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

Engineering of Engineering of xylose reductase xylose reductase and and

• verexpression of
• verexpression of xylitol dehydrogenase

xylitol dehydrogenase and and xylulokinase xylulokinase improves improves xylose alcoholic xylose alcoholic fermentation fermentation in the thermotolerant yeast in the thermotolerant yeast Hansenula polymorpha Hansenula polymorpha

Speaker: Jui-Yun Weng Advisor: Ching-Tsan Huang PhD 2008/10/14

Olena V Dmytruk, Kostyantyn V Dmytruk, Charles A Abbas, Andriy Y Voronovsky and Andriy A Sibirny Microbial Cell Factories 7 (21), 2008. (on-line)

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Bio-ethanol

polysaccharides polysaccharides lignocelluloses lignocelluloses starch starch cellulose hemicellulose lignin xylose glucose mannose galactose arabinose

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xylose glucose mannose galactose arabinose

Main glucose Hemicellulase (Xylanase) Cellulase

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

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Xylose metabolism

xylose xylose xylitol xylitol

XR XR = Xylose reductase NADPH NADPH

xylulose xylulose

XDH XDH = Xylitol dehydrogenase NAD NAD+

+

• 1. Co-factor imbalance
• 2. Xylitol accumulation

XK XK = Xylulose kinase

Xylulose Xylulose-

• 5

5-

• phosphate

phosphate Pentose Phosphate Pathway

XR XR XDH XDH XK XK XYL1 gene XYL2 gene XYL3 gene NADP NADP+

+

NADH NADH

The ways they used

• 1. Engineering of xylose reductase (XR)
• 2. Over-expression of xylitol dehydrogenase (XDH)
• 3. Over-expression of xylulose kinase (XK)

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Engineering of Engineering of xylose reductase xylose reductase and overexpression and overexpression

• f
• f xylitol dehydrogenase

xylitol dehydrogenase and and xylulokinase xylulokinase improves improves xylose alcoholic fermentation xylose alcoholic fermentation in the thermotolerant in the thermotolerant yeast yeast Hansenula polymorpha Hansenula polymorpha

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• 1. Protein engineering of XR

cofactor binding site sequences

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Hansenula polymorpha

• Simultaneous Saccharification and Fermentation

(SSF)

• Thermotolerant
• Protein factory
• Comparing to S. cerevisiae
• CBS4732 (wild type)

CBS4732 (wild type)

• Δ

Δxyl1 xyl1— —deficient in XR (Host) deficient in XR (Host)

1.

• 1. Protein engineering of XR

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Δxyl1 Δxyl1

XR XRm m XRn XRn

Activity and KM of XR Strain NADPH NADH Activity [U (mg protein‐1)] KM (μM) Activity [U (mg protein‐1)] KM (μM) XRn 0.091 ± 0.005 9 ± 0.5 0.016 ± 0.001 100 ± 5.5 XRm 0.01 ± 0.001 152 152 ± 8.4 0.014 ± 0.001 112 ± 6.5

CBS4732

0.034 ± 0.025 7 ± 0.4 0.012 ± 0.001 85 ± 4.7

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2.

• 2. Over-expression of XDH

Over Over-

• expression

expression

cofactor binding site sequences

2.

• 2. Over-expression of XDH

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Δxyl1 Δxyl1

XR XRm m/XDH /XDH XRn XRn/XDH /XDH

Activity [U (mg protein-1)] Strain XDH XRn 0.551 ± 0.035 XRm 0.504 ± 0.028

XRn/XDH

1.3 1.3 ± 0.07

XRm/XDH

1.485 1.485 ± 0.087

CBS4732

0.697 ± 0.049

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3.

• 3. Over-expression of XK

Over-expression

Over Over-

• expression

expression

3.

• 3. Over-expression of XK

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XRn/XDH XRn/XDH

Activity [U (mg protein-1)] Strain XK

XRn/XDH/XK

0.375 0.375 ± 0.021

XRm/XDH/XK

0.366 0.366 ± 0.019

CBS4732

0.156 ± 0.009

XRn/XDH/XK XRn/XDH/XK

XR XRm m/XDH /XDH

XR XRm m/XDH/XK /XDH/XK

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 XRn 6.3 ± 0.3 2.75 ± 0.11 3.6 ± 0.2 1.56 ± 0.1 XRm 9.8 ± 0.4 4.45 ± 0.19 3.2 ± 0.2 1.45 ± 0.07 XRn/XDH 12 ± 0.5 5.7 ± 0.3 3.1 ± 0.2 1.48 ± 0.07 XRm/XDH 18.4 ± 0.9 8.76 ± 0.35 1.6 ± 0.1 0.76 ± 0.04 XRn/XDH/XK 13.8 ± 0.6 6.4 ± 0.3 2.8 ± 0.1 1.3 ± 0.06 XRm/XDH/XK XRm/XDH/XK 54.7 54.7 ± 2.7 21.9 21.9 ± 1.1 0.9 0.9 ± 0.04 0.36 0.36 ± 0.02 CBS4732 7.5 ± 0.4 3.3 ± 0.2 4.2 ± 0.2 1.8 ± 0.09

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Xylose fermentation

CBS4732 XRn n/XDH/XK XRm m/XDH/XK

Ethanol Ethanol Ethanol Xylitol Xylitol Xylitol Xylose Xylose Xylose Biomass Biomass Biomass

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• 1. Protein engineering of XR
• 1. Protein engineering of XR

KM of NADPH ↑

↑

KM of NADH unchanged

• 2. over
• 2. over-
• expression of XDH

expression of XDH Activity of XDH ↑

↑

• 3. over
• 3. over-
• expression of XK

expression of XK Activity of XK ↑

↑

Productivity of ethanol ethanol ↑

↑

Productivity of xylitol ↓ ↓ Xylose fermentation

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Thank you for your attention