Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.

The two forms of karyogamy transcription factor Kar4p are regulated by differential initiation of transcription, translation, and protein turnover.

Kar4p is a transcription factor in Saccharomyces cerevisiae that is required for the expression of karyogamy-specific genes during mating, for the efficient transit from G1 during mitosis, and for essential functions during meiosis. Kar4p exists in two forms: a constitutive slower-migrating form, which predominates during vegetative growth, and a faster-migrating form, which is highly induced by mating pheromone. Transcript mapping of KAR4 revealed that the constitutive mRNA was initiated upstream of two in-frame ATG initiation codons, while the major inducible mRNA originated between them. Thus, the two forms of Kar4p are derived from the translation of alternative transcripts, which possess different AUG initiation codons. Site-directed mutations were constructed to inactivate one or the other of the initiation codons, allowing the expression of the two Kar4p forms separately. At normal levels of expression, the constitutive form of Kar4p did not support wild-type levels of mating. However, the two forms of Kar4p could also be expressed separately from the regulatable GAL1 promoter, and no functional difference was detected when they were expressed at equivalent levels. Pulse-chase experiments showed that the induced form of Kar4p was highly expressed and stable during mating but rapidly turned over in vegetative cells. In contrast, the constitutively expressed longer form showed the same rate of turnover regardless of the growth condition. Furthermore, overexpression of either form of Kar4p in vegetative cells was toxic. Thus, the elaborate regulation of the two forms of Kar4p at the levels of transcription, translation, and protein turnover reflects the requirement for high levels of the protein during mating and for low levels during the subsequent phases of the cell cycle.

Kar4p, a karyogamy-specific component of the yeast pheromone response pathway.

Karyogamy is the process whereby two haploid nuclei fuse to form a diploid nucleus during mating in Saccharomyces cerevisiae. Here, we describe the characterization of the KAR4 gene, previously identified in a screen for new nuclear fusion-defective mutants. During mating, kar4 mutants were defective for the microtubule-dependent movement of nuclei, a phenotype identical to that of mutations in KAR3 and CIK1. Consistent with its mutant phenotype, we found that the kar4 mutation resulted in failure to induce KAR3 and CIK1 mRNA during mating. Expression of KAR3 and CIK1 under independent regulatory control suppressed the kar4 defect, indicating that KAR4 is required primarily for the induction of KAR3 and CIK1. KAR4 was also required for meiosis, during which it may regulate KAR3; however, mitotic expression of KAR3 and CIK1 during S/G2 phase was independent of KAR4. A 30-bp region upstream of KAR3 conferred both KAR4- and STE12-dependent induction by mating pheromone. This region contained one moderate and two weak matches to the consensus pheromone response element to which the Ste12p transcriptional activator binds and five repeats of the sequence CAAA(A). Overproduction of Ste12p suppressed the kar4 defect in KAR3 induction and nuclear fusion. In contrast, Ste12p-independent expression of Kar4p did not alleviate the requirement for Ste12p during KAR3 induction. We propose that Kar4p assists Ste12p in the pheromone-dependent expression of KAR3 and CIK1. KAR4 defines a novel level of regulation for the pheromone response pathway, acting at a subset of Stel2p-inducible genes required for karyogamy.