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,

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Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc–FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.

The catB and catC genes encode cis,cis-muconate lactonizing enzyme I (EC 5.5.1.1) and muconolactone isomerase (EC 5.3.3.4), respectively. These enzymes are required for the dissimilation of benzoate to 13-ketoadipate by Pseudomonas putida and are under coordinate transcriptional regulation. By deletion analysis and the use of pKT240 as a promoter probe vector, we located a single promoter region for the catBC operon upstream of catB. RNA-DNA hybridization studies, together with reverse transcriptase mapping, demonstrated that this promoter must be activated in the presence of an inducer molecule for effective transcription of the operon. In addition, the transcription initiation site was located 64 base pairs upstream of the catB initiation codon, and sequences upstream of -43 were required for promoter function. The catBC promoter was compared with other positively regulated procaryotic promoters to identify possible consensus sequences.Pseudomonads and other soil microorganisms are able to utilize a variety of natural and synthetic compounds as sole sources of carbon. Their ability to dissimilate synthetic chlorinated compounds such as 3-chlorobenzoic acid, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid to nontoxic components (12) makes them attractive candidates for use in the environment to detoxify chemical wastes. Presently, there is no safe, economical means available for the removal of such hazardous chemicals. Having detailed information about the structural and regulatory genes involved in degradative processes increases the likelihood that organisms with these capabilities can be used for the purpose of chemical detoxification and gain approval for release into the environment.

A long open reading frame (ORF) closely linked to the Cephalosporium acremonium gene cefEF was identified by DNA sequencing. The cefEF gene encodes the enzyme involved in cephalosporin C (CPC) biosynthesis known as expandase/hydroxylase. Complementation of a C. acremonium cefG mutant, as well as expression of the gene in Aspergillus niger, showed this ORF to be the cefG gene, encoding cephalosporin C acetyltransferase, which catalyzes the last step in CPC biosynthesis. Analysis of transformants containing additional copies of this gene showed that a direct relationship exists between cefG copy number, cefG message levels, and CPC titers. This gene encodes an enzyme for what may be a rate-limiting step in CPC production.

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