Construction of Lycopene overproducing E'coli strains by combining systematic and combinatorial gene

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Construction of Lycopene overproducing E.coli
strains by combining systematic and combinatorial
gene knockout targetsNature Biotechnology10
April 2005

• Hal Alper, Kohei Miyaoku Gregory Stephanopoulos
• Department of Chemical Engineering, Massachusetts
  Institute of Technology
• USA

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Introduction

• Lycopene- Bright red carotenoid pigment, a
  phytochemical found in tomatoes and other red
  fruits
• Lycopene is a terpene assembled from 8 isoprene
  units
• Another source of lycopene is the fungus
  Blakeslea trispora

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Nutritional Benefits

• Anti-oxidant- most powerful carotenoid quencher
  of singlet oxygen
• Used against skin ageing
• Cardiovascular disease, cancer, diabetes,
  osteoporosis, and male infertility
• Food coloring
• Biosynthesis
• Mevalonate and non- mevalonate pathway

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Mevalonate and non- mevalonate pathway

• Mevalonate Pathway - all higher eukaryotes and
  many bacteria
• Condensation of three molecules of acetyl
• coenzyme A (acetyl CoA) into 3-hydroxy-3-
• methylglutaryl coenzyme A (HMG-CoA),
• which is reduced to mevalonate by HMGCoA
• reductase

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Non- Mevalonate pathway
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Parental Recombinant Strain E. coli K12
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Two Methodology to search for gene knock out
targets

• Systematic Approach( Model Based)
• Combinatorial Approach (Transposons based)

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Systematic Approach

• Specific enzymes are perturbed
• Increase yield by precursor or cofactor
  availability
• Totally stoichiometric based, deviod of any
  kinectic or regulatory characteristics
• Together with parental strain accompanies a total
  of 8 types of strains

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Combinatorial Approach (Transposons based)

• Transposons Library was used
• 3 target genes obtained- rssB (also known as
  hnr), yjfP and yjiD
• rssB- Response regulator responsible for
  recruiting the proteolysis of the stationary
  phase sigma factor, sS
• Together with parent recombinant strains, 8
  strains were prepared with the gene knockout

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Pictorial representation
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Results

• A total of 64 strains screened
• Two global maxima exist- 11,000 p.p.m. (mg/g dry
  cell weight)
• Genotypes ?gdhA ?aceE ?fdhF and ?gdhA ?aceE
  ?pyjiD
• Several local maximum points yeild between
  8400-7500 p.p.m
• Combination of more than one combinatorial
  knockout target greatly reduces lycopene levels

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Lycopene Production landscape
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Clustering Analysis
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Relative Production Profile
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Conclusion

• Rationally selected stoichiometric gene knockout
  targets have the potential of generating serious
  contenders in the quest for maximally producing
  strains
• Metabolic genes seem to have a linear impact in
  the overall cellular phenotype whereas the effect
  of regulatory targets is definitely nonlinear and
  more complex

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• Thank
• You!!!!!!!!!