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Cellular Processes and Mechanisms in Saccharomyces cerevisiae Influencing Anaerobic Xylose Fermentation

Bergdahl, Basti LU (2013)
Abstract (Swedish)
Popular Abstract in Swedish

Ett viktigt steg i strävan att ersätta fossila bränslen med biobränslen är ett effektivt utnyttjande av förnyelsebar lignocellulosa. Råmaterialet är restprodukter från skogs- och jordbruksindustrin och består bl. a. av komplexa kolhydrater som cellulosa. Efter nedbrytning till enkla sockermolekyler kan dessa omvandlas till bioetanol genom jäsning. Sockret som extraheras från vissa lignocellulosa material består upp till 30% av xylos. Xylos är en sockermolekyl med fem kolatomer och för en ekonomiskt lönsam process krävs fullständig förjäsning av xylos till etanol.



Kol är den grundläggande byggstenen i alla biologiska molekyler. Alla sockerarter, även xylos, är både viktiga... (More)
Popular Abstract in Swedish

Ett viktigt steg i strävan att ersätta fossila bränslen med biobränslen är ett effektivt utnyttjande av förnyelsebar lignocellulosa. Råmaterialet är restprodukter från skogs- och jordbruksindustrin och består bl. a. av komplexa kolhydrater som cellulosa. Efter nedbrytning till enkla sockermolekyler kan dessa omvandlas till bioetanol genom jäsning. Sockret som extraheras från vissa lignocellulosa material består upp till 30% av xylos. Xylos är en sockermolekyl med fem kolatomer och för en ekonomiskt lönsam process krävs fullständig förjäsning av xylos till etanol.



Kol är den grundläggande byggstenen i alla biologiska molekyler. Alla sockerarter, även xylos, är både viktiga energikällor och kolkällor. Vid produktion av bioetanol används vanligtvis bagerijäst, Saccharomyces cerevisiae. Tyvärr saknar S. cerevisiae flera gener som behövs för att tillgodogöra sig xylos. Under de senaste 20 åren har nya jäststammar utvecklats genom att introducera dessa gener i S. cerevisiae. En stor del av utvecklingsarbetet har lagts på att öka upptagningshastigheten av xylos och utbytet av etanol. Produktionshastigheten av etanol från xylos är dock fortfarande mycket lägre än den som erhålls vid jäsning av socker med sex kolatomer, som t.ex. glukos.



Målet med arbetet som presenteras i denna avhandling var att öka produktiviteten av etanol vid jäsning av glukos och xylos tillsammans. Tillväxten av jästceller har stort inflytande på etanolproduktiviteten. De jäststammar som finns idag växer tämligen dåligt på xylos. Många av de signaler som jästcellen använder för att reglera tillväxten beror på förändringar i koncentrationen av små molekyler inne i cellen, s.k. metaboliter. För att förstå xylos-metabolismen undersökte jag hur koncentrationer av metaboliter förändras vid jäsning av xylos och jämförde dessa med förändringar som sker vid jäsning av glukos. I mitt arbete dentifierade och utvärderade jag cellulära processer och mekanismer som krävs för celltillväxt. Arbetet gav upphov till en ny hypotes: den långsamma tillväxten på xylos kan bero på begränsningar i veckningen av proteiner inuti i cellen. En essentiell process som ger proteinerna deras aktiva form.



Hexokinas 2 (Hxk2p) är ett bi-funktionellt protein som deltar i regleringen av processer specifika för produktionen av etanol. Enzymet inaktiveras av xylos med minskad kontroll som följd. Genom att kombinera metoder för att modifiera proteiner och mikroorganismer med jäsningsteknologi har jag identifierat en mutation i HXK2-genen som gör enzymet mindre känsligt för xylos. Det muterade enzymet medförde snabbare konsumtion av glukos i närvaro av xylos, men hade ingen uppenbar effekt på jäsningen av xylos. Det innebär att Hxk2p inte agerar på egen hand och att andra proteiner också påverkar regleringen. Vilka dessa proteiner är återstår att utforska.



I min avhandling har jag sammanfattat min kunskap om de ntracellulära signaler som uppkommer vid jäsning av xylos. Jag visar också hur jag har använt dessa signaler för att identifiera biokemiska processer som potentiellt begränsar förmågan hos S. cerevisiae att växa på kol- och energikällan xylos. (Less)
Abstract
In 2009 the EU approved two directives as a first initiative towards reducing greenhouse gas emissions and becoming independent of fossil fuels: the Renewable Energy Directive and the Fuel Quality Directive. As a result, the demand for biofuels will increase enormously over the next decade, both nationally and in the entire EU. This huge demand will require a more advanced type of biofuels, produced from cellulosic and lignocellulosic raw materials that do not compete with the supply of food crops. These biofuels are referred to as second generation (2G) fuels. The production of 2G bioethanol at a commercial scale requires yeast strains capable of producing ethanol at high yield and high productivity from all sugars (hexoses and pentoses)... (More)
In 2009 the EU approved two directives as a first initiative towards reducing greenhouse gas emissions and becoming independent of fossil fuels: the Renewable Energy Directive and the Fuel Quality Directive. As a result, the demand for biofuels will increase enormously over the next decade, both nationally and in the entire EU. This huge demand will require a more advanced type of biofuels, produced from cellulosic and lignocellulosic raw materials that do not compete with the supply of food crops. These biofuels are referred to as second generation (2G) fuels. The production of 2G bioethanol at a commercial scale requires yeast strains capable of producing ethanol at high yield and high productivity from all sugars (hexoses and pentoses) extracted from the raw material.



The aim of the work presented in this thesis has been to increase the ethanol productivity of recombinant xylose-fermenting strains of the yeast Saccharomyces cerevisiae during batch fermentation of a glucose/xylose mixture. A parameter that has a big influence on productivity is cellular growth and the yeast strains currently used today grow rather poorly on xylose. Many of the signals cells use to regulate growth originate from changes in the concentrations of metabolites inside the cells. To increase our knowledge of xylose metabolism the dynamic changes in intracellular metabolite concentrations were measured during batch fermentation of a glucose/xylose mixture using LC-MS/MS. This study gave meaningful insights about important intracellular signals, biological phenomena and mechanism. The analysis of the metabolite data pointed toward limitations in the folding of proteins inside the ER, which might be the underlying cause of the slow growth on xylose.



Another important factor is the regulation of expression of genes required for sugar transport and those related to fermentative metabolism. Hexokinase 2 (Hxk2p) is an important bi-functional protein that acts both as a catalytic enzyme and a global transcription factor. This protein plays a role in the regulation of the above mentioned genes and becomes inactivated in the presence of xylose. As a consequence it loses its’ regulatory function. In an effort to improve repression signals during xylose fermentation this protein was engineered to become immune towards inactivation by xylose. By combining methods for protein and genetic engineering with fermentation technology a mutation in the gene was identified which increased the catalytic activity by 64% in the presence of xylose. The new variant allowed faster glucose consumption in the presence of xylose, but had no obvious impact on xylose fermentation. These results indicate that Hxk2p does not act alone and other proteins are involved in the regulation. These proteins remain to be identified.



This thesis describes the cellular processes required for balanced anaerobic microbial growth and the intracellular signals that regulate them. The aim has been to identify biochemical mechanisms that limit anaerobic growth of recombinant S. cerevisiae strains on xylose. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Prof. Förster, Jochen, Novo Nordisk Foundation Center for Biosustainability, Hørsholm, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Saccharomyces cerevisiae, Bioethanol, Xylose metabolism, Metabolomics, Regulation, Protein folding, Microbial growth
pages
254 pages
publisher
Applied Microbiology (LTH)
defense location
Lecture hall K:B, Kemicentrum, Getingevägen 60, Lund University, Faculty of Engineering
defense date
2013-05-17 10:15
ISBN
978-91-7422-318-7
language
English
LU publication?
yes
id
541cbdd6-184a-4ad0-be78-a2563db9b034 (old id 3633658)
date added to LUP
2013-04-23 13:14:42
date last changed
2016-09-19 08:45:05
@phdthesis{541cbdd6-184a-4ad0-be78-a2563db9b034,
  abstract     = {In 2009 the EU approved two directives as a first initiative towards reducing greenhouse gas emissions and becoming independent of fossil fuels: the Renewable Energy Directive and the Fuel Quality Directive. As a result, the demand for biofuels will increase enormously over the next decade, both nationally and in the entire EU. This huge demand will require a more advanced type of biofuels, produced from cellulosic and lignocellulosic raw materials that do not compete with the supply of food crops. These biofuels are referred to as second generation (2G) fuels. The production of 2G bioethanol at a commercial scale requires yeast strains capable of producing ethanol at high yield and high productivity from all sugars (hexoses and pentoses) extracted from the raw material. <br/><br>
<br/><br>
The aim of the work presented in this thesis has been to increase the ethanol productivity of recombinant xylose-fermenting strains of the yeast Saccharomyces cerevisiae during batch fermentation of a glucose/xylose mixture. A parameter that has a big influence on productivity is cellular growth and the yeast strains currently used today grow rather poorly on xylose. Many of the signals cells use to regulate growth originate from changes in the concentrations of metabolites inside the cells. To increase our knowledge of xylose metabolism the dynamic changes in intracellular metabolite concentrations were measured during batch fermentation of a glucose/xylose mixture using LC-MS/MS. This study gave meaningful insights about important intracellular signals, biological phenomena and mechanism. The analysis of the metabolite data pointed toward limitations in the folding of proteins inside the ER, which might be the underlying cause of the slow growth on xylose. <br/><br>
<br/><br>
Another important factor is the regulation of expression of genes required for sugar transport and those related to fermentative metabolism. Hexokinase 2 (Hxk2p) is an important bi-functional protein that acts both as a catalytic enzyme and a global transcription factor. This protein plays a role in the regulation of the above mentioned genes and becomes inactivated in the presence of xylose. As a consequence it loses its’ regulatory function. In an effort to improve repression signals during xylose fermentation this protein was engineered to become immune towards inactivation by xylose. By combining methods for protein and genetic engineering with fermentation technology a mutation in the gene was identified which increased the catalytic activity by 64% in the presence of xylose. The new variant allowed faster glucose consumption in the presence of xylose, but had no obvious impact on xylose fermentation. These results indicate that Hxk2p does not act alone and other proteins are involved in the regulation. These proteins remain to be identified. <br/><br>
<br/><br>
This thesis describes the cellular processes required for balanced anaerobic microbial growth and the intracellular signals that regulate them. The aim has been to identify biochemical mechanisms that limit anaerobic growth of recombinant S. cerevisiae strains on xylose.},
  author       = {Bergdahl, Basti},
  isbn         = {978-91-7422-318-7},
  keyword      = {Saccharomyces cerevisiae,Bioethanol,Xylose metabolism,Metabolomics,Regulation,Protein folding,Microbial growth},
  language     = {eng},
  pages        = {254},
  publisher    = {Applied Microbiology (LTH)},
  school       = {Lund University},
  title        = {Cellular Processes and Mechanisms in Saccharomyces cerevisiae Influencing Anaerobic Xylose Fermentation},
  year         = {2013},
}