Construction of cellulosic ethanol production process via Bacillus-yeast co-culturing strategy

Kefir酵母菌Kluyveromyces marxianus KY3具有很廣泛的基質利用性、很好的高溫發酵潛力及優異的異源基因表現能力。我們建構了一個以酵母菌為宿主的合成生物學技術，可以同時轉入多個基因於宿主染色體中，我們稱它為Promoter-based Gene Assembly and Simultaneous Overexpression〔PGASO〕，此方法利用特定的推動子重疊區域作為多基因同源重組的依據，PGASO已成功應用於酵母菌K. marxianus KY3，當我們同時轉入五個基因片段時，呈現相當高的轉形效率約63%。另外，K. marxianus KY3-NpaBGS帶有牛胃真菌β-glucosidase (NpaBGS)基因，這兩個基因轉殖菌種將被用在後續乙醇生產系統中。 『可設計的纖維素分解酵素複合體』是一個製作人工纖維素分解酵素複合體的重要議題，可以不同的酵素比例來面對不同的木質纖維素降解情況，參考前人針對Clostridium thermocellum ATCC27405的蛋白質體學之研究，設計了面對結晶狀纖維素（Avicel）與纖維雙糖（cellobiose）的兩種操作組以仿生式模擬C. thermocellum分解纖維質時的原始表現狀態於枯草桿菌168中表現，八個C. thermocellum的纖維素分解酵素複合體相關基因：一個支架蛋白(cipA)、一個細胞膜上連接蛋白(sdbA)、兩個纖維外切酵素(celK and celS)、兩個內切酵素(celA and cel R)、兩個聚木糖分解酵素(xynC and xynZ)，利用方法Ordered Gene Assembly in B. subtilis method (OGAB)共同表現於枯草菌中。 為達成高效纖維乙醇生產製程，本研究建構了一個新的Bacillus /yeast共培養系統，兩個基轉酵母菌（KY3-NpaBGS and KR5）可以分泌纖維素分解酵素，用來與B. subtilis type I共培養，以提高纖維素糖化及發酵效率。所有Bacillus /yeast共培養系統皆可表現優於單獨CBP菌種KR5的同步纖維素糖化及乙醇轉化效率。由結果顯示，以β-glucan為碳源時，共培養組合KY3-NpaBGS＋Bacillus Type I可以分別呈現6倍、34%、2.5倍的生質乙醇產量之增加。由我們的結果顯示參與菌種之特性賦予Bacillus /yeast共培養系統之纖維乙醇生產能力，且具有很好的潛力應用於同步糖化發酵的製程。The kefir yeasts that produce ethanol and higher aromatic alcohols not only create kefir’s distinctive characteristics but could also serve as a good cell-based bioethanol production and biorefinery platform. This Kluyveromyces marxianus KY3 strain possesses a broad spectrum of substrate utilization, has a high potential for bioethanol production at elevated temperatures and suitable for expressing heterologous genes. We developed one synthetic biology techniques to transform genes into the host genome, named Promoter-based Gene Assembly and Simultaneous Overexpression 〔PGASO〕, which employed overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual unique promoters. As an example of application, PGASO was used to engineer yeast K. marxianus KY3. Our data showed high transformation efficiency and accuracy because ~63% of the transformants were found to carry the correct five-gene cassette assembly. And K. marxianus KY3-NpaBGS, which carries a β-glucosidase (NpaBGS) gene from rumen fungus. The two transgenic strains were employed to process ethanol production. “Designer cellulosomes” is a concept for making an artificial cellulosome that can be proposed as a tool for regulating the cellulosomal enzyme ratio to apply on different lignocellulose degradation. Two types of “designer operon” were expressed in Bacillus subtilis 168 via biomimetic approach according to a proteome-wide analysis of Clostridium thermocellum ATCC27405, which induced by avicel and cellobiose respectively. Eight celulosomal genes including one scaffolding protein gene (cipA), one cell-surface anchor gene (sdbA), two exoglucosidase genes (celK and celS), two endoglucanase genes (celA and cel R), and two xylanase genes (xynC and xynZ) of C. thermocellum were cloned and co-expressed on the polycistronic operons in desire order via an Ordered Gene Assembly in B. subtilis method (OGAB). A novel dual-microbe Bacillus/yeast co-culture system is developed for cellulosic bioethanol production. Two engineered yeasts, KY3-NpaBGS and KR5, which possess secretive cellulolytic enzymes, were used to co-cultivate with the B. subtilis type I strain for improving the cellulose digestion and fermentation efficiency. All Bacillus/yeast co-culture systems could achieve the cellulose saccharification and ethanol conversion simultaneously better than KR5 alone. In our result, the co-culturing of KY3-NpaBGS with Bacillus Type I appeared 6 times, 34% and 2.5 times ethanol production increasing than KY3+TypeI, KR5+ Type I and KY3-NpaBGS+host when utilized β-glucan as carbon source. Our results suggest that the dual-microbe Bacillus/yeast co-culturing system could leverage the advantages from both microbes and have a great potential for integrating into consolidated bioprocessing system.摘要 i Abstract ii Chapter I: Introduction 1 Chapter II: Material and Methods 13 2.1. Yeast strains, culture conditions and isolation 13 2.2. PCR–DGGE Analysis 13 2.3. Gene sequencing and phylogeny reconstruction 14 2.4. Inhibitor tolerance and substrate spectrum assay 15 2.5. Heterologous β-glucosidase gene transformation 15 2.6. Multiple-gene cassette construction 16 2.7. Yeast transformation and clone screening 17 2.8. The designer operon construction 18 2.9. Quantitative PCR analysis 19 2.10. Quantitative assays of enzyme activity 20 2.11. Cellulosome complex purification 21 2.12. Gel electrophoresis and Zymogram 22 2.13. Fluorescence microscope observation 22 2.14. Co-culture and cell counting assay 23 2.15. Carbon source utilization, ethanol production assay and accumulating reducing sugar assay 24 2.16. Co-culturing cellulosic ethanol production assay 25 Chapter III: Results and Discussions 27 3-1 Isolation and characterization of a thermo-tolerant kefir yeast Kluyveromyces marxianus KY3: a potential strain for developing co-cultural consolidated bioprocess 27 3-1.1. Analysis of the microbial community ofkefir grains 27 3-1.2. Yeast isolates and their thermo-tolerance 28 3-1.3. Identification of KY3 29 3-1.4. Aromatic compounds 30 3-1.5. Toxin tolerance assay 32 3-1.6. Ethanol productivity 33 3-1.7. Improvement of cellobiose consumption ability by genetic manipulation 35 3-1.8. Direct fermentation of beta-glucan to ethanol using K. marxianus KY3 co-cultured with Bacillus. 37 3-1.9. Monitoring of Co-culture 38 3-2 Synthetic Biological Technique for Yeast Genome Engineering: Promoter-based Gene Assembly and Simultaneous Overexpression Method 42 3-2.1. The technical concept 42 3-2.2. Insertion of fivegene cassettes 43 3-2.3. Expression of the five heterologous promoters 47 3-2.4. Performance of the secreted cellulases of KR5 48 3-2.5. Sugar utilization and ethanol production assay 50 3-3 Construct an artificial cellulosome in Bacillus subtilis via biomimetic expression of the cellulosomal genes of Clostridium thermocellum 52 3-3.1. Plasmid construction of “designer operon” 52 3-3.2. Expression assay of the “designer cellulosomes” 53 3-3.3. Quantitative enzyme assay of the “designer cellulosomes” 56 3-3.4. Assembly of the “designer cellulosomes” 57 3-3.5. Functional assay of the “designer cellulosomes” 59 3-4 Development of cellulosic ethanol production process via co-culturing of artificial cellulosomal Bacillus and kefir yeast 62 3-4.1. Microbe co-culture with cellobiose 62 3-4.2. The cellulolytic ability assay of cellulosomal B. subtilis 63 3-4.3. Dual-microbe co-culture for napier grass cellulosic ethanol conversion 64 3-4.4. The cellulolytic activities of engineered yeasts 65 3-4.5. Direct fermentation of β-glucan to ethanol using K. marxianus strains co-cultured with the designer cellulosome, Bacillus 66 Chapter IV: Conclusion 69 Reference 71 Tables and Figures 88 Table 1. The primer pairs used in the study. 88 Table 2. The yields of 2-phenylethylalcohol and 2-phenylethylacetate from yeast in YPAD medium. 89 Table 3. The primer pairs used in the PGASO construction. 90 Fig. 1 The microbes community assay. 93 Fig. 2 Metabolite profiling of K. marxianus KY3 fermentation by mass spectrometric techniques using an HP-5MX column. 94 Fig. 3 Toxin tolerance assay. 96 Fig. 4 Beta-glucosidase activity assays using 97 Fig. 5 Bio-ethanol production with beta-glucan as a carbon source via co-culture of KY3-196 and Bacillus, E. coli or L.kefiri. 98 Fig. 6 Total OD of samples containing KY3 only, Bacillus subtilis only or a co-culture in media 100 Fig. 7. Genomic integration of five gene cassettes into KR3. 102 Fig. 8. Gene insertion confirmation and copy-number quantification. 103 Fig. 9. The relative ratios of the five promoter transcripts are shown in comparison to the alg9 gene in KR5 at different temperatures. 104 Fig. 10. Cellulolytic enzyme assays of K. marxianus transformants. 106 Fig. 11. The growth patterns on plates with different carbon sources and simultaneous saccharification and fermentation (SSF) ability assays. 107 Fig. 12. The structure of the designer cellulosome and designer operon in B. subtilis. 109 Fig. 13. The expression at transcription level of the 8 genes on the type I “designer cellulosomes” was quantitatively determine by qRT-PCR assay. 110 Fig. 14. Expression assay of the “designer cellulosomes. 111 Fig. 15. The specific and quantitative activity enzyme assay of the designer cellulosome. Both the supernatant and intracellular materials which derived from the three clones (Type I, Type II, and Control) were tested. 112 Fig. 16. A 5-15 % gradient native PAGE gel electrophoresis was preformed for cellulosomes complex zymogram assay. 113 Fig. 17. Comparsion the cellulosomal cellulolytic activity of the two clones with native C. thermocellum, both the supernatant materials and intact cells of the culturing were harvested. 114 Fig. 18. Comparsion the cellulosomal cellulolytic ability of the three B. subtilis clones, Type I, Type II, and Control. 115 Fig. 19. The fluorescence microscope observation of the dual-microbe co-culture system via the specific dye BacLight for Bacillus staining (green) and Calcofluor fluorescence for yeast staining (blue). 116 Fig. 20. The cellulolytic enzyme activity assay. 117 Fig. 21. Dual-microbe co-culturing test of cellulosic ethanol conversionusing napier grass at different temperatures following a 7-day incubation. 118 Fig. 22. Yeast cellulolytic activity assay. 119 Fig. 23. Ethanol production using β-glucan as the sole carbon source by coculturing of KY3, KY3-NpaBGS or KR5 with a designer cellulosome Bacillus at 42°C. Eachmean (±SD) was obtained from three independent experiments. 120 Fig. S1 Growth temperature assay of kefir yeast strains. 121 Fig. S2 Ethanol productivity and glucose consumption of KL, SC and KY3. 122 Fig. S3 Carbon source utilization assay of KY3, KL and SC. 12

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本發明提供一種利用微生物產氫之方法,其包含下列步驟:取Clostridium屬及Bacillus屬之微生物;及將前述Clostridium屬及Bacillus屬之微生物共同培養於發酵培養系統,藉此產生氫氣。本發明並提供一種生物產氫系統,其特徵在於:該系統包含Clostridium屬及Bacillus屬之微生物,且該系統係使用有機廢料培養基作為基質,來進行具有高效率、高穩定性及高再現性之產氫發酵