Fatty acid biosynthesis in actinomycetes

All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation for elucidating the type II FAS pathways in other bacteria (White et al., 2005). However, fatty acid biosynthesis is more diverse in the phylum Actinobacteria: Mycobacterium, possess both FAS systems while Streptomyces species have only the multienzyme FAS II system and Corynebacterium species exclusively FAS I. In this review, we present an overview of the genome organization, biochemical properties and physiological relevance of the two FAS systems in the three genera of actinomycetes mentioned above. We also address in detail the biochemical and structural properties of the acyl-CoA carboxylases (ACCases) that catalyzes the first committed step of fatty acid synthesis in actinomycetes, and discuss the molecular bases of their substrate specificity and the structure-based identification of new ACCase inhibitors with antimycobacterial properties.     

Approaches to intensification and prolongation of antibiotic biosynthesis

World Journal of Microbiology and Biotechnology -     

Extracellular protease activities in relation to xylanase secretion in an alkalophilic Bacillus sp.

The proteolytic activity of an alkalophilic Bacillus sp. (NCL 87-6-10) correlates with xylanase secretion. Addition of DL-norvaline, glycine or Casamino acids to a medium formulated for xylanase production resulted in 2-5-fold enhancement of xylanase secretion (8 to 45 IU/ml). Inhibition of proteolytic activity is a possible mechanism for enhanced xylanase activity.     

Lysine epsilon-aminotransferase, the initial enzyme of cephalosporin biosynthesis in actinomycetes

Streptomyces clavuligerus, Streptomyces lipmanii and Nocardia (formerly Streptomyces) lactamdurans are Gram-positive mycelial bacteria that produce medically important beta-lactam antibiotics (penicillins and cephalosporins including cephamycins) that are synthesized through a series of reactions starting from lysine, cysteine and valine. L-lysine epsilon-aminotransferase (LAT) is the initial enzyme in the two-step conversion of L-lysine to L-alpha-aminoadipic acid, a specific precursor of all penicillins and cephalosporins. Whereas S. clavuligerus uses LAT for cephalosporin production, it uses the cadaverine pathway for catabolism when lysine is the nitrogen source for growth. Although the cadaverine path is present in all examined streptomycetes, the LAT pathway appears to exist only in beta-lactam-producing strains. Genetically increasing the level of LAT enhances the production of cephamycin. LAT is the key rate-limiting enzyme in cephalosporin biosynthesis in S. clavuligerus strain NRRL 3585. This review will summarize information on this important enzyme.     

Plasmalemma structure in relation to microfibril biosynthesis in Oocystis

Summary The plasmalemma of Oocystis apiculata, W. West when freezeetched has been shown to bear granules of several sizes. At the earliest stage of development the outer face of the plasmalemma of the naked autospore has small (8.5 nm diameter) granules aligned in rows, in pairs. These rows are stacked together forming extensive granule-bands over the plasmalemma surface. The orientation of these granule-bands corresponds exactly to one of the major microfibril directions. Occasionally, the bands are reduced to patches, some of which are at right angles to each other. Banding of granules on the inner plasmalemma face of naked autospores is also seen. During development the plasmalemma is seen to change so that in the final stages it bears reticulate invaginations, the granule bands occurring within them. The significance of the granulebands in terms of cellulose microfibril biosynthesis is discussed.     

Lipid biosynthesis in relation to chloroplast development in barley

During greening of detached leaves from dark-grown barley seedlings, the linolenic acid content of the lipids increases in the early stages of the formation of the chloroplast lamellar system. Primarily the fraction containing monogalactosyl diglyceride is enriched with linolenic acid. Incorporation of (14)C-labeled acetate into the leaf lipids of detached whole leaves is low, but increases 10- to 20-fold during greening. Increasing percentages of label appear in linolenic acid during the first 15 hr of greening, whereafter they remain constant. A constant, relatively high amount of acetate is incorporated into lipids when slices of leaves at various stages of greening are incubated by submersion in acetate solution, a treatment that blocks further chlorophyll synthesis during incubation. At the initial greening stages 75% of the label is channeled into steroids and other unsaponifiable lipids, but in advanced stages of chloroplast development 75% of the incorporated acetate is built into phospho-, sulfo- and galacto-lipids, and only 25% is channeled into unsaponifiable lipids. Experimental variation of the physiological conditions of the tissue during incubation resulted in differences in the amount of label found in the various phospho- and galacto-lipids. The amounts of labeling of the individual fatty acids in the lipid classes studied differ markedly and could be changed by varying the conditions of incubation. Labeling of linolenic acid was found to be highest in the monogalactosyl diglyceride fraction at all stages of greening.     

Primary metabolism of Aspergillus parasiticus in relation to aflatoxin biosynthesis

Summary The uptake of various ¹⁴C labelled compounds like (1-¹⁴C) glucose, (1-¹⁴C) acetate, (2-¹⁴C) uracil, (1-¹⁴C) leucine and (¹⁴C–CH₃) methionine was studied in Aspergillus parasiticus. A comparative study of asparagine deficient, zinc deficient and SLS cultures revealed different growth patterns. High lipid levels under zinc and asparagine deficiency were observed. During the stationary phase the synthesis of proteins and DNA declined. The uptake of ¹⁴C labelled glucose, methionine and acetate was maximum in asparagine deficient cultures during the transitional and stationary phase of growth. Maximum uptake of labelled methionine and glucose occured during the exponential growth phase (45 h). The uptake of labelled leucine was highest under asparagine deficiency during the exponential and transitional phases but reached a minimum during stationary phase. The uptake of labelled uracil remained high throughout in the asparagine deficient cultures. The mechanism of inhibition of aflatoxin biosynthesis in the absence of zinc and asparagine seems to be different.     

In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes

Abstract The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched-chain fatty acids, which constitute the majority of the fatty acid pool, were confirmed to be biosynthesized using the amino acid degradation products methylbutyryl-CoA and isobutyryl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using butyryl-CoA as a starter unit. Isomerization of the valine catabolite isobutyryl-CoA was shown to be only a minor source of this butyryl-CoA.     

Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger

The production of extracellular glucose oxidase in a submerged culture by a number of auxotrophic, 2-deoxy-D-glucose resistant and protease-less mutants of Aspergillus niger was evaluated. Among the auxotrophic strains, no evident dependence was found between the kind of the nutritional requirements and the level of the glucose oxidase activity. However, the majority of auxotrophs, requiring serine or niacin, showed a higher enzyme activity (from 16 to 680%) than the parent strain. The dynamics of the glucose oxidase synthesis by the free and immobilized mycelium of the most active niacin^? mutant of A. niger was also investigated.     

Ethylene biosynthesis in oilseed rape pods in relation to pod shatter

Ethylene production was studied during the development and senescence of seeds and pericarp tissues of oilseed rape (Brassica napus L.) pods (siliquae). In the course of the rise to a pre-senescence climacteric, little change in 1-aminocyclopropane-1-carboxylic acid (ACC) was recorded in the seeds, indicating a rapid conversion to ethylene. In contrast, very small amounts of ethylene were produced by the pod wall (PW) tissues, which included the dehiscence zone (DZ), while levels of free and conjugated ACC in the PW increased consistently. As climacteric thylene production by the seeds declined, biosynthesis of ethylene by the PW increased. Effects of reducing ethylene production by various means were examined in relation to cell separation in the dehiscence zone. Aminoethoxyvinylglycine (AVG) applied during the pre-senescence climacteric reduced ACC levels and ethylene production by the seeds, but did not affect subsequent values in the PW. The production of -1,4-glucanase and the separation of the cells of the DZ were delayed for 3-4 d by AVG, but the force required to open fully mature pods was unaltered. In parthenocarpic (seedless) pods, ethylene was produced during senescence. Cell separation in the DZ took place as in seeded pods, although it was also delayed by 3-4 d. The results are related to changes in indole-3-acetic acid (IAA) levels in oilseed rape pods which decline in PW and DZ tissues during senescence. It is concluded that separation in the cells of the dehiscence zone requires only small amounts of ethylene to trigger the process when IAA levels are low.     

Proteoglycan biosynthesis in relation to differentiation of cord blood monocytes in vitro

Human monocytes were obtained from umbilical cord blood and cultured in vitro. By morphological criteria, the neonatal monocytes developed into macrophage-like cells in the course of 3-5 days in culture. The cells were exposed to [35S]sulphate for 24 h, either from day 0-1 or day 9-10 in vitro. The 35S-labelled macromolecules recovered were mainly associated with the medium fraction (approximately 75%) in both day 1 and day 10 cultures. These secretory macromolecules were demonstrated by the use of chondroitinase ABC-digestions to contain predominantly chondroitin sulphate proteoglycan (CSPG). [35S]galactosaminoglycan chains from day 10 cultures were more highly sulphated than the corresponding day 1 species due to the appearance of (glucuronosyl-4,6-diS-N-acetylgalactosamine) disulphated disaccharide units. The galactosaminoglycan chains in neonatal CSPG were found to increase in Mr during cultivation in vitro; from mean Mr of 20,400 to 30,200 (n = 5) in day 1 and day 10 medium proteoglycans, respectively. The corresponding Mr values for adult monocyte [35S]galactosaminoglycan chains were 21,300 and 22,800. On the basis of the concomitant changes in cellular morphology and glycosaminoglycan structure, it is concluded that neonatal monocytes, like monocytes from adults, differentiate into macrophage-like cells in vitro.     

Interaction between QTLs induces an advance in ethylene biosynthesis during melon fruit ripening

The coexistence of both climacteric and non-climacteric genotypes and the availability of a set of genetic and genomic resources make melon a suitable model for genetic studies of fruit ripening. We have previously described a QTL, ETHQB3.5, which induces climacteric fruit ripening in the near-isogenic line (NIL) SC3-5 that harbors an introgression on linkage group (LG) III from the non-climacteric melon accession PI 161375 in the, also non-climacteric cultivar, “Piel de Sapo” genetic background. In the current study, a new major QTL, ETHQV6.3, on LG VI was detected on an additional introgression in the same NIL. These QTLs are capable, individually, of inducing climacteric ripening in the non-climacteric background, the effects of ETHQV6.3 being greater than that of ETHQB3.5. The QTLs interact epistatically, advancing the timing of ethylene biosynthesis during ripening and, therefore, the climacteric responses. ETHQV6.3 was fine-mapped to a 4.5 Mb physical region of the melon genome, probably in the centromeric region of LG VI. The results presented will be of value in the molecular identification of the gene underlying ETHQV6.3