Engineering of xylose reductase xylose reductase and and - PowerPoint PPT Presentation

Engineering of xylose reductase xylose reductase and and Engineering of overexpression of xylitol dehydrogenase xylitol dehydrogenase and and overexpression of xylulokinase improves improves xylose alcoholic xylose alcoholic xylulokinase fermentation in the thermotolerant yeast in the thermotolerant yeast fermentation Hansenula polymorpha Hansenula polymorpha Olena V Dmytruk, Kostyantyn V Dmytruk, Charles A Abbas, Andriy Y Voronovsky and Andriy A Sibirny Microbial Cell Factories 7 (21), 2008. (on-line) Speaker: Jui-Yun Weng Advisor: Ching-Tsan Huang PhD 2008/10/14

Bio-ethanol starch starch polysaccharides polysaccharides cellulose xylose lignocelluloses hemicellulose lignocelluloses glucose mannose lignin galactose arabinose 2

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4 Main arabinose galactose mannose glucose xylose Hemicellulase (Xylanase) glucose Cellulase

Cellulose Hemicellulose lignin Simultaneous saccharification Simultaneous saccharification Cellulose hydrolysis Hemiellulose hydrolysis Cellulose hydrolysis Hemiellulose hydrolysis and fermention and fermention Alcoholic fermentation Alcoholic fermentation Bio-ethanol Bio-ethanol 5

Xylose metabolism 1. Co-factor imbalance xylose xylose 2. Xylitol accumulation NADPH NADPH XR XR � XYL1 gene XR = Xylose reductase XR + NADP + NADP xylitol xylitol NAD + + NAD XDH = Xylitol dehydrogenase � XYL2 gene XDH XDH XDH NADH NADH xylulose xylulose � XYL3 gene XK = Xylulose kinase XK XK XK Xylulose- -5 5- -phosphate phosphate Xylulose 6 Pentose Phosphate Pathway

The ways they used 1. Engineering of xylose reductase (XR) 2. Over-expression of xylitol dehydrogenase (XDH) 3. Over-expression of xylulose kinase (XK) Engineering of xylose reductase xylose reductase and overexpression and overexpression Engineering of of xylitol dehydrogenase xylitol dehydrogenase and and xylulokinase xylulokinase improves improves of xylose alcoholic fermentation in the thermotolerant in the thermotolerant xylose alcoholic fermentation yeast Hansenula polymorpha Hansenula polymorpha yeast 7

8 1. Protein engineering of XR cofactor binding site sequences

Hansenula polymorpha • Simultaneous Saccharification and Fermentation (SSF) • Thermotolerant • Protein factory • Comparing to S. cerevisiae • CBS4732 (wild type) CBS4732 (wild type) • • Δ Δ xyl1 xyl1— — deficient in XR (Host) deficient in XR (Host) • 9

1. Protein engineering of XR 1. Δ xyl1 XRm m XR Δ xyl1 XRn XRn Activity and K M of XR NADPH NADH K M ( μ M) K M ( μ M) Strain Activity Activity [U (mg protein ‐ 1 )] [U (mg protein ‐ 1 )] 0.091 ± 0.005 9 ± 0.5 0.016 ± 0.001 100 ± 5.5 XR n 0.01 ± 0.001 152 ± 8.4 0.014 ± 0.001 112 ± 6.5 XR m 152 0.034 ± 0.025 7 ± 0.4 0.012 ± 0.001 85 ± 4.7 CBS4732 10

2. Over-expression of XDH 2. cofactor binding site sequences Over- -expression expression Over 11

2. Over-expression of XDH 2. XRm m/XDH /XDH XR Δ xyl1 XRn/XDH /XDH XRn Δ xyl1 Activity [U (mg protein -1 )] Strain XDH 0.551 ± 0.035 XR n 0.504 ± 0.028 XR m 1.3 ± 0.07 XR n /XDH 1.3 XR m /XDH 1.485 ± 0.087 1.485 0.697 ± 0.049 CBS4732 12

3. Over-expression of XK 3. Over-expression Over- -expression expression Over 13

3. Over-expression of XK 3. XRn/XDH XRm m/XDH /XDH XRn/XDH XR XRn/XDH/XK XRm m/XDH/XK /XDH/XK XRn/XDH/XK XR Activity [U (mg protein -1 )] Strain XK 0.375 ± 0.021 XR n /XDH/XK 0.375 0.366 ± 0.019 XR m /XDH/XK 0.366 0.156 ± 0.009 CBS4732 14

Productivity of ethanol and xylitol Strain Ethanol Xylitol mg (L h) -1 mg (L g h) -1 mg (L h) -1 mg (L g h) -1 6.3 ± 0.3 2.75 ± 0.11 3.6 ± 0.2 1.56 ± 0.1 XRn 9.8 ± 0.4 4.45 ± 0.19 3.2 ± 0.2 1.45 ± 0.07 XRm 12 ± 0.5 5.7 ± 0.3 3.1 ± 0.2 1.48 ± 0.07 XRn/XDH 18.4 ± 0.9 8.76 ± 0.35 1.6 ± 0.1 0.76 ± 0.04 XRm/XDH 13.8 ± 0.6 6.4 ± 0.3 2.8 ± 0.1 1.3 ± 0.06 XRn/XDH/XK 54.7 ± 2.7 21.9 ± 1.1 0.9 ± 0.04 0.36 ± 0.02 XRm/XDH/XK XRm/XDH/XK 54.7 21.9 0.9 0.36 7.5 ± 0.4 3.3 ± 0.2 4.2 ± 0.2 1.8 ± 0.09 CBS4732 15

Xylose fermentation Biomass Biomass Xylose Xylose Ethanol Ethanol Xylitol Xylitol CBS4732 XRn n/XDH/XK Biomass Xylose Ethanol 16 Xylitol XRm m/XDH/XK

1. Protein engineering of XR 1. Protein engineering of XR ↑ K M of NADPH ↑ K M of NADH unchanged 2. over- -expression of XDH expression of XDH 2. over ↑ Activity of XDH ↑ 3. over- -expression of XK expression of XK 3. over ↑ Activity of XK ↑ Xylose fermentation ethanol ↑ ↑ Productivity of ethanol 17 ↓ Productivity of xylitol ↓

18 Thank you for your attention