Application examples technology platform

 

Screening of mutant banks

(Kevin O'Connor, University College Dublin)

 

 

One of the purposes for which the group of Kevin O'Connor from the University College Dublin utilizes the system is the screening of bacterial mutant banks. For example, they have prepared a bank of 2,000 mutants of the styrene-degrading strain Pseudomonas putida CA-3, and stored them in 96-deepwell plates at -80 ºC.  One of their goals was to obtain a mutant without styrene monooxygenase activity. This enzyme is capable of transforming Indole into Indigo (blue). They sub-screened the white colonies for styrene negative and styrene oxide negative for growth as well as phenylacetladehyde positive for growth and phenylacetic acid postivie for growth. 

 

The storage of the mutant bank at -80 ºC in microtiter plates has the advantage that future screenings for other mutants can be performed very quickly. The use of the cryo-replicator system allows a rapid revival and replication of all 2000 mutants, and makes the storage of multiple copies of the mutant bank obsolete (no freeze-thaw cycles).

 

 

 

 

 

References:

 

E. Fischer E, U. Sauer (2005). Large-scale in vivo flux analysis

shows rigidity and suboptimal performance of Bacillus

subtilis metabolism NATURE GENETICS 37: 636-640 .

 

L.M. Blank, L. Kuepfer, U. Sauer (2005). Large-scale C-13-flux analysis

reveals mechanistic principles of metabolic network robustness

to null mutations in yeast. GENOME BIOLOGY 6: Art. No. R49 .

 

M. Sonderegger, M. Schumperli, U. Sauer (2005) Selection of

quiescent Escherichia coli with high metabolic activity.

METABOLIC ENGINEERING 7: 4-9 .

 

E. Fischer, N. Zamboni, U.Sauer (2004) High-throughput metabolic

flux analysis based on GC-MS derived 13C constraints (2004) Anal.

Biochem. 325: 308-316.

 

E. Fischer & U. Sauer (2003). A novel metabolic cycle catalyzes

glucose oxidation and anaplerosis in hungry E. coli.

J. Biol. Chem. 278: 46446-46451.

 

P. Steiner & U. Sauer (2003) Overexpression of the ATP-

dependent helicase RecG confers acetate tolerance

to E. coli. Appl. Microbiol. Biotechnol. 63: 293-299.

 

E. Fischer & U. Sauer (2003). Metabolic flux profiling of

E. coli mutants in central carbon metabolism by GC-MS.

Eur. J. Biochem. 270: 880-891.

 

N. Zamboni & U. Sauer (2003). Knockout of the high-

coupling cytochrome aa3 oxidase reduces TCA cycle fluxes

in B. subtilis. FEMS Microbiol. Lett. 226:121-126.

 

 

 

 

Quantitative flux analaysis in Escherichia coli, Bacillus subtilis and yeasts (Uwe Sauer, Institute of Biotechnology, ETH Zurich)

 

In the research group of Uwe Sauer, a few hundred mutants of E. coli (different enzymes overexpressed or knocked out) are grown with various substrates and media. The flux through the various metabolic pathways is determined using e.g. 13C tracer studies.

 

The 96-pins cryo-replicator is used for the parallel sampling of this library of mutants, and their inoculation into the deepwell microtiter plates for growth in suspended cultures. The use of the sandwich cover system is important for this purpose as it :

 

The resulting absence of physicochemical well-to-well variations enables small effects of certain mutations on fluxes or biomass amounts to be accurately established. These results - in their turn - enable an accurate flux analysis for the various metabolic pathways in E. coli.

 

In a similar way, the system is used to determine fluxes in mutant banks of B. subtilis and yeasts.

 

 

 

 

Set of alkane-degrading bacterial strains containing

a broad range of alkane monooxygenases.

Enzyscreen utilizes these and other strains

(totally 2000 strains) for custom screenings for

new enzyme activities

 

refs:

van Beilen JB, Holtackers R, Luscher D, et al. (2005)

Biocatalytic production of perillyl alcohol from limonene

by using a novel Mycobacterium sp cytochrome P450

alkane hydroxylase expressed in Pseudomonas putida

APPLIED AND ENVIRONMENTAL MICROBIOLOGY

71: 1737-1744 APR 2005

 

Zhang J, Duetz WA, Witholt B, et al. (2004)

Rapid identification of new bacterial alcohol dehydrogenases

for (R)- and (S)-enantioselective reduction of beta-ketoesters

CHEMICAL COMMUNICATIONS (18): 2120-2121 2004

 

Spain JC, Nishino SF, Witholt B, et al. (2003)

Production of 6-phenylacetylene picolinic acid from

diphenylacetylene by a toluene-degrading Acinetobacter strain

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69: 4037-4042 JUL 2003

 

 

 

 

 

Screening for new enzyme activities in wild type strains (Enzyscreen, and a range of pharmaceutical/fine-chemical companies)

 

At ENZYSCREEN and a number of other companies active in the field of biocatalysis (E.g. Albany Molecular Research, DOW chemicals, DSM, Lonza, Merck, Novartis), the system is used for the screening for new biocatalysts. These companies use the system to screen collections of streptomycetes, fungi, yeasts and bacteria. The manual (freely available as a pdf-file) contains protocols for this specific purpose.

 

 

A separate web-page shows examples reactions  resulting from screenings performed at the ETH Zurich and Enzyscreen