Epicurian Coli XL1-Red cells allow isolation of gene products with altered activity
Uwe T. Bornscheuer Markus M.
Institute for Technical Biochemistry, University of Stuttgart, Stuttgart, Germany
Institute for Industrial Genetics, University of Stuttgart, Stuttgart, Germany
Hartmut H. Meyer
Institute for Organic Chemistry, University of Hannover, Hannover, Germany
Mutants of an esterase (PFE) from Pseudomonas fluorescens were generated using Stratagenes mutator strain, Epicurian Coli XL1-Red. One variant (A209D / L181V) stereoselectively hydrolyzed a sterically-hindered 3-hydroxy ester, which was not accepted as substrate by the wild type. After several mutation cycles, mutants were assayed by plating the esterase-producing colonies onto minimal-media agar plates containing the 3-hydroxy ethyl or glyceryl ester and indicators.
Lipases, as well as esterases, accept a wide range of non-natural esters and also exhibit high activity in organic media.1,2 However, for sterically hindered substrates, these enzymes usually fail.
In previous articles, we were able to resolve aliphatic3 and arylaliphatic4 3-hydroxy esters using commercial lipases or esterases with good to excellent optical purities. Still, the esters could not be resolved for the 3-hydroxy ester 1 bearing two methyl groups at carbon 4 (Figure 1). None of the 18 lipases and 2 esterases tested showed any activity. We considered several options: screen for new enzymes (tedious and time consuming), alter the reaction conditions (e.g. change the solvent system or acyl donor), or evolve new enzymes by mutagenesis.
The last strategy required the gene encoding the enzyme and an efficient expression system. Fortunately, our lab had a gene encoding a PFE from Pseudomonas fluorescens, which was expressed well in E. coli using a rhamnose-inducible promoter. Since the structure of PFE is not known, positions for site-directed mutagenesis are difficult to predict. Alternatively, a random mutagenesis of the gene combined with an assay system also allows detection of the desired variants.
Recent methods are described for the directed evolution of enzymes by random mutagenesis using error-prone PCR or DNA-shuffling.5,6 For both of these methods, ligation of the PCR products is critical. In addition, the mutation bias must be optimized. We chose to use the mutator strain, Epicurian Coli XL1-Red (lacking DNA repair mechanisms), which should result in random mutations generated within a clone of interest.7
To identify the desired variants from the numerous clones produced by random mutagenesis, we supplemented agar plates containing E. coli colonies with rhamnose, substrate 1, and indicators. A color change, caused by the release of substrate 1s corresponding acid, indicated hydrolysis. To further select for the mutants, the corresponding glycerol ester 2 was introduced. When this ester is hydrolyzed, the carbon source glycerol is released, thereby speeding up bacterial growth and producing active esterases on minimal media. With this strategy, we identified an esterase variant capable of stereoselectively hydrolysing the sterically-hindered 3-hydroxy ester 1.8 This compound can serve as a building block in the synthesis of Epothilones, a new class of macrolides showing taxol-like biological activity.9
In Figure 2, plasmids isolated from E. coli colonies produced during mutation cycles using XL1-Red competent cells were transferred into a non-mutator E. coli host, then plated and incubated on LB/Amp agar plates. From these master plates, colonies were replica plated onto minimal-media agar containing rhamnose (to induce esterase production), substrates 1 or 2, and indicators (crystal violet and neutral red). Upon hydrolysis of substrates 1 or 2, the pH in the microenvironment of the colonies decreased and red spots are formed (from the indicator).
We achieved a good compromise between fast bacterial growth and reliable detection of positive variants by using plates with minimal media. From approximately 750 colonies, several putative mutant clones were identified based on the red color that developed on the agar plates after the colonies were incubated for 2 to 6 days at 37C.
The plasmids were isolated using colonies from the master plates and then transformed back into a non-mutator host. After the colonies were cultivated for 3 hours and induced with rhamnose for 4 hours, the esterase produced was isolated by sonification and subjected to preparative biotransformation. When the esterases capable of hydrolysing the 3-hydroxy ester 1 were sequenced, one variant contained two point mutations (A209D and L181V). In the indicator assay, the clone that produced this variant also gave the strongest red color and largest colony size.
The esterase gene is 843 bp long, and the double mutant was isolated after three mutation cycles. Each cycle is approximately 30 generations and the spontaneous mutation rate should yield approximately 1 mutation per 2,000 base pairs every 30 generations. This PFE mutant was subjected to another mutation cycle using XL1-Red competent cells, and approximately 9500 colonies were investigated. From these, 12 clones were selected, and the esterase was produced and subjected to biotransformation reactions; however, the stereoselectivity did not improve.
The esterases (wild type and selected mutants) were subjected to the preparative hydrolysis of substrate 1 in phosphate buffer (50 mM, pH 7.5) at 40C. As expected, no reaction was observed either with the wild type (PFE) or in the absence of enzyme. In contrast, by using variant A209D/L181V, stereoselective hydrolysis of substrate 1 occurred, resulting in what is 25% enantiometric excess (%ee) (determined by gas chromatography using a chiral column) for the remaining ester. The stereoselectivity was further confirmed by determining the optical rotation of the substrate and the reaction product. The remaining ester substrate gave an [a]D20 of + 0.97 , and the produced acid, as expected, rendered a contrary sense of rotation of [a]D20= - 11.01 after a 10-day reaction time. Mutants derived from the second mutation cycle exhibited less stereoselectivity compared to the variant, A209D/L181V. The low stereoselectivity observed in the esterase-catalyzed resolution of substrate 1 relates to the generally observed weak stereoselectivity of PFE.10
The results clearly demonstrate that the XL1-Red mutator strain is efficient as an alternative method to direct the evolution of enzymes. Besides being easy to use, XL1-Reds mutation rate compares well to error-prone PCR, while excluding the problems associated with PCR ligation. Because the assay does not require special substrates, such as chromophoric esters, a large number of clones can be quickly identified by their surrounding red color. Additionally, only a few mutation cycles are necessary to identify an esterase variant, which hydrolyzes a sterically-hindered substrate. We expect that direct ed evolution using the XL1-Red mutator strain will easily transfer to other experiments with problems related to biotransformation.