Molecular cloning and expression of α-acetolactate synthase and α- acetolactate decarboxylase for 2,3-butanediol production in Pdc-deficient Saccharomyces cerevisiae

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부(식품생명공학전공), 2015. 8. 서진호.2,3-Butanediol (2,3-BD) is a chemical compound with extensive industrial applications. Especially, 2,3-BD could be converted to valuable chemicals by dehydrogenation, esterification, ketalization and dehydration. It has been used as drugs, solvents, flavoring agents, cosmetic and food products. Although biotechnological production of 2,3-BD mainly uses bacteria, most bacteria used for 2,3-BD production are classified as potentially pathogenic microbes, which makes difficult industrial-scale production of 2,3-BD in terms of safety regulations. As an alternative, 2,3-BD production by a GRAS (Generally Regarded As Safe) microorganism Saccharomyces cerevisiae would be suitable. As S. cerevisiae naturally produces ethanol, a pyruvate decarboxylase (Pdc)-deficient S. cerevisiae (SOS4) was constructed to eliminate ethanol production. The 2,3-BD biosynthetic pathway was intensified for redirecting pyruvate toward 2,3-BD production. Since the (Pdc)-deficient S. cerevisiae has a low 2,3-BD yield and productivity, it is necessary to improve both 2,3-BD yield and productivity by optimizing the biosynthetic pathway from pyruvate to 2,3-BD. First, bacteria which produce a high 2,3-BD yield were investigated. Klebsiella pneumoniae, K. oxytoca, and Enterobacter aegrogenes which can produce 2,3-BD at high yield and productivity were selected. The genes coding for acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC) from K. pneumoniae, K. oxytoca, and E. aegrogenes were cloned and introduced to the SOS4 strain which is also able to overexpress the BDH1 gene. The yeast strain containing both the ALS gene and ALDC gene from B. subtilis and the innate BDH1 gene (BD_BS) was used as the control strain. A batch fermentations with 100 g/L glucose was carried out under oxygen-limited conditions. The 2,3-BD yield and productivity of the control strain were still higher than other strains. Second, the SOS4 strains containing the ALS gene from B. subtilis and the ALDC gene from K. pnuemoniae, K. oxytoca, and E. aerogenes and overexpression of the innate BDH1 gene were constructed and tested for performance of 2,3-BD production in batch fermentations with 100 g/L glucose under oxygen-limited conditions. The 2,3-BD yield and productivity of the BD_BS_EA strain containing ALS from B. subtilis and ALDC from E. aerogenes and overexpressing the innate BDH1 gene were slightly higher than the control strain. To explore the potential ability of 2,3-butanediol production between the control and the BD_BS_EA strains as a 2,3-butanediol producer, fed-batch fermentations was carried out through intermittent addition of glucose under the optimum aeration condition. The final concentration of 2,3-butanediol of the BD_BS_EA strain was 132.4 g/L with a 2,3-butanediol yield (0.34 g 2,3-butanediol/g glucose) and volumetric productivity (0.41 g 2,3-butanediol/L?h), corresponding to 28% increase in yield and 24% increase in productivity. Finally, in order to analyze why the BD_BS_EA strain is better than the control strain, the specific activity of ALDC from E. aerogenes was compared with that ALDC from B. subtilis. The ALDC was purified by affinity chromatography using the histidine tag. Specific activity of ALDC from E. aerogenes was found to be 250.4 mU/mg protein, which is higher by 2.5-folds than ALDC from B. subtilis. Although ALDC from E. aerogenes possessed a low substrate affinity (Km), Vmax and kcat/Km of ALDC were higher by 5-folds, 1.5-folds than ALDC from B. subtilis respectively. These results suggested that the BD_BS_EA strain is suitable for producing 2,3-BD for industrial applications.ABSTRACT i CONTENTS vi LIST OF TABLES x LIST OF FIGURES xi I. INTRODUCTION 1 1. 2,3-Butanediol 1 2. Metabolic pathway 3 3. 2,3-Butanediol production in bacteria 5 4. 2,3-Butanediol production in S. cerevisiae 8 5. 2,3-Butanediol production in pyruvate decarboxylase-deficient S. cerevisiae 11 6. α-Acetolactate decarboxylase 15 7. Research objectives 16 II. MATERIALS AND METHODS 17 1. Reagents 17 2. Strains and plasmids 18 2.1. Strains 18 2.2. Plasmids 20 3. DNA manipulation and transformation 25 3.1. Enzymes 25 3.2. Polymerase chain reaction (PCR) 25 3.3. Preparation of plasmid DNA and bacteria genomic DNA 25 3.4. Transformation of E. coli 26 3.5. Yeast transformation 27 4. Media and culture conditions 27 4.1. Media 27 4.2. Batch fermentation 28 4.3. Fed-batch fermentation 28 5. Analysis 30 5.1 Dry cell weight 30 5.2 Metabolite detection 30 6. Protein purification 31 6.1 Sample preparation 31 6.2 Affinity chromatography 31 7. α-Acetolactate decarboxylase activity assay 32 7.1 Enzyme activity assay 32 7.2 Bradford assay 34 III. RESULTS AND DISCUSSIONS 35 1. Construction of the efficient 2,3-butanediol biosynthetic system 35 1.1. Investigation of 2,3-butanediol biosynthetic genes from various microorganisms 35 1.1. Enhancement of 2,3-butanediol production by combination of ALS and ALDCs 39 2. Promoter replacement to increase 2,3-butanediol biosynthetic genes expression level. 43 3. 2,3-Butanediol production by the BD_BS_EA strain in fed-batch fermentation. 47 4. Specific activity assay and Kinetic analysis of α-acetolactate decarboxylase 51 IV. CONCLUSIONS 56 V. REFERENCES 57 국 문 초 록 64Maste