A _ Forrest Gump _ Story on How the Ho - Purdue University by jlhd32

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"Forrest Gump" is a novel under the same name, American films and novels of Winston Groom temperature, the film won the Best Picture Oscar for 1994, the Oscar for Best Actor, Best Director Oscar awards 6 Awards.

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									    A “Forrest Gump” Story on
How the Ho-Purdue Yeast Came About

                 Nancy W. Y. Ho
         School of Chemical Engineering
            Molecular Genetics Group
  Laboratory of Renewable Resources Engineering
                Purdue University

              Founder and President
               Green Tech America
              West Lafayette, Indiana

        Presented at LORRE 30 Years Celebration
                      April 3, 2008
My First Professional Break
    My Encounter with
Recombinant DNA Techniques
                     D-Xylose
            NADPH
            (NADH)         Xylose Reductase
             NADP
             (NAD)
                        Xylitol
                                  NAD +
Xylitol Dehydrogenase
              (XD)                NADH + H +

                     D-Xylulose
              ATP
                            Xylulokinase
              ADP             (XK)

            D-Xylulose-5-Phosphate




          Pentose Phosphate Pathway -> -> -> Ethanol

The xylose metabolic pathway in yeast
Joining LORRE
                     D-Xylose
            NADPH
            (NADH)         Xylose Reductase
             NADP
             (NAD)
                        Xylitol
                                  NAD +
Xylitol Dehydrogenase
              (XD)                NADH + H +

                     D-Xylulose
              ATP
                            Xylulokinase
              ADP             (XK)

            D-Xylulose-5-Phosphate




          Pentose Phosphate Pathway -> -> -> Ethanol

The xylose metabolic pathway in yeast
Successfully cloned the
Xylose Isomerase gene
     from E. coli
Cloning and Characterization of the
  xyl Genes from Escherichia coli

 Stuart A. Rosenfeld, Panayiotis E. Stevis,
            and Nancy W. Y. Ho
   Mol. Gen. Genet., 194, 410-415 (1984)
                     D-Xylose
            NADPH
            (NADH)         Xylose Reductase
             NADP
             (NAD)
                        Xylitol
                                  NAD +
Xylitol Dehydrogenase
              (XD)                NADH + H +

                     D-Xylulose
              ATP
                            Xylulokinase
              ADP             (XK)

            D-Xylulose-5-Phosphate




          Pentose Phosphate Pathway -> -> -> Ethanol

The xylose metabolic pathway in yeast
  Successful Cloning and
   Overexpression of the
     xylulokinase gene
in the Saccharomyces yeast
Cloning of Yeast Xylulokinase Gene by
Complementation of E. coli and Yeast
              Mutations

 Nancy W. Y. Ho and Sue-Fen Chang

Enzyme Microbial Technology, 11, 417-
            421 (1989)
                     D-Xylose
            NADPH
            (NADH)         Xylose Reductase
             NADP
             (NAD)
                        Xylitol
                                  NAD +
Xylitol Dehydrogenase
              (XD)                NADH + H +

                     D-Xylulose
              ATP
                            Xylulokinase
              ADP             (XK)

            D-Xylulose-5-Phosphate




          Pentose Phosphate Pathway -> -> -> Ethanol

The xylose metabolic pathway in yeast
                                            Cofermentation of Glucose and Xylose by 1400(pLNH32)
                                           100




                                                                  Glucose
                                                                  Xylose
                                                                  Ethanol
                                            80
                                                                  Glycerol
                                                                  Xylitol



Co-fermentation of
glucose and xylose                          60

By 1400(pLNH32)

                     Concentration (g/L)
                                            40




                                            20




                                             0
                                                 0   10      20          30         40   50   60

                                                             Fermentation time (hours)
                        Cofermentation of Glucose and Xylose by Untransformed Strain 1400
                                                   100




                                                                                      Glucose
                                                   80                                 Xylose
                                                                                      Ethanol


Co-fermentation of
Glucose and xylose by



                             Concentration (g/L)
                                                   60

transformed 1400 only
Containing XR and XD
                                                   40




                                                   20




                                                    0
                                                         0   20          40           60        80

                                                              Fermentation time (hours)
Genetically Engineered Saccharomyces
      Yeast Capable of Effective
Cofermentation of Glucose and Xylose

    Nancy W. Y. Ho, Zhengdao Chen
        and Adam P. Brainard

   Applied and Environmental Microbiology
        Vol. 64 (5) 1852-1859 (1998)
              1993-1997

 Development of stable yeast containing
high-copy-number XR, XD, and XK genes
 integrated into the yeast chromosomes
                              80
                                                                                                        80
                                                                                  Glucose                                                               Glucose
                                                                                  Xylose                                                                Xylose
                                                                                  Ethanol                                                               Ethanol




                              60
                                                                                                        60




                        g/L




                                                                                                  g/L
                              40
                                                                                                        40




                              20
                                                                                                        20




Ho’ s new integration          0
                                                                                                         0
                                   0   10     20            30          40              50



methods can stepwise                        Fermentation Time (Hours)
                                                                                                             0   10      20            30


                                                                                                                       Fermentation Time (Hours)
                                                                                                                                                   40             50




Integrate genes into          80

                                                                             Glucose
                                                                             Xylose
                                                                                                        80                                              Glucos
                                                                                                                                                        Xylose
                                                                             Ethanol                                                                    Ethanol


the yeast chromosome
                              60
                                                                                                        60
                        g/L




                                                                                                  g/L
                              40
                                                                                                        40




                              20                                                                        20




                                                                                                         0
                               0                                                                             0    10          20            30           40            50
                                   0   10      20            30              40              50

                                                                                                                       Fermentation Time (Hours)
                                            Fermentation Time (Hours)




                        The integration of genes into the yeast chromosome by the method of Ho et. al. is
                         a continuous process. (A) represents the beginning of the integration; (B, C) is
                              the middle stage of integration; and (D) is the final stage of integration.
                                                Cofermentation of glucose and xylose by 1400 (LNH-ST)


                                           80


                                                                         Glucose
                                                                         Xylose
                                                                         Ethanol
                                                                         Xylitol
                                                                         Glycerol

                                           60




Co-fermentation of

                     Concentration (g/l)
glucose and xylose
by 1400(LNH-ST)                            40




                                           20




                                            0
                                                0                                   20

                                                                  Fermentation Time (hr)
                                            Cofermentation of Glucose and Xylose by 1400(pLNH32)
                                           100




                                                                  Glucose
                                                                  Xylose
                                                                  Ethanol
                                            80
                                                                  Glycerol
                                                                  Xylitol



Co-fermentation of
glucose and xylose                          60

By 1400(pLNH32)

                     Concentration (g/L)
                                            40




                                            20




                                             0
                                                 0   10      20          30         40   50   60

                                                             Fermentation time (hours)
                     1997-1999

Large-scale screening for better yeasts with no legal
constraints for converting cellulosic sugars (mixed
sugars recovered from cellulosic biomass) to ethanol
                              70                                                    500




                              60

                                                                    Glucose
                                                                    Xylose          400
                                                                    Glycerol
                                                                    Ethanol
                              50                                    O.D.
New stable yeast
424A(LNH-ST) for                                                                    300
co-fermentation of            40




                                                                                        O.D. (Klett unit)
glucose and xylose to
ethanol                 g/L
                              30
                                                                                    200




                              20



                                                                                    100


                              10




                              0                                                     0
                                   0   5   10      15          20    25        30

                                                Time (hours)
    9                     9                      9                      9                     9                     Glucose


                                                                                                                    Xylose
    8                     8                      8                      8                     8

                                                                                                                      Ethanol
    7                     7                      7                      7                     7


    6                     6                      6                      6                     6


    5                     5                      5                      5                     5
%




    4                     4                      4                      4                     4


    3                     3                      3                      3                     3


    2                     2                      2                      2                     2


    1                     1                      1                      1                     1


    0                     0                      0                      0                     0
        0     20    40        0     20    40         0      20   40         0     20    40        0     20    40
            Time (hour)           Time (hour)            Time (hour)            Time (hour)           Time (hour)
            1st-cycle             2nd re-cycle           5th re-cycle       12th re-cycle         20th re-cycle


    Genetically Enginnered Stable Glucose-Xylose-Cofermenting Saccharomyces
        Yeast 424A(LNH-ST) Repeatedly Cofermenting Glucose and Xylose
               2000-Present

• Further genetic engineering of the best yeast,
 424A(LNH-ST), to improve its xylose fermentation
 and to make it able to ferment two other minor
 sugars , L-arabinose and galactose, effectively.
                                                    10




                                                     9

                                                                              Mannose
                                                                              Galactose
                                                     8
                                                                              Xylose
                                                                              Glucose

                                                     7

Co-fermentation of Glucose,
 Mannose, and Xylose by




                              Concentration (g/L)
                                                     6


      424A(pLNH32)
                                                     5




                                                     4




                                                     3




                                                     2




                                                     1




                                                     0
                                                         0   10      20            30     40

                                                                  Time (hr)
Making yeast co-ferment glucose
     xylose and arabinose
Expression of E. coli araBAD Operon
Encoding Enzymes for Metabolizing
          L-arabinose in
    Saccharomyces cerevisiae

       Miroslav Sedlak, Nancy W.Y. Ho

Enzyme and Microbial Technology, 28, 16-24 (2001)
Application of Microarray Analysis to
learn how we should further improve
              our yeast
      70                                                            500
           10             13     14                      15
                     12

                11
      60


                                                                    400

                                              Glucose
                                              Xylose
                                              Glycerol
      50
                                              Ethanol
                                              Xylitol
                                              O.D.


                                                                    300




                                                                          O.D. (Klett unit)
      40
g/L




      30
                                                                    200




      20




                                                                    100


      10




      0                                                             0
           0         5    10      15          20          25   30

                               Time (hours)
  Expression profile of S.
 cerevisiae 424A(LNH-ST)
   co-fermenting glucose
and xylose. Clustering by K-
    means and measure
   similarity by Pearson
         correlation
Industrial cellulosic ethanol
   production using our
     Ho-Purdue Yeast
Iogen has used our yeast in their
facility to produce cellulosic
ethanol since 2004.

ADM has also used our yeast
to develop its process for the
conversion of corn fiber to ethanol.
 The Ho-Purdue Yeast Produces 30-60% more ethanol
             from cellulosic biomass
                              Fermentation with
                               Ordinary Yeast       48-60
                                                  Gallons of
                                                   Ethanol


Feedstock      Pretreatment
 1 dry ton         And
Cellulosic      Hydrolysis
 Biomass        processes



                                                   80-100
                                                  Gallons of
                        Fermentation with          Ethanol
                      Ho-Purdue Engineered
                             Yeast
Demonstrations of the
   performance of
 the Ho-Purdue yeast
Ho-Purdue Yeast Fermenting glucose   Ho-Purdue Yeast Fermenting xylose
Comparison of 424A(LNH-ST) with Leading Commercial corn
Ethanol Yeast for co-fermenting glucose and xylose to ethanol
Leading Commercial yeast fermenting   Ho-Purdue yeast fermenting wheat straw
wheat straw hydrolysates              hydrolysates
Converting MSW to ethanol
             Acknowledgement
      MAJOR PARTICIPANTS SINCE 1985

      Stuart A. Rosenfeld      Panayiotis E. Stevis
       Sue-Fan Chang               S. K. Liu
       Zhengdao Chen            Adam P. Brainard

                     Miroslav Sedlak

Our current work is supported by DOE and USDA
Our early work, prior to 1990, was mostly supported by DOE
and USDA. EPA also provided us a grant for three years for
screening the most effective yeast for xylose fermentation.
Overall, we are most indebted to the NRI program of USDA,
particularly its Bio-based Products and Bio-energy Research
Program, that has persistently supported our research on the
development of recombinant yeast since the early 1980s.
The unexpected recognitions
The DOE 3 million plus support
       For improving:

      xylose transport
     inhibitor tolerance
     cofactor imbalance
             etc
Current major participants

        Nancy Ho

      Miroslav Sedlak

      Nathan Mosier

       Jiri Adamec
Scientists currently working in
        Nancy Ho’s lab

        Miroslav Sedlak
          Aloke Beta
          Aftab Khan
        Chia-Ling Wu
         Shanging Tao

								
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