Systems-level characterisation and improvement of Clostridium autoethanogenum metabolism
Date
2024-09-12
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Atsetogeensete bakterite uurimine on pälvinud kasvavat tähelepanu nende võime tõttu muundada kasvuhoonegaase väärtuslikeks produktideks läbi gaasfermentatsiooni. Tehnoloogial võib olla oluline roll kliimamuutuste ja jäätmekäitluse probleemide leevendamisel kuid globaalsel rakendusel seisavad ees väljakutsed. Esiteks on arusaam atsetogeenide metabolismist piiratud ning genotüüp-fenotüüp suhted on lõpuni mõistmata. Lisaks on geneetiline inseneerimine võrreldes mudelorganismidega algusjärgus, mida raskendab fakt, et enamike geenide funktsioonid on teadmata või ebaselged.
Doktoritöö keskendus nende väljakutsete lahendamisele, ühendades gaasfermentatsiooni süsteemibioloogia, adaptiivse laboratoorse evolutsiooni (ALE) ja geneetilise inseneerimisega. Eesmärk oli uurida kasvuerikiirusest sõltuvat metabolismi ning kasutada ALE-i et, omandada täiustatud omadustega C. autoethanogenum tüvesid. Lisaks oli eesmärgiks parendada genotüüp-fenotüüp seoste mõistmist.
Töö sisaldab kvantitatiivseid andmeid kasvuerikiiruse mõjust atsetogeenide metabolismile ja gaasfermentatsiooni protsessile. Antud tulemused näitavad, et C. autoethanogenumi kiirendatud kasv suurendab tööstuslikult oluliste ühendite tootlikkust ning edendavad arusaamist atsetogeenide transkriptsioonilisest regulatsioonist. Lisaks järeldab doktoritöö, et ALE-i abil on võimalik saada tüvesid, mis omavad oluliselt parenenud kasvuerikiirust, suudavad kasvada minimaalsöötmel ning näitavad head võimekust autotroofsetes läbivoolukultuurides. Muuhulgas uuriti ALE jooksul tekkinud kolme mutatsiooni mõju, konstrueerides kolm metsiktüvel põhinevat tüve ja iseloomustati neid pudeli- ning läbivoolukultuurides. Tulemused näitavad, et ALE-st tuletatud geneetiline inseneerimine saab anda oluliselt parendatud omadustega C. autoethanogenum tüved. Lisaks tuvastati konvergentse evolutsiooni olemasolu ning mitmeid uusi geneetilise inseneerimise sihtmärke, et luua parendatud omadustega rakuvabrikuid.
Acetogenic bacteria have become attractive biocatalysts for their ability to convert greenhouse gases into valuable products through gas fermentation. This technology has the potential to mitigate climate change and waste management problems, but challenges remain for global deployment. Firstly, our understanding of acetogen metabolism is limited and genotype-phenotype links are not clearly resolved. In addition, genetic engineering is in its relative infancy compared to model organisms and is hampered by the fact that the functions of most genes are currently unclear. This thesis focused on overcoming these challenges by combining gas fermentation with systems biology, adaptive laboratory evolution (ALE), and genetic engineering. The aim was to investigate specific growth rate-dependent metabolism and to use ALE to obtain C. autoethanogenum strains with improved characteristics. The thesis also aimed to improve the understanding of genotype-phenotype links. The work presents quantitative data on specific growth rate-dependent acetogen metabolism and the gas fermentation process. Results show that accelerated growth of C. autoethanogenum increases the productivity of industrially relevant compounds and advances our understanding of transcriptional regulation in acetogens. In addition, the thesis concludes that ALE can be used to engineer strains that grow faster, grow on minimal medium, and are robust in autotrophic continuous cultures. In addition, effects of three mutations from ALE were investigated in-depth by construction of three wild-type based strains and characterisation in bottle and bioreactor cultures. The findings show that ALE-inspired genetic engineering can yield C. autoethanogenum strains with notably improved traits. In addition, convergent evolution was detected and several novel targets for genetic engineering to acquire improved microbial cell factories were identified.
Acetogenic bacteria have become attractive biocatalysts for their ability to convert greenhouse gases into valuable products through gas fermentation. This technology has the potential to mitigate climate change and waste management problems, but challenges remain for global deployment. Firstly, our understanding of acetogen metabolism is limited and genotype-phenotype links are not clearly resolved. In addition, genetic engineering is in its relative infancy compared to model organisms and is hampered by the fact that the functions of most genes are currently unclear. This thesis focused on overcoming these challenges by combining gas fermentation with systems biology, adaptive laboratory evolution (ALE), and genetic engineering. The aim was to investigate specific growth rate-dependent metabolism and to use ALE to obtain C. autoethanogenum strains with improved characteristics. The thesis also aimed to improve the understanding of genotype-phenotype links. The work presents quantitative data on specific growth rate-dependent acetogen metabolism and the gas fermentation process. Results show that accelerated growth of C. autoethanogenum increases the productivity of industrially relevant compounds and advances our understanding of transcriptional regulation in acetogens. In addition, the thesis concludes that ALE can be used to engineer strains that grow faster, grow on minimal medium, and are robust in autotrophic continuous cultures. In addition, effects of three mutations from ALE were investigated in-depth by construction of three wild-type based strains and characterisation in bottle and bioreactor cultures. The findings show that ALE-inspired genetic engineering can yield C. autoethanogenum strains with notably improved traits. In addition, convergent evolution was detected and several novel targets for genetic engineering to acquire improved microbial cell factories were identified.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone