The results show that the nonaketide and diketide portions of lovastatin are synthesized by separate large multifunctional PKSs. Elucidation of the primary structure of the PKS that forms the lovastatin nonaketide, as well as characterization of blocked mutants, provides new details of lovastatin biosynthesis.

Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source. This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon. The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies. However, the inducer is required for transcriptional activation of the catBC operon. The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240. This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start. Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression. Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family. Striking homology was seen between CatR and a putative regulatory protein, TfdS.

CatR, a LysR family protein, positively regulates the Pseudomonas putida catBC operon, which is required for growth on benzoate as a sole carbon source. Transcriptional studies show that the catR and catBC promoters are divergent and overlapping by 2 bp. A I8-galactosidase promoter probe vector was constructed to analyze expression from the catR and catBC promoters under induced and uninduced conditions. As predicted, the catBC promoter is expressed only under induced conditions, while the catR promoter is constitutive. CatR has been shown to specifically bind the catRBC promoter region, and this property was used to devise a purification protocol for CatR. Linear M13 DNA containing the catRBC control region was covalently bound to cyanogen bromide-activated Sepharose in order to construct a DNA ainity column. Crude extracts containing hyperproduced CatR protein were then incubated with the affinity resin under binding conditions, and the CatR protein was eluted with 1 M NaCl. CatR was also purified by heparin-agarose chromatography. This highly purified protein was used for gel retardation and hydroxyl-radical footprinting studies. From this analysis, it was shown that CatR binds upstream of the catBC promoter within the transcribed region of catR.Pseudomonads utilize many natural and man-made organic compounds. In order to develop strains capable of dissimilating recalcitrant compounds such as chlorinated aromatics, it is important to understand how biodegradative pathways evolve in nature and how they are regulated. Pseudomonads provide a good model for studying how degradative pathways evolve in order to expand the substrate range of a microorganism so that it may degrade more complex and toxic compounds. A simple model used for this analysis has been Pseudomonas putida, which is capable of utilizing benzoate and can also degrade 3-chlorobenzoate when harboring plasmid pAC27 (10). While the structural genes encoding enzymes for the dissimilation of benzoate and 3-chlorobenzoate have been fairly well characterized (1,2,13,15,43), the mechanism for regulation of these genes is only now being investigated.The catBC operon encodes the genes for two enzymes: cis,cis-muconate lactonizing enzyme I (EC 5.5.1.1) and muconolactone isomerase (EC 5.3.3.4), respectively. Both of these genes are required for the dissimilation of benzoate (28). The operon is coordinately regulated and requires the product of a regulatory gene for induction (42). As described previously (32), the regulatory gene catR maps upstream of the catBC operon and is divergently transcribed from the catBC operon (Fig. 1). The catR gene encodes a 32.2-kDa polypeptide that binds to the catBC promoter region in vitro in the presence or absence of the inducer cis,cis-muconate. The inducer, however, is required for in vivo transcriptional activation of the catBC operon. CatR was shown to be a member of a large class of procaryotic regulatory proteins,