Yeast Saccharomyces cerevisiae strain isolated from lager beer shows tolerance to isobutanol.

Linköping University | Department of Physics, Chemistry and Biology Bachelor thesis, 16 hp | Educational Program: Physics, Chemistry and Biology Spring term 2016 | LITH-IFM-G-EX—16/3184--SE

Yeast Saccharomyces

cerevisiae strain isolated from

lager beer shows tolerance to

isobutanol.

Datum

Date 2016-06-02

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Division, Department

Department of Physics, Chemistry and Biology Linköping University

URL för elektronisk version

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ISRN: LITH-IFM-G-EX--16/3184--SE

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Yeast Saccharomyces cerevisiae strain isolated from lager beer shows tolerance to isobutanol.

Författare

Author Linnéa Gerebring

Nyckelord

KeywordBiofuels, Butanol tolerance, isobutanol, metabolic engineering, RPN4, Saccharomyces cerevisiae

Sammanfattning

Abstract

The development of biofuels has received much attention due to the global warming and limited resources associated with fossil fuels. Butanol has been identified as a potential option due to its advantages over ethanol, for example higher energy density, compatibility with current infrastructure and its possibility to be blended with gasoline at any ratio. Yeast Saccharomyces cerevisiae can be used as a producer of butanol. However, butanol toxicity to the host limits the yield produced. In this study, four strains of yeast isolated from the habitats of lager beer, ale, wine and baker´s yeast were grown in YPD media containing isobutanol concentrations of 1.5 %, 2 %, 3 % and 4 %. Growth was measured to determine the most tolerant strain. Gene expression for the genes RPN4, RTG1 and ILV2 was also measured, to determine its involvement in butanol stress. The genes have in previous studies seen to be involved in butanol tolerance or production, and the hypothesis was that they all should be upregulated in response to butanol exposure. It was found that the yeast strain isolated from lager beer was most tolerant to isobutanol concentrations of 2 % and 3 %. It was also found that the gene RPN4 was upregulated in response to isobutanol stress. There was no upregulation of RTG1 or ILV2, which was unexpected. The yeast strain isolated from lager beer and the gene RPN4 is proposed to be investigated further, to be able to engineer a suitable producer of the biofuel butanol.

Content

1 Abstract ... 1

2 Introduction ... 1

2.1 The need for biofuels ... 1

2.2 Advantages of butanol ... 2

2.3 Butanol production in Saccharomyces cerevisiae ... 2

2.4 Butanol toxicity ... 3

2.5 Changes in gene expression due to butanol stress ... 4

2.6 Aim of the study ... 5

3 Material & methods ... 5

3.1 YPD-medium and agar plates preparation ... 5

3.2 Strains and growth conditions ... 5

3.3 Spot plating for isobutanol tolerance ... 6

3.4 OD measurements of growth ... 6

3.5 Sequencing of the isolated strains to determine species ... 6

3.6 Measurements of gene expression ... 7

3.7 Statistical analyses ... 9

4 Results ... 9

4.1 Growth measurements by spot tests ... 9

4.2 OD measurements of growth ... 10

4.3 Measurements of gene expression ... 12

4.4 Sequencing for determining the identity of the strains ... 14

5 Discussion ... 14

6 Acknowledgement ... 20

7 References ... 20

1 Abstract

The development of biofuels has received much attention due to the global warming and limited resources associated with fossil fuels. Butanol has been identified as a potential option due to its advantages over ethanol, for example higher energy density, compatibility with current infrastructure and its possibility to be blended with gasoline at any ratio. Yeast Saccharomyces cerevisiae can be used as a producer of butanol. However, butanol toxicity to the host limits the yield produced. In this study, four strains of yeast isolated from the habitats of lager beer, ale, wine and baker´s yeast were grown in YPD media containing

isobutanol concentrations of 1.5 %, 2 %, 3 % and 4 %. Growth was measured to determine the most tolerant strain. Gene expression for the genes RPN4, RTG1 and ILV2 was also measured, to determine its

involvement in butanol stress. The genes have in previous studies seen to be involved in butanol tolerance or production, and the hypothesis was that they all should be upregulated in response to butanol exposure. It was found that the yeast strain isolated from lager beer was most tolerant to isobutanol concentrations of 2 % and 3 %. It was also found that the gene RPN4 was upregulated in response to isobutanol stress. There was no upregulation of RTG1 or ILV2, which was unexpected. The yeast strain isolated from lager beer and the gene RPN4 is proposed to be

investigated further, to be able to engineer a suitable producer of the biofuel butanol.

2 Introduction

2.1 The need for biofuels

The global climate change and the limited availability of fossil fuels have inspired for a search for renewable and environmentally friendly

alternatives (Zhou et al. 2012). Biofuels, for example ethanol, butanol and biodiesel, are considered to play an important role when it comes to replacing fossil fuels as transportation fuels (Li et al. 2014). The Swedish government have a vision to have all transportation free from fossil fuels by 2030, and a long-term goal is to produce energy without any

contribution to the greenhouse effect by 2050 (Swedish government 2012). The US Department of Energy have estimated that at least 30 % of petroleum consumption can be replaced with renewable biofuels in 2030 (U.S. Department of Energy 2011). Other sources, for example fuel cells, hydrogen and electricity have also been investigated to replace fossil fuels, but their low energy density and limited capacity makes them

(International Energy Agency 2010). When replacing fossil fuels, the whole spectrum of fuels must be replaced, everything from gasoline to the jet fuel kerosene (Buijs et al. 2013). The most commonly used biofuel today is ethanol, which can be produced from sugar or starch-based sugar cane, corn and wheat. The quandary with using ethanol as a biofuel is that it is a feedstock in food production, and our increasing population size and demand for food supply prevents the expanding of ethanol as a biofuel further (Fairley 2011). Biodiesel, produced from vegetable oil, is also used as a biofuel, but its low energy density and limited resource of feedstock prevents it from being used at a large scale (Röttig et al. 2010). 2.2 Advantages of butanol

Butanol is renewable and environmentally friendly, and can also be used as a biofuel (Xue et al. 2013). Compared to ethanol, butanol has a longer carbon chain and therefore a higher energy density. It is also less

hygroscopic, less volatile and is compatible with current infrastructure for gasoline distribution because it is less corrosive (Durre 2007, Fischer et al. 2008, Buijs et al. 2013, Gonzales-Ramos et al. 2013). Butanol can be blended with gasoline or diesel fuels at any ratio, but it can also be used as 100 % biofuel (Hönig et al. 2014), in comparison to ethanol that can only be blended up to 85 % (Durre 2007). There are four isomers of butanol; 1-butanol, isobutanol, 2-butanol and tert-butanol. Isobutanol has the most similar octane number and energy density to gasoline, making it the best option as replacement for fossil fuels (Connor & Liao 2009). 2.3 Butanol production in Saccharomyces cerevisiae

Butanol has previously been naturally produced by bacterium family

Clostridium through ABE-fermentation. However, it has not been

economically advantageous for industrial production due to a large

production of the byproducts acetone and ethanol (Lee et al. 2008) and its sensitivity to butanol concentrations above 2 % (Lin & Blaschek 1983). Many have tried to engineer Clostridium to overcome its weaknesses, but it has an advanced physiology and there is a lack of efficient genetic tools (Zheng et al. 2009). The ability of Escherichia coli, Bacillus subtilis and

Lactobacillus brevis to produce butanol has also been investigated

(Atsumi et al. 2008, Nielsen et al. 2009, Berezina et al. 2010), but bacteria has the disadvantages of needing to grow in neutral pH, is difficult to separate from fermentation broths (Ezeji et al. 2007), and is vulnerable to phage infections when grown in a large scale (Huffer et al. 2012).

An optimal microbial producer should produce high yields of butanol, be tolerant to high concentrations of alcohols and low pH, be easy to

separate from fermentation broths and be able to produce butanol from various feedstocks (Fischer et al. 2008, Steen et al. 2008). Saccharomyces

cerevisiae is a potentially great alternative host for higher alcohol

production, and can ferment cellulosic feedstocks in addition to sugar- and starched based crops. Yeast is a natural producer of small amounts of higher alcohols, for example isobutanol, through degradation of amino acids (Hazelwood et al. 2008). It has been used in production of

beverages, for example wine and beer, for thousands of years and is known for its high tolerance to ethanol and low pH, which is important when fermenting lignocellulosic materials (Chen et al. 2011, de Lucena et al. 2012). Due to its large size and mass, S. cerevisiae is easy to separate from fermentation broths, it can grow under various conditions

(Hasunuma & Kondo 2012) and it is also easy to genetically manipulate (Krivoruchko et al. 2011). S. cerevisiae can also be used to produce diesel substitutes farnesene, bisabolene and fatty acid ethyl esters (FAAE), but also jet fuel substitute amorphadiene, in addition to the gasoline

substitutes isobutanol and 1-butanol (Buijs et al. 2013). In that way, butanol production in yeast can cover the whole spectrum of fuels. Yeast produces butanol through the Ehrlich pathway. Isobutanol production consists of anabolic synthesis of pyruvate into

α-ketoisovalerate, an intermediate in the valine biosynthesis, by ILV2, ILV3 and ILV5 in the mitochondria. The ketoacid is then transported to the cytosol where it is converted into isobutanol by enzymes 2-ketoacid decarboxylase (Kdc) and alcohol dehydrogenase (Adh) (Hazelwood et al. 2008).

Despite all positive properties with S. cerevisiae, the naturally produced butanol yield is very small, 0.16 mg/g glucose (Chen et al. 2011). To achieve higher yields, many attempts have been made to metabolically engineer S. cerevisiae to increase butanol production (Chen et al. 2011, Kondo et al. 2011, Brat et al. 2012, Lee et al. 2012, Avalos et al. 2013). 2.4 Butanol toxicity

The major problem with reaching high yields of butanol is that the butanol itself is toxic to the host cell (Gonzalez-Ramos et al. 2013). Butanol changes the structure and fatty acid composition of the cell

membrane and increases its fluidity (Osborne et al. 1990). The membrane function is inhibited by butanol because the cell cannot maintain its pH due to increased proton permeability and loss of intracellular molecules such as RNA, ATP and proteins (Osborne et al. 1990).

It has been proven that S. cerevisiae tolerates alcohol well compared to other Saccharomyces species, and it has been noticed that it can tolerate concentrations of ethanol as high as 20 % (Chen et al. 2011). Butanol is more toxic to the host than ethanol (Fisher et al. 2008), so a tolerant strain must be found to achieve high yields. It is reported that in Clostridium spp., an addition of 7-13 g/l of butanol inhibits growth by 50 %, whereas an addition of 40 g/l acetone or ethanol inhibits growth by 50 % (Jones & Woods 1986).

One study has shown that 1-butanol concentrations of up to 1 % had modest inhibitory effects on S. cerevisiae strains BY4741 and

CEN.PK113-7D, but there was no growth at 1.45 % or 1.57 %

respectively (Gonzalez-Ramos et al. 2013). In a study from 2014, 90

Saccharomyces spp. strains were screened for their tolerance to

1-butanol. S. cerevisiae strains DBVPG1788, DBVPG6044 and YPS128 were the most tolerant strains, and were able to tolerate 3 % 1-butanol (Zaki et al. 2014). It was also shown that all yeast strains had a reduced metabolic output in presence of 3 % 1-butanol, compared to unstressed yeast (Zaki et al. 2014). S. uvarum species were shown to be the most sensitive, and had no metabolic output at all in 3 % 1-butanol (Zaki et al. 2014).

2.5 Changes in gene expression due to butanol stress

Yeast S. cerevisiae has been shown to be adjustable and has evolved mechanisms to adapt to changing environments, such as shifting temperature, nutrient supply, osmolarity and acidity, by changing its genomic expression (Gasch et al. 2000). A few genes have previously been described as upregulated when S. cerevisiae is exposed to butanol stress. In a study from 2013, both the genes RPN4 and RTG1 contributed to an increased butanol tolerance, and overexpression of the two genes generated a strain that tolerated a higher concentration of butanol (Gonzalez-Ramos et al. 2013). RTG1 is a gene encoding transcription factors involved in communication between the mitochondria,

peroxisomes and the nucleus, and is important for the expression of peroxisomal genes (Chelstowska & Butow 1995). RPN4 is a gene encoding a transcription factor involved in proteasome genes and is

responsible for protein degradation (Wang et al. 2008). In a study done in 2014 by Zaki et al., it was also shown that the gene RPN4 was

upregulated in the tolerant strain YPS128, in exposure to both 1.5 % and 3 % 1-butanol, compared to one of the sensitive strains, UWOPS05-227.2. Knock-out of RPN4 made the strain sensitive to 1-butanol compared to the control (Zaki et al. 2014). ILV2, which is involved in conversion of pyruvate into α-ketoisovalerate in the mitochondrial

isobutanol pathway, is a gene that has been shown to increase butanol yield. Overexpression of ILV2 generated a 13-fold increase in isobutanol production in S. cerevisiae (Kondo et al. 2011).

2.6 Aim of the study

The first aim of this experimental study was to investigate the butanol tolerance in four strains of yeast S. cerevisiae, isolated from the different habitats lager beer, ale, wine and baker´s yeast, to determine which strain that was most tolerant to butanol and would be most suitable as a

producer of biofuels. The unknown yeast strains from the different

habitats were then sequenced, to determine which strain they belonged to. The second aim of this study was to measure the gene expression for three different genes, RPN4, RTG1 and ILV2, and the hypothesis was that all three genes would be upregulated in exposure to 2 % isobutanol, compared to the reference gene UBC6.

3 Material & methods

3.1 YPD-medium and agar plates preparation

Two different YPD mediums were made, one in a liquid form and one containing agar. 10 g/l yeast extract, 20 g/l peptone and 20 g/l glucose was added to 1 litre of distilled water. For the agar plates, 20 g/l agar was added to the YPD medium. The medium was then sterilized for 15

minutes at 121 °C. YPD medium and 4 %, 3 % or 1.5 % isobutanol were then added to the agar plates. Agar plates without isobutanol were also prepared, as a control.

3.2 Strains and growth conditions

Four different yeast strains were isolated from the habitats of wine, ale, lager beer and baker´s yeast. The lager yeast was isolated from ”Folkes Fodgeöl” from Centralbryggeriet in Linköping. The ale yeast was isolated from ”Single Hop Ale” from Oppigårds. 100 µl from the ale and lager was transferred to a YPD agar plate each. Yeast cells were identified with a microscope and one yeast colony from each habitat was streaked on a new YPD agar plate. Isolated yeast cells from wine (Italian Red) were ordered from WYEAST, and the baker´s yeast (Kronjäst) was ordered from Jästbolaget, and streaked on an YPD agar plate. Yeast from the different habitats was then inoculated and grown in 5 ml liquid YPD medium for 72 hours in 30 degrees, while shaken in 190 rpm.

3.3 Spot plating for isobutanol tolerance

To measure the butanol tolerance for the four different strains, yeast was spotted onto YPD agar plates containing 1.5 %, 3 % or 4 % isobutanol. Control plates without isobutanol were also prepared. The different cultured strains were diluted to an initial OD600 (optical density, 600 nm) of 1. Sterile water was then added to the cultures to obtain samples with 102, 103 and 104 dilution factors. 5 µl aliquot from each concentration of yeast was spotted onto agar plates. After 24 and 48 hours, the growth of the different strains was measured to determine their tolerance to

isobutanol, and the plates were made in triplicate. The spot plating test for isobutanol tolerance was repeated twice, with similar results.

3.4 OD measurements of growth

To measure growth in isobutanol, the different yeast strains were inoculated into 10 ml liquid YPD medium overnight. Enough yeast culture was then inoculated to reach an initial OD600 of 0.1 in 50 ml YPD medium. 3 %, 2 % or 1.5 % isobutanol was added to the samples. A control without isobutanol was also made. All of the samples were prepared in duplicates. The yeast was incubated in 30 °C and shaken at 190 rpm throughout the measurements. OD600 was measured

approximately every other hour in a WPA Lightwave II

Spectrophotometer (Biochrom, Cambridge, England) to measure the growth of the different strains in isobutanol. To determine if there was any difference in percentage growth between the strains, the mean OD-values obtained at the last measurement was divided with the initial OD. 3.5 Sequencing of the isolated strains to determine species

Two different methods were used to extract the DNA.

1. DNA was extracted from the yeast cells according to the protocol of FastDNA Spin Kit (MPBiomedicals, Solon, OH 44139).

2. 200 µl liquid yeast (OD600=0.4) from every strain was suspended in 100 µl of 0.2 M LiOAc with 1 % SDS solution. The samples were incubated at 70 °C for 5 minutes before 300 µl of 99 % ethanol was added. The samples were vortexed and then centrifuged at 15000 x g for 3 minutes. The pellet was then washed with 70 % ethanol and dissolved in 100 µl sterile water. The cell debris was spun down at 15000 x g for 15 seconds and the supernatant (extracted DNA) was used for the

Polymerase Chain Reaction (PCR).

A master mix for each strain and for both primer combinations (COX1 and ITS) was then made with 1 µl 10 nM dNTP Mix, 0.4 µl DreamTaq DNA polymerase (5 U/µl), 5 µl 10X DreamTaq Buffer, 2 µl forward

primer (10 pmol/µl), 2 µl reverse primer (10 pmol/µl) and 38.6 µl water. Then 49 µl master mix was added to 1 µl extracted DNA in a PCR tube. A PCR was run in a S1000 Thermo Cycler, with the following program: 95 °C for 3 minutes, then 35 cycles of 95 °C for 30 seconds, 55 °C for 60 seconds and 72 °C for 45 seconds. The PCR ended with 72 °C for 10 minutes. The PCR samples were then kept in 4 °C before the gel electrophoresis.

To prepare the gel, 0.5 g agarose was melted in 50 ml 0.5X TBE gel electrophoresis buffer by incubating in a microwave for 2-3 minutes. The gel was then let to cool down to 50-60 °C before adding 5µl of 10000X SYBR Safe stain. The gel was poured into a gel tray and was let to cool down for at least 15 minutes. Then 10 µl of each PCR sample was mixed with 2 µl 6X loading dye, and the samples were loaded onto the gel. The gel was run at 100 V for 1 hour and a picture was taken in UV-light to determine which samples to send in for sequencing.

Purification of the DNA in the PCR products was done according to the protocol of GeneJET PCR Purification Kit (Fermentas, 2009). 15 µl of the purified DNA with a concentration of 5 ng/µl and 2 µl of primer with a concentration of 10 pmol/µl were then mixed and the samples were sent in to Eurofins Genomics (Eurofins Genomics 2016) for sequencing. Sequencing was performed for the genes ITS and COX1. COX1 has previously been proven a successful region when determining species of yeast in a study from 2014 (Schroeder & Shadel 2014). In a study from 2015, the ITS-region was used to discriminate 18 different strains of S.

cerevisiae, isolated from fermented foods (Keshani et al. 2015). Primers

(Appendix Table 1) were ordered from Invitrogen ThermoFisher (ThermoFisher 2015).

3.6 Measurements of gene expression

Gene expression was measured for the genes RPN4, RTG1 and ILV2, in lager yeast that was stressed in 2 % isobutanol and lager yeast that was unstressed. The reference gene UBC6, to which the different gene

expressions were compared to, has previously been proven to be reliable in a study from 2015 where it showed no up- or downregulation when exposed to different conditions (Llanos et al. 2015). Lager yeast was chosen because it showed the most tolerance to 2 % isobutanol. 10 ml of yeast cells with an OD600 of 0.6 were taken from an overnight culture, where yeast had grown without or in 2 % isobutanol. RNA was isolated by using the protocol of FastRNA Pro Red Kit (Biomedicals). The

concentration of Q-RNA was then measured on the nanodrop spectrophotometer ND-1000.

To remove the genomic DNA from the RNA of the stressed and

unstressed lager yeast, 1 µg RNA, 1 µl 10X reaction buffer with MgCl2, 1 µl DNase (1 U/µl) and DEPC-treated water to reach a final volume of 10 µl was added to a RNase-free tube. The samples were then incubated at 37 °C for 30 minutes before 1 µl of 50 mM EDTA was added, and the samples were incubated again at 65 °C for 10 minutes.

To convert RNA into cDNA, 11 µl of the template RNA, 1 µl random hexamer primer (100 pmol/µl), 4 µl 5X reaction buffer, 2 µl 10 mM dNTP Mix, 1 µl RiboLock RNase Inhibitor (40 U/µl) and 1 µl RevertAid M-MuLV Reverse Transcriptase (200 U/µl) were added to a 0.25 ml PCR reaction tube on ice and mixed gently. The samples were then inserted into the thermal cycler S1000 and were run with the program: 5 minutes at 25 °C, 60 minutes at 42 °C and then 5 minutes at 70 °C. 10 µl of cDNA was then diluted 4-fold by an addition of 30 µl water.

To measure the gene expression, a qPCR was performed. 8 µl of cDNA was mixed with 12.5 µl 2X PCR Master Mix, 1 µl forward primer (10 pmol/µl), 1 µl reverse primer (10 pmol/µl) and 2.5 µl sterile water in a 0.1 ml tube. This was done for all four primer pairs for the four genes (ILV2, RTG1, RPN4, UBC6) investigated. The samples were prepared in technical duplicates and a non-template-control, containing only PCR Master Mix and water, was also made. The tubes were kept on a chilled metal rack before loaded into a qPCR instrument (Rotor-gene 6000) and run with the program: 95 °C for 10 minutes and then 40 cycles of 95 °C for 15 seconds, 60 °C for 30 seconds and 72 °C for 30 seconds. Ct-values were obtained and the change in gene expression was calculated with the formula (Deltadelta Ct method or Livak method): 2(-ΔΔCt) (Livak &

Schmittgen 2001). Ct values is the number of cycles in the PCR that is required for the fluorescent signal to exceed the background noise, and is inversely proportional to the amount of DNA. The lower Ct value, the higher DNA content in the sample. Delta-Ct values is the difference in Ct values between the target gene and the reference gene.

The open-reading frames of the genes RPN4, RTG1 and ILV2 were obtained from SGD (Saccharomyces Genomic Database) and the primer pair for each gene was obtained from NCBI Primer Blast. The melting temperature was set to a minimum of 55°C, a maximum of 65 °C and an optimal of 60 °C. The product length was set to between 150 and 200 base pairs. Primers (Appendix Table 1) were ordered from Invitrogen ThermoFisher (ThermoFisher 2015).

3.7 Statistical analyses

To determine if there was any statistical difference in delta-Ct values between lager yeast that was stressed in 2 % isobutanol and lager yeast that was unstressed, a t-test was performed for each gene, using SPSS Statistics software version 24.0.0.0. The significance level was set to 0.05.

4 Results

4.1 Growth measurements by spot tests

All strains had grown well on the control agar plates, without isobutanol, after 48 hours (Figure 1A). The growth of each strain, without isobutanol, after 24 hours can be seen in Appendix Figure 1.

In 1.5 % isobutanol, the yeast strains from lager, wine and baker´s yeast had grown well after 48 hours, proven to be tolerant to 1.5 % isobutanol. The yeast strain from ale could be seen to not grow as well as the others (Figure 1B). The growth of each strain in 1.5 % isobutanol after 24 hours can be seen in Appendix Figure 2.

In 3 % isobutanol, yeast from the habitat of lager was seen to be most tolerant, after 48 hours. Yeast strains from the habitat of baker´s yeast and wine also grew on 3 %, but not as well as the lager yeast. Yeast from the habitat of ale was proven to be sensitive, when it didn´t grew at all on 3 % isobutanol (Figure 1C). After 24 hours, there was not yet any growth of the strains in 3 % isobutanol. (Appendix Figure 3).

All four strains of yeast seemed sensitive to 4 % isobutanol, when there was no growth of any of the strains on the agar plates containing 4 % isobutanol (Figure 1D and Appendix Figure 4).

Figure 1. Yeast strains from different habitats grown for 48 hours on A.

without isobutanol, B. 1.5 % isobutanol, C. 3 % isobutanol, D. 4 % isobutanol. 1. Wine. 2. Ale. 3. Lager. 4. Baker´s yeast. The upper row of dots on each plate is non-diluted yeast with an OD of 1. The second row of dots contains yeast with a dilution factor of 102. The third row of dots contains yeast with a dilution factor of 103, and the bottom row of dots contain yeast with a dilution factor of 104_.

4.2 OD measurements of growth

In 3 % isobutanol, all four strains of yeast had trouble growing, and they all seemed sensitive to 3 % isobutanol. The growth of all the strains started to decline after 4-6 hours of incubation (Figure 2A). In 26 hours, lager yeast had reached a percentage growth of 134.4 %, while ale yeast, wine yeast and baker´s yeast had reached a percentage growth of 115.7 %, 131.9 % and 129.9 % respectively (Figure 3A). All strains grew well

in the control, without isobutanol, reaching an OD above 2.5 (a growth of 2500 %) within 22 hours (Appendix Figure 5).

Yeast strains were then grown in 1.5 % isobutanol, and after 71 hours there was no big difference in growth between the strains that were

incubated without isobutanol and the strains that were incubated in 1.5 % isobutanol. This shows that all of the strains from the four different

habitats were tolerant to 1.5 % isobutanol (Figure 2B). The growth percentage for all the strains were above 13000 % (Figure 3B). Finally, the strain’s tolerance to 2 % isobutanol was measured. Importantly, the yeast strain isolated from lager beer was the most

tolerant to 2 % isobutanol (Figure 2C). After 32 hours, the lager yeast had reached a percentage growth of 1218.8 %. The wine yeast, ale yeast and baker´s yeast had reached a percentage growth of 363.6 %, 137.3 % and 194.1 % respectively (Figure 3C). All four yeast strains grew very well in the control, without isobutanol, reaching an OD above 2.5 (2500 %

growth) after 26 hours (Appendix Figure 6).

Figure 2. The growth, measured in OD600, in different concentrations of isobutanol, for the four strains isolated from different habitats. A. 3 % isobutanol. B. 1.5 % isobutanol. C. 2 % isobutanol. The results show sensitivity to 3 % isobutanol for all four strains, and tolerance to 1.5 %

isobutanol for all strains. Yeast strain isolated from lager beer showed highest tolerance to 2 % isobutanol.

Figure 3. Percentage growth for the different strains in isobutanol. A. 3 % isobutanol for 26 hours. B. 1.5 % isobutanol for 71 hours. C. 2 % isobutanol for 32 hours. Yeast strain from lager beer shows best growth in 2 %

isobutanol.

4.3 Measurements of gene expression

Importantly, RPN4 was the only gene that showed an upregulation in response to isobutanol (Table 1). Ct-values for the qPCR, with technical duplicates, can be seen in Appendix Table 2. For the gene RPN4, yeast that had been stressed in 2 % isobutanol had significant lower mean delta-Ct values compared to unstressed yeast (Figure 4A).

For the gene RTG1, yeast that had been stressed in 2 % isobutanol had significant higher mean delta-Ct values compared to unstressed yeast (Figure 4B).

For the gene ILV2, there was no significant difference in mean delta-Ct values between yeast that was stressed in 2 % isobutanol and unstressed yeast (Figure 4 C).

Table 1. The gene expression of the genes RPN4, ILV2 and RTG1 in response to 2 % isobutanol stress, compared to the reference gene UBC6. The gene expression was calculated with the Livak method.

Gene Normalized Gene expression

RPN4 3.36-fold upregulation

ILV2 1.12-fold upregulation

RTG1 0.60-fold downregulation

Figure 4. Difference in mean delta-Ct values, between lager yeast that had been stressed in 2 % isobutanol and unstressed lager yeast, for three different genes. A. RPN4. B. RTG1. C. ILV2. In RPN4, yeast that was

stressed in 2 % isobutanol had significant lower mean delta-Ct values (-1.78 +/- 0.14) compared to unstressed yeast (-0.03 +/- 0.13), t(2)= -19.33, p=0.003. In RTG1, yeast that was stressed in 2 % isobutanol had significant higher mean delta-Ct values (0.9 +/- 0.01), compared to unstressed yeast (0.17 +/- 0.18), t(2)= 5.86, p= 0.028. There was no significant difference, t(2)= -1.74, p= 0.225, between stressed (-2.95 +/- 0.0)and unstressed yeast (-2.79 +/- 0.13) for the gene ILV2. 95 % CI. Asterisk indicate significant values.

4.4 Sequencing for determining the identity of the strains

It was found that this method was not precise enough to distinguish the different strains from each other. When blasting was done on the

sequences of each strain, the results were the same for all the four

different strains; S. cerevisiae S288c, which indicate a close similarity in gene sequences (Appendix Table 3). By contacting the different

breweries, the factory names of the all of the strains could be determined. The yeast strain from the ale beer was Fermentis Safale S-04. The yeast strain from the lager beer was Fermentis Saflager W-34/70, the strain from the wine was Wyeast Italian Red 4244 and the strain from the baker´s yeast was commercial Kronjäst for bread doughs.

5 Discussion

When the four different yeast strains were grown on agar plates containing isobutanol, the lager yeast was the one that seemed most tolerant. It grew best out of the four strains both on 1.5 % and 3 % isobutanol. When diluted to a 103 dilution factor, the lager yeast was the only strain that managed to grow on 3 % isobutanol. Yeast from the habitats of wine and baker´s yeast also grew well on 1.5 % isobutanol and, in higher concentrations, managed to grew on 3 % isobutanol as well. Yeast from the habitat of ale did not grow as well as the other strains on the plates containing 1.5 % isobutanol, and did not grow at all on the plates containing 3 % isobutanol, proven to be the most sensitive strain to isobutanol. None of the four yeast strains managed to grow on 4 % isobutanol. The results agree with a previous study from 2014, where spot tests were done and yeast strains were grown on 1.5 % and 3 % 1-butanol, instead of isobutanol (Zaki et al. 2014). Several strains had the ability to grow on both 1.5 % and 3 % 1-butanol, so it could be suggested that the strains tolerance is similar to both 1-butanol and isobutanol. The same study also showed that yeast was inhibited on plates containing concentrations above 3 % 1-butanol (Zaki et al. 2014).

When growth of the different strains was measured in OD600 in liquid media, the results were similar. The strains in 1.5 % isobutanol grew well and at the same rate as the strains in the control, without isobutanol, proven that all four strains are tolerant to 1.5 % isobutanol. The result is expected, since previous studies have shown many strains that are

tolerant to 1.5 % butanol (Zaki et al. 2014).

In 2 % isobutanol, the lager yeast was the most tolerant strain, reaching an OD600 of 1.426 after 32 hours, while the strains from wine, ale and baker´s yeast only reached an OD of 0.360, 0.114 and 0.196 respectively.

In 3 % isobutanol, none of the four yeast strains grew well. A decline in growth after 4-6 hours of incubation shows that all four strains are sensitive to isobutanol concentrations of 3 %. This result differs some from the spot tests, where growth was measured on solid media at 3 % isobutanol. The conclusion is that it is harder for the yeast to grow in liquid media, compared to on solid media.

The fact that tolerance to isobutanol varies between the strains was not surprising. Studies have shown both strains that are tolerant to butanol concentrations of 1.5 % and 3 % (Zaki et al. 2014), and strains that are sensitive to butanol concentrations above 1.5 % (Gonzalez-Ramos et al. 2013). Out of the four strains tested, the yeast isolated from the habitat of lager beer seems to be the most tolerant against isobutanol. It had the highest percentage growth in both 2 % and 3 % liquid isobutanol and it grew best of the strains on the agar plates containing 3 % isobutanol. The most sensitive yeast strain seems to be from the habitat of ale. Due to non-existing previous studies, more studies need to be done to confirm the lager yeast´s tolerance to butanol. However, in a study from 2013 it has been shown that strains from lager beer had better performance during growth in 10 °C, and produced more alcohol yield than the ale strain. The lager strains (Frohberg and Saaz strains) reached an alcohol yield between 4.2 % and 6.5 %, while the ale strain (A60) only reached an alcohol yield of 2.2 % (Gibson et al. 2013). Ale strains also showed to be least tolerant to 12 % ethanol, when compared with other strains from different habitats, for example wine, sake and baker´s yeast (Mukherjee et al. 2014).

Because of two failing attempts to determine the strains of the unknown yeast by sequencing, it could be an interesting study for the future to find a method that can distinguish the different strains from each other. In the two attempts to sequence the yeast strains, all of the four strains seemed to belong to S. cerevisiae S288c. This is probably due to a close similarity in gene sequences between the strains. By determine to which strain the yeast from the habitat of lager beer belongs to, more research can be done on that strain, to further evaluate its potential to become a producer of biofuels. By contacting the different breweries, the factory names for the different strains could be determined. One study has shown that the lager yeast strain Fermentis Saflager W-34/70 was more sensitive to ethanol stress than the ale strain W210. However, it showed a higher tolerance to being stored at 6 °C in ethanol. W-34/70 tolerated storage in 7.5 %

ethanol, while W210 was sensitive to storage in 5 % ethanol (Bleoanca et al. 2013). No previous studies have been done on W-34/70 concerning

but more studies are needed to confirm its tolerance qualities. No previous studies on Fermentis Safale S-04 or Wyeast Italian Red 4244 regarding tolerance to alcohols have been found.

Other strains besides W-34/70, that have previously been found to be butanol tolerant, are DBVPG1788, DBVPG6044 and YPS128, that tolerated concentrations of 1-butanol up to 3 % (Zaki et al. 2014).

Even if tolerant strains have been discovered, the butanol yield still must be increased to make S. cerevisiae a suitable producer of biofuels. The yields of butanol produced by unmodified S. cerevisiae strains are still very low, 0.16 mg/g glucose (Chen et al. 2011). The metabolic pathway of S. cerevisiae has previously been engineered, and genes involved in the metabolic pathway and also in the protein degradation system have been overexpressed, in attempts to increase butanol yield (Kondo et al. 2011, Gonzalez-Ramos et al. 2013, Zaki et al. 2014).

The genomic expression of S. cerevisiae has been proven to change due to environmental shifts, and it has been shown that hundreds of genes change in transcript levels directly after an environmental shift (Gasch et al. 2000). Most of the genes that were upregulated in response to heat-shock, hyper-osmotic shock and exposure to hydrogen peroxide, were involved in processes like protein degradation, DNA damage repair, protein folding, detoxification of reactive oxygen species and cellular redox reactions (Gasch et al. 2000).Many researchers have previously tried to increase the butanol tolerance in yeast, by modifying genes involved in cell wall integrity, high osmolarity responses, amino acid starvation and ubiquitin-proteasome system (Gonzalez-Ramos et al. 2013). It is important to recognize genes that contribute to an increased butanol tolerance, so that a suitable host for butanol production can be found.

In this study, the gene expression of RPN4, RTG1 and ILV2 was measured, when they previously have been proven to contribute to a higher butanol tolerance or production (Kondo et al. 2011, Gonzalez-Ramos et al. 2013, Zaki et al. 2014).

It could be determined that RPN4 was upregulated in response to 2 % isobutanol, compared to the reference gene UBC6. It was also found that yeast that had been stressed in 2 % isobutanol had lower delta-Ct values compared to the unstressed yeast. The results support the hypothesis, and agrees with previous studies where similar results have been seen in 2013 and 2014, where overexpression of the gene RPN4 contributed to a

upregulated in response to 1-butanol (Gonzalez-Ramos et al. 2013, Zaki et al. 2014). In the study done by Zaki et al., it was shown that the gene RPN4 was upregulated in the tolerant strain YPS128, in exposure to both 1.5% and 3 % 1-butanol, compared to the sensitive strain UWOPS05-227.2 (Zaki et al. 2014). Knock-out of RPN4 made the tolerant strain sensitive to 1-butanol compared to the control (Zaki et al. 2014). The tolerance could be due to an amino acid substitution in the gene RPN4. Sensitive strains seem to have a substitution of leucine for a histidine at residue 444, and the importance of this residue for the tolerance is currently being investigated (Zaki et al. 2014).

The gene ILV2 showed barely no upregulation in response to isobutanol. The study also showed that there was no difference in delta-Ct values between stressed and unstressed yeast. An upregulation was predicted, and the upregulation was thought to be larger because of the result in a previous study where ILV2 was found to contribute to an increased isobutanol production when overexpressed (Kondo et al. 2011).

Overexpression of ILV2 increased isobutanol titer from 11 mg/l to 143 g/l and the yield to 6.6 mg/g glucose (Kondo et al. 2011). ILV2 has also in other studies shown to increase butanol yield. When Chen et al. overexpressed ILV2, ILV3 and ILV5, there was an increase in butanol yield by 6-fold, from 0.16 mg/g glucose to 0.97 mg/g (Chen et al. 2011). Overexpression of ILV2 together with BAT2 also increased isobutanol titer by 4.4-fold compared to the control strain (Zhang et al. 2016). It is possible that the yield could be further increased if the two other genes in the mitochondrial pathway, ILV3 and ILV5, was simultaneously

overexpressed (Zhang et al. 2016). More studies need to be done to find out if ILV2 is upregulated during butanol stress, and if the tolerance improves when ILV3 and ILV5 are overexpressed simultaneously with ILV2. It is also possible that ILV2 is only involved in the production of butanol, and not in the tolerance, which could explain the results in this study. It could also be interesting to compare the delta-Ct values in a future study, where more biological duplicates is used, instead of only two technical duplicates. For this study, the fact that there only were two samples each, for the stressed and the unstressed lager yeast, needs to be taken into consideration.

The gene RTG1 was found to be downregulated, which was not expected and did not support the hypothesis. It was also determined that yeast that had been stressed in 2 % isobutanol had significant higher delta-Ct

values, compared to unstressed yeast. In a previous study, overexpression of RTG1 resulted in a higher butanol tolerance (Gonzalez-Ramos et al.

sensitive (Gonzalez-Ramos et al. 2013). However, another study failed to observe an upregulation of RTG1 in response to 1-butanol (Zaki et al. 2014), which is more in line with this study. More studies, preferably with more biological duplicates, need to be done to determine if RTG1 has a role in butanol tolerance.

Other genes have also been identified for its contribution to butanol tolerance. In a study from 2013, they found that overexpression of the gene YLR224W, which is involved in recognition of damaged proteins, lead to an increased butanol tolerance (Gonzalez-Ramos et al. 2013). Overexpression of YLR224W in the strain IMI088 lead to twice as high growth rate compared to its reference strain, and lead to a 1-butanol tolerance of 1.75 % (Gonzalez-Ramos et al. 2013).

The genes INO1, HAL1 and DOG1 were also identified as contributors to a higher butanol tolerance. When overexpressed, they increased the

isobutanol tolerance (Hong et al. 2010). INO1 catalyses the conversion of glucose, HAL1 is involved in halo tolerance and DOG1 encodes a

phosphatase. These genes have previously been proven to increase tolerance to the stressors 2-deoxyglucose and salt (Gaxiola et al. 1992, Randez-Gil et al. 1995).

Other genes that are worth considering for engineering in S. cerevisiae is spo0A and groESL. In the bacteria C. acetobutylicum, the overexpression of the two genes lead to an increased butanol tolerance, but also an

increased production (Tomas et al. 2003, Alsaker et al. 2004). The gene groESL was previously recognized in bacteria for heat-shock response (Mogk et al. 1997).

Ghiaci et al. found that 32 genes were upregulated by at least 1.5-fold in response to butanol stress, in a mutant butanol tolerant strain, compared to a wild-type strain. The upregulated genes were primarily involved in mitochondrial activity and also glycerol biosynthesis. Overexpression of the gene Gpp2 increased the 2-butanol tolerance to 3 % (Ghiaci et al. 2013).

Many attempts have also been made to increase the butanol production in

S. cerevisiae, by engineering its metabolism. Compartmentalization of the

Ehrlich pathway into the mitochondria increased isobutanol production by 260 %, while overexpression of the same pathway in the cytoplasm only increased the yield by 10 % (Avalos et al. 2013). By moving valine synthesis to the cytosol instead, there was an increase in butanol titer from 20 mg/l to 151 mg/l (Lee et al. 2012). Brat et al. also replaced the mitochondrial pathway with a cytosolic pathway, and by overexpressing

Aro10, Adh2, ILV2, ILV3 and ILV5 in the cytosolic pathway and

deleting the first gene, ILV2, in the mitochondrial pathway they received a titer of 630 mg/l and a yield of 15 mg/g glucose (Brat et al. 2012). It is important to search for a strain that is tolerant, and to investigate genetic traits associated with butanol tolerance, to be able to develop a host that is suitable for biofuel production. Based on the results from this study, yeast strains from the habitat of lager beer should be further

examined for its use in butanol production in the biofuel industry. Plenty of genes, together with RPN4, RTG1 and ILV2, have been proposed to increase butanol tolerance and production, and they should be further examined and overexpressed in the yeast strain from lager beer to create a better butanol producer. Preferably, RPN4 should be manipulated,

because it has been proven to be upregulated in response to butanol stress (Gonzalez-Ramos et al. 2013, Zaki et al. 2014) and if overexpressed, the yeast strain from the habitat of lager beer could have the potential of being a great butanol producer.

5.1 Conclusions

To be able to increase butanol production in S. cerevisiae, strains that is tolerant to butanol, and genes that is involved in butanol tolerance, must be identified. In this study, a yeast strain isolated from the habitat of lager beer has been found to be most tolerant to isobutanol. Out of the four strains investigated, it had the highest percentage growth in isobutanol concentration of 2 % and 3 %. The most sensitive strain to butanol was isolated from ale. The gene RPN4 was found to be upregulated in response to 2 % isobutanol stress, which was expected when previous studies have shown similar results. There was no upregulation of the genes ILV2 and RTG1, which was unexpected. If ILV2 and RTG1 is involved in butanol tolerance need to be further investigated. In future studies, RPN4 should be overexpressed in the lager beer strain to possibly create a suitable host for biobutanol production.

5.2 Societal and ethical considerations

Finding a yeast strain that is tolerant to butanol, and genes that contribute to an increased butanol tolerance, is relevant because our society is in a process of reducing the use of fossil fuels as transportation fuels. It is very important to find a biofuel that is more environmentally friendly and can deliver the same amount of energy as the fossil fuels. In this study, a yeast strain from the habitat of lager beer was found to be most tolerant to isobutanol, making it the best potential producer of biofuels. There are no ethical aspects to consider in this study.

6 Acknowledgement

I want to thank my supervisor Johan Edqvist for all the assistance and suggestions with this project. I also want to thank my colleagues Rebecka Heinrup and Cecilia Hansson for the good collaboration during the study.

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8 Appendix

Table 1. Primers used for sequencing and measurements of gene expression.

Gene Forward primer Reverse primer

ITS 5´-TCCGTAGGTGAACCTGCGG-3´ 5´-TCCTCCGCTTATTGATATGC-3´ COX1 5′-CTACAGATACAGCATTTCCAAGA-3′ 5′-GTGCCTGAATAGATGATAATGGT-3´

RPN4 5´-GCTTCGATACCCCCACAACA-3´ 5´-GGGTTTCGCTAGCACCCTTA-3´

RTG1 5´-GGAACTGATGGTGAAGGCCA-3´ 5´-CCTCTTTGCTGGCGGTCTTA-3´

ILV2 5´-TGTCATGGTCAAGTCCGTGG-3´ 5´-ATCTTGTGCGCGACTGGTTA-3´

Figure 1. Yeast strains from different habitats grown for 24 hours without isobutanol. 1. Baker´s yeast. 2. Lager. 3. Ale. 4. Wine.

Figure 2. Yeast strains from different habitats grown for 24 hours on 1.5 % isobutanol. 1. Baker´s yeast. 2. Lager. 3. Ale. 4. Wine.

Figure 3. Yeast strains from different habitats grown for 24 hours on 3 % isobutanol. 1. Baker´s yeast. 2. Lager. 3. Ale. 4. Wine.

Figure 4. Yeast strains from different habitats grown for 24 hours on 4 % isobutanol. 1. Wine. 2. Ale. 3. Lager. 4. Baker´s yeast.

Figure 5. Control for 3 % isobutanol. The growth, measured in OD600, for the four control strains isolated from different habitats, when incubated without isobutanol.

Figure 6. Control for 2 % isobutanol. The growth, measured in OD600, for the four control strains isolated from different habitats, when incubated without isobutanol. 0 0,5 1 1,5 2 2,5 3 0 5 10 15 20 Gr ow th (O D600 ) Time (hours)

Wine control Ale control Bakery control Lager control

0 0,5 1 1,5 2 2,5 3 0 5 10 15 20 25 Gr ow th (O D600 ) Time (hours)

Table 2. Ct-values from qPCR. Contain technical duplicates.

Gene and treatment Ct-value

RPN4 2 % stress 19.99 RPN4 2 % stress 19.90 RPN4 control 21.27 RPN4 control 21.21 RTG1 2 % stress 22.67 RTG1 2 % stress 22.58 RTG1 control 21.43 RTG1 control 21.44 ILV2 2 % stress 18.83 ILV2 2 % stress 18.72 ILV2 control 18.51 ILV2 control 18.46 UBC6 2 % stress 21.78 UBC6 2 % stress 21.67 UBC6 control 21.39 UBC6 control 21.15

Table 3. Results from the sequencing. Shows the length of the sequences in base pairs, which primer that was used and the results with the identity and e-values. The e-value is the number of alignments expected by chance, and the identity is the highest percent identity for a set of align segments to the same subject sequence.

Sequence Length Primer Habitat Result Identity

E-value

ATTGCTGTATC

TGTAGACCCCCC 22 COX1 _Reverse Lager Saccharomyces _cerevisiae S288c

100 % 0.38

GAGGCTATCTTC

TTATCGATAACGTT 25 ITS4 _Reverse Bakery Saccharomyces _cerevisiae S288c 100 % 5e-04 CATTATCATCTATTC AGGCACACCATTAT CATCTATTCAGGCAC ACCATTATCATCTATT CAGGCACACCTTTAT CATCTATTCAGGCACA CCATTATCATCTATTC GGGCACACCATTATCA TTTAT 127 COX1 Forward Ale Saccharomyces cerevisiae S288c 100 % 1e-05 CCCATTATCATCTATTCA GGCACACCATTATCATCT ATTCAGGCACACCATTAT CATCTATTCAGGCACACC ATTATCATCTATTCAGGCA CACCATTATCATATATTCA GGCACACCAT TATCATCTATTCAGGCAC ACCATTA 144 COX1 Forward Wine Saccharomyces cerevisiae S288c 100 % 2e-05 AGTGCCTGATATATGATAA _{18 COX1} Reverse Ale Saccharomyces cerevisiae S288c 100 % 0.23 TGGGGGAGGACG _{11 ITS4} Reverse Lager Saccharomyces cerevisiae S288c 100 % 4.7