A semiquantitative highthroughput screening method

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A semi-quantitative high-throughput screening
method for microbial L -tyrosine production in
microtiter plates Tina Lütke-Eversloh Gregory
Stephanopoulos J Ind Microbiol Biotechnol
(2007) 34807811
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Industrial and pharmaceutical compounds derived
from L-tyrosine
1. stimulate brain activity 2. control depression
anxiety
3. for PKU patients
Parkinsons disease
1. Adhesive coatings 2. Bio-cosmetics 3. health
nutrition products
1. UV absorbers 2. Drug carriers 3. Semiconductors
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Biosynthetic Pathway for L-tyrosine
Appl Microbiol Biotechnol (2007) 77751762
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Pathway of aromatic amino acid biosynthesis and
allosteric regulation by end products
Appl Environ Microbiol, Nov. 2005, p.
72247228 Appl Microbiol Biotechnol (2007)
75103110 Appl Microbiol Biotechnol (2007)
77751762
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L-Tyrosine production
Strain Development

• 1. Alleviation of regulatory circuits
• 2. Over-expression of rate-
• controlling enzymes
• 3. Use of auxotrophic strains to
• reduce by-product formation
• 4. Increased supply of
• intracellular precursors

Appl Environ Microbiol, Nov. 2005, p.
72247228 Appl Microbiol Biotechnol (2007)
75103110 Appl Microbiol Biotechnol (2007)
77751762
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L-Tyrosine production
Strain Development
1. Alleviation of regulatory circuits

• ? Tyr R
• fbr derivatives of
• DAHP synthase and
• Chorismate mutase /
• Prephanate dehydrogenase

Appl Environ Microbiol, Nov. 2005, p.
72247228 Appl Microbiol Biotechnol (2007)
75103110 Appl Microbiol Biotechnol (2007)
77751762
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L-Tyrosine production
Strain Development
2. Over-expression of rate controlling
enzymes

• fbr derivatives of
• DAHP synthase and
• Chorismate mutase /
• Prephanate dehydrogenase

Appl Environ Microbiol, Nov. 2005, p.
72247228 Appl Microbiol Biotechnol (2007)
75103110 Appl Microbiol Biotechnol (2007)
77751762
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L-Tyrosine production
Strain Development
3. Use of auxotrophic strains to reduce
by-product form

• Block Trp and Phe pathways

E.coli T1 3.8 g/l ?tyrR fbr-DAHP synthase
fbr-CM /PDH
Appl Environ Microbiol, Nov. 2005, p.
72247228 Appl Microbiol Biotechnol (2007)
75103110 Appl Microbiol Biotechnol (2007)
77751762
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L-Tyrosine production
ppsA
tktA
Strain Development
4. Increased supply of intracellular
precursors

• Overexpress ppsA and tktA

E.coli T2 9.7 g/l ?tyrR fbr-DAHP synthase
fbr-CM /PDH Transketolase PEP synthase
Appl Environ Microbiol, Nov. 2005, p.
72247228 Appl Microbiol Biotechnol (2007)
75103110 Appl Microbiol Biotechnol (2007)
77751762
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L-Tyrosine production
Strain Development
Appl Environ Microbiol, Nov. 2005, p. 72247228
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E.coli T1
E.coli T2
Glucose (g/l)
Glucose (g/l)
Growth (OD600)
Growth (OD600)
Tyrosine (g/l)
Productivity
Productivity
Tyrosine (g/l)
Appl Environ Microbiol, Nov. 2005, p. 72247228
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1Nitroso2-naphthol Assay
Charge transfer complex (red, unstable)
HNO2
HNO3

55oC for 1 hr
Remove excess dye with ethylene dichloride
Incubate at 25oC for 3 - 4 hrs
Fluorescence Exitation - 485 nm Emission - 590
nm
Stable yellow compound
Calorimetrically at 450 nm
CLIN.CHEM.29/11, 1969-1971 (1983) J Lab Clin Med
50733736, (1957)
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High Throughput Screening Method

• Fluorescence based assay, employ 96 well
  microtiter
• plates
• Highly suitable for combinational approaches
• Sensitive (0.05 0.5 g/l) and rapid
• Doesnt require cell removal step by
  centrifugation and
• protein removal by TCA precipitation

J Ind Microbiol Biotechnol (2007) 34807811
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Accuracy of the Nitrosonaphthol assay
J Ind Microbiol Biotechnol (2007) 34807811
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Conclusion
The assay is suitable for screening large number
of samples to identify putative microbes with
altered L- tyrosine production
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Thank You