Josef Altenbuchner

Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones.

The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester.

Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids

A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts.

Genetic instability of the Streptomyces chromosome.

The Streptomyces wild‐type chromosome is linear in all examples studied. The ends of the chromosome or telomeres consist of terminal inverted repeats of various sizes with proteins covalently bound to their 5′ ends. The chromosome is very unstable and undergoes very large deletions spontaneously at rates higher than 0.1% of spores. Frequently, the telomeres are included in the deletions. Loss of both telomeres leads to circularization of the chromosome. The wild‐type chromosome can also be circularized artificially by targeted recombination. Spontaneously or artificially circularized chromosomes are even more unstable than the linear ones. High‐copy‐number tandem amplifications of specific chromosomal regions are frequently associated with the deletions. RecA seems to be involved in the amplification mechanism and control of genetic instability.

Directed evolution of an esterase: screening of enzyme libraries based on ph-Indicators and a growth assay

In order to resolve a sterically hindered 3-hydroxy ethyl ester, which was not accepted as substrate by 20 wild-type hydrolases, a directed evolution of an esterase from Pseudomonas fluorescens (PFE) was performed. Mutations were introduced using the mutator strain Epicurian coli XL1-Red. Enzyme libraries derived from seven mutation cycles were assayed on minimal media agar plates supplemented with pH indicators (neutral red and crystal violet), thus allowing the identification of active esterase variants by the formation of a red color caused by a pH decrease due to the released acid. A further selection criteria was introduced by using the corresponding glycerol estar, because release of the carbon source glycerol facilitates growth on minimal media. By this strategy, one double mutant (A209D and L181V) of PFE was identified, which hydrolyzed the 3-hydroxy ethyl ester in a stereoselective manner (25% ee for the remaining ester, E approximate to 5).

Plasmid‐mediated sucrose metabolism in Escherichia coli K12: mapping of the scr genes of pUR400

The scr genes located on plasmid pUR400 and responsible for sAucrose (Scr) metabolism of Escherichia coli K12 and other enteric bacteria have been cloned on a 9.3 kb DNA fragment. The different genes were mapped by transposon insertion mutagenesis, by restriction endonuclease and deletion mapping, and the corresponding gene products were identified. Besides the known structural genes scrA, coding for an EnzymellScr (45 kD) of the phosphoenolypyruvate‐dependent phosphotransferase system (PTS), and scrB, coding for a sucrose 6‐phosphate hydrolase (invertase) (55 kD), two new structural genes were discovered. Gene scrK apparently codes for an intracellular and ATP‐dependent fructokinase (39 kD), while scry seems to code for a sucrose porin (58 kD) in the outer cell membrane. No genes for an Enzyme IIIScr of the PTS or for (a) glycosyltransferase(s) were detected. The four genes form an scr operon (gene order, scrK scrY scrA scrB, transcription from K to B), regulated by a repressor (gene scrR, 37 kD) and inducible by sucrose, fructose and fructose‐containing oligosaccharides

Characterization and enantioselectivity of a recombinant esterase from Pseudomonas fluorescens

Abstract A recombinant esterase from Pseudomonas fluorescens (PFE) was produced from Escherichia coli cultures and purified to homogeneity resulting in a specific activity of 120 U mg −1 ( p -nitrophenyl acetate assay). PFE is stable in a wide range of pH values (5–10) and active from 30–70°C, but rather unstable at temperatures >50°C. PFE hydrolyzes a wide range of aliphatic and aromatic esters, but no long chain fatty acid esters. The enzyme showed high rate and enantioselectivity in the resolution of α-phenyl ethanol ( E > 100) and its acetate ( E = 58) while the closely related α-phenyl propanol was converted at very low rate and enantioselectivity.

Nucleotide sequence and role in DNA amplification of the direct repeats composing the amplifiable element AUD1 of Streptomyces lividans 66

The amplifiable unit of DNA no. 1 (AUD1) of Streptomyces lividans consists of three 1 kb repeats (left direct repeat, LDR; middle direct repeat, MDR; and the slightly different right direct repeat, RDR) and two 4.7 kb repeats alternately arranged in identical orientation to each other. Both 4.7 kb repeats have been sequenced. They are identical and contain one open reading frame (orf4.7 ). The deduced amino acid sequence has a low similarity to chitinases, and two amino acid repeats present high similarities to fibronectin type III modules. Sequencing had previously shown that the ORF corresponding to each 1 kb repeat encodes a putative DNA‐binding protein. Crude extracts of Escherichia coli overexpressing the orfRDR‐encoded protein and of S. lividans Jni1, having a high amplification of AUD1 and therefore orfMDR, were used in gel retardation assays. The orfRDR‐ and probably the orfMDR‐encoded proteins can bind to an imperfect palindromic sequence upstream from MDR and RDR and to another sequence downstream from RDR. An extrachromosomal DNA amplification system was constructed containing different combinations of the sequences composing AUD1. In mutants having a deletion of the chromosomal AUD1, the 4.7 kb repeats could be reduced in size, mutated or replaced by E. coli DNA without altering the ability to amplify when RDR was present. Therefore, the only function of the 4.7 kb repeats in amplification is to provide directly repeated DNA sequences. When RDR was lacking or mutated, no amplification was observed. This strongly suggests that the DNA‐binding protein encoded by orfRDR is required for AUD1 amplification.

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans

SummaryA broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cms), arginine auxotrophic (Arg−) mutant of Streptomyces lividan 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cms Arg− mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721

SummaryDNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72–73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two clements. A TnpR− derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.

Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762

A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tü24, was used as a probe in Southern blot hybridization of total DNA from S. aureofaciens ATCC 10762. A single SstI fragment of 5.4 kb was detected, which was cloned via an enriched gene library into Escherichia coli. The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S. lividans TK64, using the multicopy plasmid pIJ486. The enzyme was overproduced in S. lividans TK64 (up to 30,000 times compared to S. aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S. aureofaciens ATCC 10762. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M(r) and N-terminal amino acid sequence as the purified subunit of BPO-A2.