Development of xyloseutilizing and inhibitortolerant yeast strains for bioethanol production

Vrije Universiteit Brussel Katholieke Universiteit Leuven Universiteit Antwerpen Interuniversity Program Molecular Biology (IPMB) Development of xylose-utilizing and inhibitor-tolerant yeast strains for bioethanol production Thesis submitted in partial fulfillment of the requirements for the Degree of Master of Science in Molecular Biology Tung Thanh Dinh Promoter: Prof Johan Thevelein Co-promoter: Dr Franỗoise Dumortier Supervisor: Mekonnen M Demeke Laboratory of Molecular Cell Biology Department of Molecular Microbiology Faculty of Science K.U.Leuven Academic year 2010-2011 CONFIDENTIAL DOCUMENT This thesis is a piece of examination that has not been corrected after the defense The results of this thesis might be used for a patent application Therefore, all the data of this document should be considered as confidential They may by no means made public; and there should not be any reference to them To know the date of public release, the promoter of this thesis can be contacted TABLE OF CONTENTS TABLE OF CONTENTS i LIST OF FIGURES iv LIST OF TABLES vi ACKNOWLEDGEMENT vii ABSTRACT viii INTRODUCTION LITERATURE REVIEW 2.1 BIOETHANOL PRODUCTION – AN OVERVIEW 2.1.1 First generation bioethanol 2.1.2 Second generation bioethanol 2.2 INDUSTRIAL REQUIREMENTS, CURRENT STATUS AND CHALLENGES 2.2.1 Industrial requirements 2.2.2 Current status 2.2.3 Challenges 2.3 INHIBITORS IN HYDROLYSIS PRODUCTS OF LIGNOCELLULOSES 10 2.3.1 Origin 10 2.3.2 Effects and Mechanisms 11 2.3.2 Solutions for inhibitor 12 2.4 XYLOSE FERMENTATION 15 2.4.1 Xylose fermentation in yeast 15 2.4.2 Recombinant xylose-fermenting strain development 16 OBJECTIVES 20 MATERIAL AND METHODS 21 i 4.1 CULTURING MEDIA 21 4.1.1 Inoculation media 21 4.1.1 Screening media 21 4.2 YEAST STRAINS 22 4.3 YEAST MANIPULATION 23 4.3.1 Sporulation 23 4.3.2 Tetrad analysis 23 4.3.3 Mating type determination 23 4.3.4 Spot test 23 4.3.5 OD 600 measurement 24 4.3.6 Dry weight measurement 24 4.3.7 Fermentation 24 4.4 DNA MANIPULATION 25 4.4.1 DNA isolation 25 4.4.2 Polymerase chain reaction (PCR) 25 4.4.2 Agarose gel electrophoresis 25 4.5 SUGAR AND METABOLITE ANALYSIS 26 4.5.1 Rate of sugar consumption 26 4.5.2 Ethanol, Sugar and metabolite analysis 26 4.6 GENOME SHUFFLING 26 RESULTS AND DISCUSSION 28 5.1 EXPERIMENTS ON ETHANOL RED 28 5.1.1 Segregant preparation 28 5.1.2 High-throughput inoculating segregants with non-specified amounts of cells 28 5.1.3 High-throughput inoculating segregants with specified amounts of cells 29 5.1.4 Fermentation of Ethanol Red segregants 32 ii 5.2 FERMENTATION OF SACCHAROMYCES CEREVISIAE STRAINS 33 5.2.1 Effect of furan derivative on fermentation of ER, BY4742, and TMB3400 strains 33 5.2.2 Fermentation of ISO12 ISOB57 ER TMB3400 strains in 50%, 60%, and 70% Hydrolysate and containing YP and 13% glucose 35 5.3 GENETIC MAPPING BASED ON AMTEM 38 5.3.1 Genetic mapping strategy 38 5.3.2 AMTEM for inhibitor tolerance phenotype 38 5.4 GENOME SHUFFLING 40 CONCLUSION AND RECOMMENDATION 42 BIBLIOGRAPHY 44 iii LIST OF FIGURES Figure 2.1: General principle of bioethanol production Figure 2.2: Structures of cellulose and hexose components of hemicelluloses Figure 2.3: The required stages in case cellulose is the main substrate Figure 2.4: Inhibitors in the lignocellulosic biomass Figure 2.5: Schematic view of inhibition mechanisms of inhibitors Figure 2.6: Three main fermentation modes used in fermentation technology Figure 2.7: Xylose (a,b) and arabinose (c,d) utilization pathways in bacteria (a,c) and in fungi (b,d) Figure 2.8: Metabolic pathways used by yeasts: the nonoxidative pentose phosphate pathways Figure 5.1: OD 600 values of ER segregants on 65% liquid hydrolysate in days Figure 5.2: ER segregants from 1A to 44D on 60% solid hydrolysate in YPD pH=6 after days Figure 5.3: ER segregants from 1A to 44D on 70% solid hydrolysate in YPD pH=6 after days Figure 5.4: ER segregants from 1A to 44D on 80% solid hydrolysate in YPD pH=6 after days Figure 5.5: Spot test of ER segregants on 50% solid hydrolysate after days Figure 5.6: Spot test of ER segregants on 60% solid hydrolysate after days Figure 5.7: OD 600 values of ER and its 12 selected segregants and in 60% and 70% liquid hydrolysate media supplemented with YP and 2% glucose after 48hrs Figure 5.8: Sugar consumption of ISO12, ER and its segregants in 60% spruce hydrolysate + YP + 13% glucose Figure 5.9: Effect of furan derivatives on fermentation of BY4742 iv Figure 5.10: Effect of furan derivatives on fermentation of ER Figure 5.11: Effect of furan derivatives on fermentation of TMB3400 Figure 5.12: Sugar consumption of ISO12, ISOB57, ER, and TMB3400 in 50% hydrolysate supplemented with YP and 13% glucose Figure 5.13: Sugar consumption of ISO12, ISOB57, ER, and TMB3400 in 60% hydrolysate supplemented with YP and 13% glucose Figure 5.14: Sugar consumption of ISO12, ISOB57, ER, and TMB3400 in 70% hydrolysate supplemented with YP and 13% glucose Figure 5.15: Ethanol yields measured from the residual fermentation medium of ISO12, ISOB57, ER, and TMB3400 in 50%, 60%, and 70% hydrolysate supplemented with YP and 13% glucose Figure 5.16: AMTEM strategy employed to identify the genes or mutation responsible for inhibitor-tolerant and xylose-fermenting phenotype Figure 5.17: OD 600 values of ER and ISOB57 after 72hrs growth in hydrolysate supplemented with YP and 2% glucose Figure 5.18: Spot test result of F1 hybrid [ISOB57 x AMS928α] segregants on 40% hydrolysate supplemented with YP and 2% glucose solid medium after days Figure 5.19: Spot test result of F1 hybrid [ISOB57 x AMS928α] segregants on 50% hydrolysate supplemented with YP and 2% glucose solid medium after days Figure 5.20: OD 600 values of initial mating products along with MDX1051, MDX1052, MDX1053, MDX1054, ER, and TMB3400 in YP + 2% xylose after 24hrs and 48hrs (with same initial OD 600 of 1) Figure 5.21: OD 600 values of mating mix in 50% and 60% hydrolysate supplemented with YP and 2% xylose after 24hrs and 48hrs Figure 5.22: Sugar consumption of the final mating mix in YP supplemented with 2% xylose v LIST OF TABLES Table 2.1: Annual world ethanol production by country Table 2.2: The most commonly used strategies applied to S cerevisiae for pentose fermentation Table 4.1: Yeast strains used in experiments Table 4.2: Composition of buffers and medium used in genome shuffling experiment Table 5.1: Composition of the spruce hydrolysate supplied by SEKAB Company vi ACKNOWLEDGEMENT First, I would like to thank Belgian government namely BTC for financing my Master study I have really enjoyed the time living in this beautiful country I also want to show my most sincere gratitude to my promoters Professor Johan Thevelein and Dr Francoise Dumortier for giving me the opportunity to my thesis at Molecular Cell Biology Laboratory (MCB), KULeuven I really appreciate my supervisor Mekonnen M Demeke for his devoted support during my time at the lab I want to devote the thesis to my parents who brought me up with love and care My family including my younger brother has been always behind me when I was in trouble and shared happiness with me in the time of success Without my family, I would not be what I am today I would like to thank all IPMB teachers because of precious knowledge I have learnt from them, as well as administrative staffs for their support during my study period My deepest gratitude to IPMB coordinator Professor Eddy Van Driessche and Mr Rudi Willems for their enthusiastic guidance and assistance I really enjoyed the time with my IPMB classmates and the memory of our class will always be in my heart vii ABSTRACT Second generation bioethanol production has been considered as a solution for energy crisis in the future due to fossil fuel depletion Despite being promising, this technology confronts several challenges to reach its full potential One of them is the presence of inhibitors generated during the hydrolysis of lignocellulosic biomass Another which is not less important is the incapacity of the most commonly used microorganism in the industry, Saccharomyces cerevisiae, to ferment pentose sugar such as arabinose and xylose The first objective of this study was to isolate a segregant of an inhibitor tolerant industrial yeast strain, Ethanol Red, and subsequently use this segregant for mapping genomic regions responsible for inhibitor tolerance By tetrad analysis, 188 segregants were isolated and applied to microbial techniques in order to find segregants which are at least as tolerant as ER They were then spotted on solid spruce hydrolysate media (60%, 70%, and 80%) without pre-determined amount of cells Segregants which grew nearly as good as ER were subsequently subjected to Spot test for further screening and 12 segregants with best growth were kept Afterwards, these 12 segregants were inoculated in liquid hydrolysate media and only segregants which were able to grow on highly inhibiting 70% hydrolysate were chosen for fermentation evaluation As a result, we manage to get segregant (8D) that ferments better than ER in 60% spruce hydrolysate containing YPD With the aim of combining the inhibitor-tolerance and xylose-fermenting phenotypes capacity from strains possessing one of these two phenotypes, genome shuffling of Ethanol Red (inhibitor tolerant strain) along with four xylose utilizing yeast strains was performed After the spores of these strains were isolated and crossed all together, the resulting diploids were able to grow in both xylose and inhibitor containing medium The mating products were proven to grow significantly faster in YPXylose than their xylose-fermenting parents but fermented xylose with very slow rate This indicates that good growth on xylose does not mean good fermentation to ethanol and further genome shuffling cycle and evolutionary adaptation in anaerobic condition should be done to obtain strains of better xylose fermenting capacity viii 5.4 GENOME SHUFFLING The yeast strains namely MDX1051, MDX1052, MDX1053, and MDX1054 with the ability of growing on YPxylose medium (figure 5.20) along with Ethanol Red were subjected to Genome shuffling technique (see 4.8) These strains were sporulated separately in Erlenmeyer flasks and their sporulation efficiencies were checked daily in order to reach high proportion of spores (more than 80%) However, the sporulation efficiency was only high for MDX1053 (89%), moderate for ER (26%), MDX1051 (32%), MDX1052 (19%), and very low for MDX1054 (5%) even after days Afterwards, the spores of these strains were allowed to mate altogether for 48hrs Out of 75 colonies, 60 (80%) colonies were diploid and the rest were haploid To evaluate the ability of growing on xylose, a pre-culture was prepared from the mating product as well as the yeast parent strains in YPD which were then cultivated in YPXylose with initial OD 600 of TMB3400 and Ethanol Red were included as positive and negative control, respectively The OD 600 values of these strains were measured after 24hrs and 48 hrs and the result was showed in figure 5.20 The mating product grew better than their parents and even TMB3400 in YPXylose (figure 5.20) Figure 5.20: OD 600 values of initial Mating products along with MDX1051, MDX1052, MDX1053, MDX1054, ER, and TMB3400 in YP + 2% xylose after 24hrs and 48hrs (with same initial OD 600 of 1) 40 Figure 5.21: OD 600 values of mating mix in 50% and 60% hydrolysate supplemented with YP and 2% xylose after 24hrs and 48hrs (with same initial OD 600 of 1) Afterwards, the mating products were first selected for xylose-utilizing phenotype by inoculating cells in YPxylose medium, starting from OD 600 of After 24hrs, the OD 600 increased to 19.21 which proved the presence of xylose-utilizing cells These cells were then transferred to YPXylose containing 50% and 60% hydrolysate media (with the same initial OD 600 of 1) to be screened for inhibitor-tolerant phenotypes Only cell culture in 50% Hydrolysate and YPX showed growth and therefore was kept (figure 5.21) The mixture of cells was again cultivated in YPXylose (second times) with the initial OD 600 of After days, the OD 600 went up to 22.175 Part of the cells was kept in stock and another part was used in a fermentation experiment 41 Figure 5.22: Sugar consumption of the final mating mix in YP supplemented with 2% xylose The fermentation was performed in 80ml YPX at 30ºC with yeast cells of OD 600 5, velocity of magnetic stirrer was 120rpm The fermentation was carried out in 80ml YPXylose at 30ºC with yeast cells density of OD 600 It can be clearly seen that the anaerobic xylose consumption rate was extremely slow, only about 25% of xylose was used after 160hrs This indicated that in the obtaining yeast cells the xylose-to-ethanol pathway did not play an essential role among the whole biochemical processes CONCLUSION AND RECOMMENDATION One of the objectives of this study was isolation of Ethanol Red (ER) segregants (by sporulation, tetrad dissection, mating type check) and by microbial techniques like spot test, and fermentation evaluation, to isolate ER segregants which are at least as tolerant as the diploid ER Through tetrad analysis, 188 segregants of Ethanol Red (ER) were isolated and mating type determined First, these segregants were inoculated in solid hydrolysate media 42 and the results were slightly variable This could be the result of the inconsistence of inhibitor content in different hydrolysate batches (SEKAB) Moreover, the spruce hydrolysate was so thick that it was difficult to prepare ideally homogenous solid hydrolysate media The 41 segregants with good growth on solid hydrolysate media (70% and 80%) were subjected to spot tests also on solid media but with less hydrolysate (60% and 70%) The 12 segregants with tolerance almost as good as ER were then inoculated in highly inhibiting liquid media (60% and 70%) The segregants which were able to grow on 70% hydrolysate were then subjected to fermentation of mixture containing 60% hydrolysate Segregant 8D fermented faster than its parent ER although its performance in spot test and in liquid media screening was not better than ER itself This segregant could be used further for mapping of genes responsible for inhibitor tolerance in ER We also attempted to apply AMTEM technology on the two strains Ethanol Red background strains ISOB57 and ISO12 (Lund University) ISOB57 was crossed with the Artificially Marked Strain AMS928α and the resulting diploid was sporulated to get F1 hybrid segregants However, the viability of F1 segregants was significantly low Among 144 tetrad (576 segregants), only 100 segregants were obtained (about 17%) The viable segregants were then subjected to spot test of 40% and 50% hydrolysate solid media and demonstrated significant intolerance toward hydrolysate compared to ISOB57 The problem of low viability was pointed to ISOB57, which was subsequently proven by the fact that when crossing ISOB57 with either the wild type strain BY4742 or segregant 6D derived from ER, the number of hybrid segregants obtained was also very low In case of ISO12, its spores were not viable at all and therefore it was not possible to the mapping experiment In general, low viability and weak inhibitor-tolerance were the bottlenecks of applying AMTEM for inhibitor tolerance phenotype on ISOB57 and ISO12 Another aim was to develop inhibitor-tolerant and xylose-fermenting yeast strain by genome shuffling This was done between ER (inhibitor tolerant strain) and MDX1051, MDX1052, MDX1053, MDX1054 (naturally xylose-fermenting strains) The resulting diploids showed significant improvement in growth in both xylose and inhibitor containing media However, the rate of fermentation was not improved as such This indicates that good growth on xylose does not mean good fermentation to ethanol and further genome shuffling cycles and evolutionary adaptation in anaerobic condition should be done to obtain strains of better xylose 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