promoter. In addition, due to highly constitutive expression provided by synthetic expression system, it was possible to produce highly pure protein in cellulase-repressing glucose medium eliminating the secretion of unwanted background enzymes.

KW - expression system

KW - yeast

KW - fungi

KW - synthetic biology

KW - transcription factor

KW - industrial biotechnology

KW - ekspressiosysteemi

KW - hiiva

KW - home

KW - synteettinen biologia

KW - transkriptiofaktori

KW - teollinen biotekniikka

KW - expression system

KW - yeast

KW - fungi

KW - synthetic biology

KW - transcription factor

KW - industrial biotechnology

M3 - Doctoral Thesis

SN - 978-952-60-8361-2

T3 - Aalto University publication series DOCTORAL DISSERTATIONS

PB - Aalto University

ER -

Broadly applicable genetic tools for fungi

Abstrakti

Tutkimusalat

Pääsy asiakirjaan

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