Cell walls from Actinopolyspora halophila,an extremely halophilic actinomycete

Cell wall components were prepared from Actinopolyspora halophila (strain wt), an extremely halophilic actinomycete requiring a minimum 12% NaCl concentration for growth, and from an erythromycin-resistant strain of A. halophila (strain ER) that required only 6% NaCl for growth. Both cell wall preparations contained glutamic acid, alanine, and diaminopimelic acid in a 1:2:1 molar ratio. On the basis of muramic acid content, peptidoglycans from the wt and ER strains contained 255 and 245 disaccharide units per mg dry weight respectively. In addition, both cell wall preparations contained from 10 to 20% more glucosamine than muramic acid, and equimolar amounts of d-galactose and d-arabinose. Analysis of cell walls before and after digestion with Myxobacter AL-1 protease indicated that nearly all glycan disaccharide units were peptide-substituted and that peptide cross-bridging was facilitated by direct peptide linkages between N-diaminopimelic acid and C-terminal alanine. While the peptidoglycan of A. halophila wt was 50% peptide cross-linked, that from A. halophila ER was approximately 67% peptide cross-linked. Chemical modifications involving substitution of non-N-acetylated hexosamines of the cell walls greatly enhanced their sensitivity to lysozyme. Although differences in peptidoglycan structure between the two strains of A. halophila were observed, these probably do not account for the reduced salt requirement for growth of the erythromycin-resistant strain.Issued as NRCC 25165     

β-Methylmuconolactone, a key intermediate in the dissimilation of methylaromatic compounds by a modified 3-oxoadipate pathway evolved in nocardioform actinomycetes

Abstract p -Toluate-grown cells of Rhodococcus ruber N75, R. corallinus N657, R. rhodochrous N5 and Rhodococcus strains BCN1, BCN2 and 4PH1 metabolized 4-methylcatechol by a modified 3-oxoadipate pathway. Steps in the conversion of this compound to 4-methyl-3-oxoadipic acid were investigated. The conversion of 4-carboxymethyl-3-methylbut-2-en-1, 4-olide to 4-carboxymethyl-3-methylbut-2-en-1, 4-olide by a new enzyme is described.     

Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins

The regulation of nitrogen assimilation was investigated in the Gram-positive actinomycete Corynebacterium glutamicum. Biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. The genes encoding the central signal transduction protein PH (glnB) and the primary nitrogen sensor uridylyltransferase (glnD) were isolated and sequenced. Additionally, genes putatively involved in the degradation of ornithine (ocd) and sarcosine (soxA), ammonium uptake (amtP) and protein secretion (ftsY, srp) were identified in C. glutamicum. Based on these observations, the mechanism of N regulation in C. glutamicum is similar to that of the Gram-negative Escherichia coli. As deduced from data base searches, the described regulation may also hold true for the important pathogen Mycobacterium glutamicum.     

Amylolytic activity of Thermomonospora fusca

Amylases which produce maltotriose as the major end-product from starch are relatively rare. The thermophilic actinomycete, Thermomonospora fusca, produced an extracellular -amylase which generated maltotriose as 61% of the identified products. The addition of maltotriose to a glucose-adapted exponential phase culture at 55°C in mineral salts medium caused rapid induction of amylase biosynthesis. Addition of glucose to cells growing on starch did not repress amylase biosynthesis because the actinomycete had a marked preference for maltotriose over glucose. The pH and temperature optima for the amylase activity of concentrated, washed extracellular protein were 6.0 and 65°C, respectively, with an energy of activation of 59kJ/mol. The thermostability of the concentrated, washed amylase was increased by the presence of its starch reaction products, but not by added Ca2+.     

Bioconversion of the sodium salt of Simvastatin (MK-733) to 6-desmethyl-6-α-hydroxymethyl Simvastatin

Summary An actinomycete (MA 6474, ATCC 53828) isolated from a soil sample (Mutare, Zimbabwe) was found to biotransform the sodium salt of Simvastatin (MK-733) to 6--hydroxymethyl MK-733, 6--hybroxymethyl MK-733, and 6-ring-hydroxy MK-733. The bioconversion efficiency to the desired compound, 6--hydroxymethyl MK-733, was enhanced by optimizing the physico-chemical parameters of the process. In shake flask cultures, addition of magnesium (0.125 mg/l Mg SO₄·7H₂O) to the medium resulted in a five-fold increase in the rate of bioconversion to the diastereomer. The ratio of bioconversion products (6--hydroxymethyl, 6--hydroxymethyl, and 6-ring-hydroxy MK-733) was regulated by pH. Process improvements and scale up in 23-1 fermentors, which consisted of a controlled addition of substrate (MK-733), resulted in a 2-fold increase in alpha diastereomer Production (42 vs. 79 U/ml) and a 23-fold rate increase in the formation of -diastereomer. A high diastereomeric ratio (: =91) facilitated downstream processing.     

Metabolic engineering of Amycolatopsis japonicum for optimized production of [S,S]-EDDS,a biodegradable chelator

The actinomycete Amycolatopsis japonicum is the producer of the chelating compound [S,S]-ethylenediamine-disuccinc acid (EDDS). [S,S]-EDDS is an isomer of ethylenediamine-tetraacetic acid (EDTA), an economically important chelating compound that suffers from an extremely poor degradability. Frequent use of the persistent EDTA in various industrial and domestic applications has caused an accumulation of EDTA in soil as well as in aqueous environments. As a consequence, EDTA is the highest concentrated anthropogenic compound present in water reservoirs. The [S,S]-form of EDDS has chelating properties similar to EDTA, however, in contrast to EDTA it is readily biodegradable. In order to compete with the cost-effective chemical synthesis of EDTA, we aimed to optimize the biotechnological production of [S,S]-EDDS in A. japonicum by using metabolic engineering approaches. Firstly, we integrated several copies of the [S,S]-EDDS biosynthetic genes into the chromosome of A. japonicum and replaced the native zinc responsive promoter with the strong synthetic constitutive promoter SP44*. Secondly, we increased the supply of O-phospho-serine, the direct precursor of [S,S]-EDDS. The combination of these approaches together with the optimized fermentation process led to a significant improvement in [S,S]-EDDS up to 9.8 g/L with a production rate of 4.3 mg/h/g DCW.     

A new actinomycete strain SK4-6 producing secondary metabolite effective against methicillin-resistant Staphylococcus aureus

A new actinomycete strain designated SK4-6, was isolated. This organism exhibited strong activity against bacteria including methicillin-resistant Staphylococcus aureus and Micrococcus luteus, in addition to the causative agents of Candidiasis and Aspergillosis diseases, Candida albicans and Aspergillus species respectively. Morphological and chemical studies indicated that this organism belongs to the genus Streptomyces. Analysis of the 16S rRNA sequence of strain SK4-6 showed a high similarity, 99%, with S. qinlingensis. Optimization of cultural conditions was carried out using Plackett–Burman statistical design where seven variables were examined. Starch, pH and inoculum size showed a positive effect on the production of the active substances, however, sucrose, (NH₄)₂SO₄ and yeast extract repress the production.     

Isolation and analysis of mutants of the methylotrophic actinomycete Amycolatopsis methanolica blocked in aromatic amino acid biosynthesis

Abstract Mutants of the actinomycete Amycolatopsis methanolica blocked in aromatic amino acid biosynthesis were isolated using brief ultrasonic treatments to obtain single cells. After UV irradiation, auxotrophic mutants were selected as pinpoint colonies on mineral agar with only 1 mg 1⁻¹ of amino acid supplements. Mutant characterization provided unambiguous evidence that l-tyrosine is synthesized via arogenate and that l-phenylalanine is synthesized via phenylpyruvate. The efficiency of chromosomal DNA marker exchange was highest in matings with mutant strains that lacked the previously characterized 13.3-kb integrative plasmid pMEA300.     

Mutational analysis of primary alcohol metabolism in the methylotrophic actinomycete Amycolatopsis methanolica

Abstract Mutants of the methylotrophic actinomycete Amycolatopsis methanolica unable to grow on methanol as carbon source were isolated and characterized. Mutants specifically affected in methanol utilization were deficient in formaldehyde assimilation. Mutants blocked in the first step of primary alcohol oxidation (C1–C4) had lost activity of the tetrazolium-dependent alcohol dehydrogenase, a three-component enzyme complex. This complex, or individual components, thus play a crucial role in utilization of primary alcohols in A. methanolica .