Method to Enhance Alcohol Production

1
Method to Enhance Alcohol Production
By- Nitin Kumar M.Sc. Biotechnology 06530013
2
Ethanol Production

• 80 by microbes
• Bacteria Zymomonas mobilis, Closteridium
  thermocellum
• Fungi S. cervisae, Candida utilis
• Ethanol production under microaerophillic/anaerobi
  c condition and high sugar concentration(30-150g/l
  itre)

3
Ethanol Production

• Growth of yeast ceases at ethanol concentration
  above 5
• Production rate reduces to zero at 6-11 ethanol
  concentration
• pH 4-6

4
Pathway of Ethanol production

• Glucose
• 
  Glycolysis
• Pyruvate
• 
  Pyruvate decarboxylase
• Acetaldehyde
• 
  Alcohol dehydrogenase
• Ethanol

5
Uses

• Manufacture of gasohol
• Industrial solvent
• Synthesis of acetone, acetaldehyde, chloroform,
  diethyl ether
• Pharmaceuticals
• Drinks

6
Engineering Yeast Transcription Machinery for
Improved Ethanol Tolerance and Production

• Stephanopoulous et.al.
• Science VOL 314, 1565(2006)

7
Global Transcription Machine Engineering

• Complete reprogramming of metabolism
• Modulation of simultaneous multiple gene
  expression at the highest level with profound
  implications for phenotype improvement
• Alteration of key proteins of transcriptone

8
Background Information

• S. cervisiae strain BY4741
• Nearly 75 general transcription factors
• THIID include TATA-binding protein(spt15) and 14
  other TAFs
• High sugar concentration and high ethanol
  concentration
• Tolerance to ethanol multi-genic trait

9
Procedure

• Two gTME libraries were created
• 1- SPT15
• 2- TAF25
• Identification of dominant mutations using
  haploid yeast carrying one copy of wild type gene
  also
• Libraries transformed in presence of elevated
  levels of ethanol and glucose

10
Results
Altered genes
11
Growth Yield of Clones
6 Ethanol
12
Cellular Viability Curve at Various Ethanol
Concentration
13
Gene Ontology Comparison Between E.coli and yeast
14
Gene Knockout Analysis(5 ethanol and 60g/liter
glucose)
15
Gene Over Expression Analysis(6 ethanol)
16
Relation Between SPT15 and SPT3
17
Role of Mutations
18
Discussion

• Y195H caused negative cofactor 2 element (NC2)
  repression
• 7/10 most highly expressed genes were SPT3
  dependent genes
• Spt15-300 mutant, unable to impart tolerance to
  spt3 knockout stain
• Spt15-300pmutant-Spt3p-DNA interaction
• SAGA(Spt-Ada-Gcn5-acetyltransferase)

19
References

• Biotechnology a textbook of industrial
  microbiology
• Engineering yeast transcription machinery for
  improved ethanol tolerance and production,
  Stephanopoulous et.al.
• Science VOL 314, 1565(2006)

20

• Thank
• You

21
Various Combinations of
22
(No Transcript)
23
(No Transcript)
24
(No Transcript)
25
(No Transcript)
26
(No Transcript)
27
(No Transcript)
28

• BY4741(his3?1, leu2?0, met15?0, ura3?0)