Mutator strain

Esterases have a catalytic function and mechanism similar to those of lipases, but some structural aspects and the nature of substrates differ [4]. One can expect that the lessons learned from the directed evolution of lipases also apply to esterases. However, few efforts have been made in the directed evolution of enantioselective esterases, although previous work by Arnold had shown that the activity of esterases as catalysts in the hydrolysis of achiral esters can be enhanced [49]. An example regarding enantioselectivity involves the hydrolytic kinetic resolution of racemic esters catalyzed by Pseudomonasfluorescens esterase (PFE) [50]. Using a mutator strain and by screening very small libraries, low improvement in enantioselectivity was... 

Following several cycles of mutagenesis using the E. coli XLl-Red mutator strain and transformation of the plasmid library into E. coli, a total of about 150 000 bacterial colonies were assayed for activity using a colorimetric prescreen [100]. The best mutant Asn336Ser showed a 47-fold increase in activity and a 5.8-fold enhancement... 

For these reasons we have developed a different approach that measures differential expression of intact proteins.21 In this approach the proteins are extracted from the cell, separated on an HPLC column, ionized via electrospray, and automatically deconvoluted into their respective uncharged nominal masses. By this methodology it is then possible to obtain accurate, intact protein profiles of the individual strains of bacteria. Because the masses of the detected proteins are accurate to +2 Da from run to run, it is possible to subtract protein profiles from known strains to quickly identify differences in protein expression among newly mutated strains. 

Epothilones are a class of molecules that show anticancer activity. Production of a synthetic intermediate was investigated through the action of an esterase on various sterically hindered 3-hydroxy esters [76]. No initial activity was observed, so a Pseudomonasfluorescens esterase was transformed into a mutator strain Epicurian coli and screened using an indicator in the growth plates that would produce a red color if hydrolysis occurred. An ee of 25% was achieved from a variant containing two mutations. 

A BDS patent [106] was awarded for the use of biocatalysts belonging to the group of Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus, or Corynebac-terium. One of the strains P. putida was further developed by mutation of the parent strain to obtain organic solvent-resistant mutants [107], The mutated strains were screened by selective cultivation in the presence of 0.1% to 10% by volume (v/v) of concentrations of a toxic organic solvent. The specific mutated strains obtained were P. putida No. 69-1 (PERM BP-4519), P. putida No. 69-2 (PERM BP-4520), and P. putida No. 69-3 (PERM BP-4521). 

In a more detailed study, the same esterase P. fluorescens) was again subjected to mutagenesis using the same mutator strain, but also by saturation mutagenesis at selected positions 133a). In addition to 3-phenylbutyric acid ethyl ester (27), 3-bromo-2-methyl-propionic acid methyl ester rac-31) was chosen for the hydrolytic kinetic resolution, with the WT PFE showing an E factor of 12 in favor of the (5)-32. 

Irvine, D. M., Puhan, Z. and Gruetzner, V. 1969. Protease complex from a mutated strain of Bacillus subtilis as a milk coagulant for cheese manufacture. J. Dairy Sci. 52, 889-889. 

Mutator strain XLl-Red (mutD, mutS, mutT Stratagene). 

The protocol for mutator strain passage and subsequent amplification of mutated plasmids is described in detail in the supplier s protocol. However, a condensed version is given here ... 

The DNA of interest can be amplified by transformation of a non-mutator strain (standard protocol) and cultivation on a 1-5 mL scale. The resulting culture can also be used directly for screening/selection purposes. Alternatively, the plasmid can be isolated by a standard procedure [21]. 

The mutator strain passage may be repeated for increasing the mutation frequency (see section 2.4, note 6). 

Although the overall mutation frequency exhibited by a mutator strain is much lower than that of error-prone PCR, there are some advantages of this procedure. (1) A complete, supercoiled expression plasmid that has already been tested for suitability may be submitted to mutagenesis. (2) The loss of DNA material is minimal, compared to ligation and transformation of relaxed plasmid. (3) The mutation frequency (yv 1 mutation per 2000 bp after one passage) can produce sufficient diversity for many optimization problems. 

Repeated mutator strain passage may promote recombination events, especially if the gene of interest exhibits toxic activity. Try chosing a lower number of passages. 

Mutator strain deficient DNA repair, stability of host strain is problematic Greener, 1996... 

A. Greener, M. Callahan, and B. Jerpseth, An efficient random mutagenesis technique using an E. colt mutator strain, Methods Mol. Biol. 1996, 57, 375-385. 

Evolution has been shown to be an efficient tool for the improvement of enzymes and pathways. Stochastic approaches to introduce point mutations in genes include error prone polymerase chain reaction (ePCR),11 the use of low-fidelity (mutator) strains,12 and chemical mutagens.13... 

Low, N. M., ffolliger, P. H., and Winter, G. (1996). Mimicking somatic hypermutation affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J. Mol. Biol., 260, 359-368. 

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