Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.

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Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli.

Streptomycetes are Gram-positive bacteria with a unique capacity for the production of a multitude of varied and complex secondary metabolites They also have a complex life cycle including differentiation into at least three distinct cell types Whilst much attention has been paid to the pathways and regulation of secondary metabolism, less has been paid to the pathways and the regulation of primary metabolism, which supplies the precursors With the imminent completion of the total genome sequence of Streptomyces coelicolor A3(2), we need to understand the pathways of primary metabolism if we are to understand the role of newly discovered genes This review is written as a contribution to supplying these wants Streptomycetes inhabit soil, which, because of the high numbers of microbial competitors, is an oligotrophic environment Soil nutrient levels reflect the fact that plant-derived material is the main nutrient input; ie it is carbon-rich and nitrogen- and phosphate-poor Control of streptomycete primary metabolism reflects the nutrient availability The variety and multiplicity of carbohydrate catabolic pathways reflects the variety and multiplicity of carbohydrates in the soil This multiplicity of pathways has led to investment by streptomycetes in pathway-specific and global regulatory networks such as glucose repression The mechanism of glucose repression is clearly different from that in other bacteria Streptomycetes feed by secreting complexes of extracellular enzymes that break down plant cell walls to release nutrients The induction of these enzyme complexes is often coordinated by inducers that bear no structural relation to the substrate or product of any particular enzyme in the complex; eg a product of xylan breakdown may induce cellulase production Control of amino acid catabolism reflects the relative absence of nitrogen catabolites in soil The cognate amino acid induces about half of the catabolic pathways and half are constitutive There are reduced instances of global carbon and nitrogen catabolite control of amino acid catabolism, which again presumably reflects the relative rarity of the catabolites There are few examples of feedback repression of amino acid biosynthesis Again this is taken as a reflection of the oligotrophic nature of the streptomycete ecological niche As amino acids are not present in the environment, streptomycetes have rarely invested in feedback repression Exceptions to this generalization are the arginine and branched-chain amino acid pathways and some parts of the aromatic amino acid pathways which have regulatory systems similar to Escherichia coli and Bacillus subtilis and other copiotrophic bacteria

Primary metabolism and its control in streptomycetes: a most unusual group of bacteria.

Although microorganisms are extremely good in presenting us with an amazing array of valuable products, they usually produce them only in amounts that they need for their own benefit; thus, they tend not to overproduce their metabolites. In strain improvement programs, a strain producing a high titer is usually the desired goal. Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by mutagenesis and screening/selection for higher producing microbial strains and the application of recombinant DNA technology.

Genetic improvement of processes yielding microbial products

Hormone-sensitive lipase is closely related to several bacterial proteins, and distantly related to acetylcholinesterase and lipoprotein lipase: Identification of a superfamily of esterases and lipases

Various aerobic Gram-negative bacteria have been examined for their ability to use 4-hydroxybutyrate and 1,4-butanediol as carbon source for growth. Alcaligenes eutrophus strains H16, HF39, PHB-4 and Pseudomonas denitrificans 'Morris' were not able to grow with 1,4-butanediol or 4-hydroxybutyrate. From A. eutrophus HF39 spontaneous primary mutants (e.g. SK4040) were isolated which grew on 4-hydroxybutyrate with doubling times of approximately 3 h. Tn5::mob mutagenesis of mutant SK4040 led to the isolation of two phenotypically different classes of secondary mutants which were affected in the utilization of 4-hydroxybutyrate. Mutants exhibiting the phenotype 4-hydroxybutyrate-negative did not grow with 4-hydroxybutyrate, and mutants exhibiting the phenotype 4-hydroxybutyrate-leaky grew at a significantly lower rate with 4-hydroxybutyrate. Hybridization experiments led to the identification of a 10-kbp genomic EcoRI fragment of A. eutrophus SK4040, which was altered in mutants with the phenotype 4-hydroxybutyrate-negative, and of two 1-kbp and 4.5-kbp genomic EcoRI fragments, which were altered in mutants with the phenotype 4-hydroxybutyrate-leaky. This 10-kbp EcoRI fragment was cloned from A. eutrophus SK4040, and conjugative transfer of a pVDZ'2 hybrid plasmid to A. eutrophus H16 conferred the ability to grow with 4-hydroxybutyrate to the wild type. DNA-sequence analysis of this fragment, enzymic analysis of the wild type and of mutants of A. eutrophus as well as of recombinant strains of Escherichia coli led to the identification of a structural gene encoding for a 4-hydroxybutyrate dehydrogenase which was affected by transposon mutagenesis in five of six available 4-hydroxybutyrate-negative mutants. Enzymic studies also provided evidence for the presence of an active succinate-semialdehyde dehydrogenase in 4-hydroxybutyrate-grown cells. This indicated that degradation of 4-hydroxybutyrate occurs via succinate semialdehyde and succinate and that the latter is degraded by the citric acid cycle. NMR studies of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) accumulated from 4-hydroxy [1-13C]butyrate or 4-hydroxy[2-13C]butyrate as substrate gave no evidence for a direct conversion of 4-hydroxybutyrate into 3-hydroxybutyrate and therefore supported the results of enzymic analysis.

Metabolic Pathway for Biosynthesis of Poly (3-Hydroxybutyrate-co-4-Hydroxybutyrate) from 4-Hydroxybutyrate by Alcaligenes eutrophus

A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5′ termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus.

Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1.

SUMMARY: This work describes isolation and characterization of Streptomyces clavuligerus mutants resistant to the lysine analogue S-(2-aminoethyl)-L-cysteine (AEC). The mutation to AEC resistance was shown to affect the feedback regulation of aspartokinase; 70% of the mutants isolated had aspartokinase activity insensitive to concerted feedback inhibition by lysine plus threonine. Among these mutants, 70% (about 50% of the total AEC-resistant strains isolated) showed significant overproduction of β-lactam antibiotics.

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Simona Mendelovitz

Papers

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus.

Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1.

beta-lactam antibiotic production by Streptomyces clavuligerus mutants impaired in regulation of aspartokinase.