Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.     

Improved production of A40926 by Nonomuraea sp. through deletion of a pathway-specific acetyltransferase

Nonomuraea strain ATCC 39727 produces the glycopeptide A40926, used for manufacturing dalbavancin, currently in advanced clinical trials. From the gene cluster involved in A40926 biosynthesis, a strain deleted in dbv23 was constructed. This mutant can produce only the glycopeptides lacking the O-linked acetyl residue at position 6 of the mannose moiety, while, under identical fermentation conditions, the wild-type strain produces mostly glycopeptides carrying an acetylated mannose. Furthermore, the total amount of glycopeptides produced by the mutant strain was found to be approximately twice that of the wild type. The reduced level of glycopeptides observed in the wild-type strain may be due to an inhibitory effect exerted by the acetylated compound on the biosynthesis of A40926. Indeed, spiking production cultures with ≥1 μg/ml of the acetylated glycopeptide inhibited A40926 production in the mutant strain.     

Protein profiles of Streptomyces aureofaciens producing tetracyclines: Reappraisal of the effect of benzyl thiocyanate

Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 m benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.     

Effects of zinc, iron, cobalt, and manganese on Fusarium moniliforme NRRL 13616 growth and fusarin C biosynthesis in submerged cultures.

The influence of zinc, iron, cobalt, and manganese on submerged cultures of Fusarium moniliforme NRRL 13616 was assessed by measuring dry weight accumulation, fusarin C biosynthesis, and ammonia assimilation. Shake flask cultures were grown in a nitrogen-limited defined medium supplemented with various combinations of metal ions according to partial-factorial experimental designs. Zinc (26 to 3,200 ppb [26 to 3,200 ng/ml]) inhibited fusarin C biosynthesis, increased dry weight accumulation, and increased ammonia assimilation. Carbohydrate was found to be the principal component of the increased dry weight in zinc-supplemented cultures. Zinc-deficient cultures synthesized more lipid and lipidlike compounds, such as fusarin C, than did zinc-supplemented cultures. Microscopic examination showed that zinc-deficient hyphae contained numerous lipid globules which were not present in zinc-supplemented cultures. Addition of zinc (3,200 ppb) to 2- and 4-day-old cultures inhibited further fusarin C biosynthesis but did not stimulate additional dry weight accumulation. Iron (10.0 ppm) and cobalt (9.0 ppm) did not affect fusarin C biosynthesis or dry weight accumulation. Manganese (5.1 ppm) did not affect dry weight accumulation but did increase fusarin C biosynthesis in the absence of zinc. Maximum fusarin C levels, 32.3 micrograms/mg (dry weight), were produced when cultures were supplied manganese, whereas minimum fusarin C levels, 0.07 micrograms/mg (dry weight), were produced when zinc, iron, cobalt, and manganese were supplied. These results suggest a multifunctional role for zinc in affecting F. moniliforme metabolism.     

Effects of zinc, iron, cobalt, and manganese on Fusarium moniliforme NRRL 13616 growth and fusarin C biosynthesis in submerged cultures

The influence of zinc, iron, cobalt, and manganese on submerged cultures of Fusarium moniliforme NRRL 13616 was assessed by measuring dry weight accumulation, fusarin C biosynthesis, and ammonia assimilation. Shake flask cultures were grown in a nitrogen-limited defined medium supplemented with various combinations of metal ions according to partial-factorial experimental designs. Zinc (26 to 3,200 ppb [26 to 3,200 ng/ml]) inhibited fusarin C biosynthesis, increased dry weight accumulation, and increased ammonia assimilation. Carbohydrate was found to be the principal component of the increased dry weight in zinc-supplemented cultures. Zinc-deficient cultures synthesized more lipid and lipidlike compounds, such as fusarin C, than did zinc-supplemented cultures. Microscopic examination showed that zinc-deficient hyphae contained numerous lipid globules which were not present in zinc-supplemented cultures. Addition of zinc (3,200 ppb) to 2- and 4-day-old cultures inhibited further fusarin C biosynthesis but did not stimulate additional dry weight accumulation. Iron (10.0 ppm) and cobalt (9.0 ppm) did not affect fusarin C biosynthesis or dry weight accumulation. Manganese (5.1 ppm) did not affect dry weight accumulation but did increase fusarin C biosynthesis in the absence of zinc. Maximum fusarin C levels, 32.3 micrograms/mg (dry weight), were produced when cultures were supplied manganese, whereas minimum fusarin C levels, 0.07 micrograms/mg (dry weight), were produced when zinc, iron, cobalt, and manganese were supplied. These results suggest a multifunctional role for zinc in affecting F. moniliforme metabolism.     

Genetic characterization of the carotenoid biosynthetic pathway in Methylobacterium extorquens AM1 and isolation of a colorless mutant

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.     

Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: significance of the stringent response (ppGpp) and GTP content in relation to A factor.

I investigated the significance of the intracellular accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and of the coordinated decrease in the GTP pool for initiating morphological and physiological differentiation of Streptomyces griseus, a streptomycin-producing strain. In solid cultures, aerial mycelium formation was severely suppressed by the presence of excess nutrients. However, decoyinine, a specific inhibitor of GMP synthetase, enabled the cells to develop aerial mycelia in the suppressed cultures at concentrations which only partially inhibited growth. A factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone) added exogenously had no such effect. Decoyinine was also effective in initiating the formation of submerged spores in liquid culture. The ability to produce streptomycin did not increase but decreased drastically on the addition of decoyinine. This sharp decrease in streptomycin production was accompanied by a decrease in intracellular accumulation of ppGpp. A relaxed (rel) mutant was found among 25 thiopeptin-resistant isolates which developed spontaneously. The rel mutant had a severely reduced ability to accumulate ppGpp during a nutritional shift-down and also during postexponential growth and showed a less extensive decrease in the GTP pool than that in the rel+ parental strain. The rel mutant failed to induce the enzymes amidinotransferase and streptomycin kinase, which are essential for the biosynthesis of streptomycin. The abilities to form aerial mycelia and submerged spores were still retained, but the amounts were less, and for both the onset of development was markedly delayed. The decreased ability to produced submerged spores was largely restored by the addition of decoyinine. This was accompanied by an extensive GTP pool decrease. The rel mutant produced A factor normally, indicating that synthesis of A factor is controlled neither by ppGpp nor by GTP. Conversely, a mutant defective in A-factor synthesis accumulated as much ppGpp as did the parental strain. It was concluded that morphological differentiation of S. griseus results from a decrease in the pool of GTP, whereas physiological differentiation results from a more direct function of the rel gene product (ppGpp). It is also suggested that A factor may render the cell sensitive to receive and respond to the specified signal molecules, presumably ppGpp (for physiological differentiation) or GTP (for morphological differentiation).     

Isolation and morphological characterization of a conidia-formingClaviceps paspali mutant

A morphological mutant ofClaviceps paspali can produce a significant amount of conidia in the production phase of the fermentation process. The mutant was isolated after γ- irradiation of a high-production mycelial strain. Differences in the morphology between the isolate and the parent strain were significant especially in submerged cultures. It was also proved that conidiation is not favorable for alkaloid production which was markedly reduced.     

Growth Inhibition of Streptomyces Species by l-Serine and Its Effect on Tetracycline Biosynthesis

The addition of serine to minimal medium inhibited the growth of Streptomyces aureofaciens and Streptomyces rimosus. Both the outgrowth of spores and the growth of vegetative cells were inhibited by l-serine. This effect was independent of the carbon source used. In rich nutrient medium, however, the serine effect was not observed. The presence of glycine and methionine in minimal medium reversed the growth inhibition imposed by serine, suggesting that a metabolic block related to the synthesis of these two amino acids was involved. A serine-tolerant mutant of S. aureofaciens isolated after ultraviolet irradiation showed a level of serine deaminase comparable to that of the wild-type strain, which indicated that tolerance to serine was not due to the presence of a more active deaminating enzyme in the mutant. Serine markedly reduced tetracycline and oxytetracycline biosynthesis with the parental strains of Streptomyces spp. The serine-tolerant mutant, however, produced almost the same amount of tetracycline in the presence or absence of serine. The final cell population in fermentation broth was not significantly reduced by l-serine, and the addition of glycine and methionine did not increase the tetracycline yields, which suggested that l-serine inhibition of antibiotic biosynthesis was by a mechanism different from that related to growth inhibition.     

Isolation and characterization of phytase isozymes produced by Aspergillus oryzae

A mutant strain (KL-38) of Aspergillus oryzae was obtained by UV irradiation. Phytase activity of KL-38 in molded rice (koji rice) was about 2.7-fold of that obtained from the parent strain (BP-1). Phytase activity of KL-38 in the submerged culture was similar to that of BP-1. Two types of phytase were produced from koji culture: phytase I (Phy I) was produced during incubation of both koji and submerged cultures, and phytase II (Phy II) was obtained only from koji culture. Phy II production was increased in KL-38 compared with BP-1, whereas the production of Phy I was similar for both KL-38 and BP-1. This finding indicates that A. oryzae has at least two types of phytase isozyme.     

Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities.

Pseudomonas aeruginosa is considered a strict aerobe that possesses several enzymes important in the disposal of toxic oxygen reduction products including iron- and manganese-cofactored superoxide dismutase and catalase. At present, the nature of the regulation of these enzymes in P. aeruginosa Is not understood. To address these issues, we used two mutants called A4 and C6 which express altered Fur (named for ferric uptake regulation) proteins and constitutively produce the siderophores pyochelin and pyoverdin. Both mutants required a significant lag phase prior to log-phase aerobic growth, but this lag was not as apparent when the organisms were grown under microaerobic conditions. The addition of iron salts to mutant A4 and, to a greater extent, C6 cultures allowed for an increased growth rate under both conditions relative to that of bacteria without added iron. Increased manganese superoxide dismutase (Mn-SOD) and decreased catalase activities were also apparent in the mutants, although the second catalase, KatB, was detected in cell extracts of each fur mutant. Iron deprivation by the addition of the iron chelator 2,2'-dipyridyl to wild-type bacteria produced an increase in Mn-SOD activity and a decrease in total catalase activity, similar to the fur mutant phenotype. Purified wild-type Fur bound more avidly than mutant Fur to a PCR product containing two palindromic 19-bp "iron box" regions controlling expression of an operon containing the sodA gene that encodes Mn-SOD. All mutants were defective in both ferripyochelin- and ferripyoverdin-mediated iron uptake. Two mutants of strain PAO1, defective in pyoverdin but not pyochelin biosynthesis, produced increased Mn-SOD activity. Sensitivity to both the redox-cycling agent paraquat and hydrogen peroxide was greater in each mutant than in the wild-type strain. In summary, the results indicate that mutations in the P. aeruginosa fur locus affect aerobic growth and SOD and catalase activities in P. aeruginosa. We postulate that reduced siderophore-mediated iron uptake, especially that by pyoverdin, may be one possible mechanism contributing to such effect.     

Studies on biosynthesis of hydrolases by trichoderma sp. M7 on submerged and solid-state cultivation conditions

The biosynthesis of cellulases and xylanase by the mould strain Trichoderma sp. M₇ on submerged and solid-state cultivation conditions has been studied. The effect of different inducers on the enzyme biosynthesis on the conditions used was determined. The relation between the enzyme biosynthesis and the morphological state of the producing strain was studied. The advantages of the submerged cultivation conditions with regards to the efficiency of the enzyme inducer are shown.     

Fatty acid and lipid composition in mycelia from submerged or surface culture of Streptomyces viridochromogenes

Abstract Streptomyces viridochromogenes was grown both as submerged and surface culture. Mycelia from these cultures were analysed for the composition of lipids and fatty acids. An increase in ornithinolipid content according to incubation time was observed. The addition of phosphate inhibited the ornithinolipid synthesis. A mutant strain with bald phenotype did not exhibit the phosphate inhibition. At the same time, the mutant strain had a higher content of 12-methyltetradecanoic acid.     

Dependence of the hydrolytic exoenzyme production level on the way of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> culturing

An 867 mutant has been derived from a B. subtilis 1560 strain capable of biofilm formation during stationary culturing. The mutant was able to form more elastic and resistant to mechanical stress biofilms that could be used in fermentation more than once. Analysis of the hydrolytic activity of the 1560 and 867 strains film and submerged cultures showed that the biofilm culturing permitted to enhance the levels of amylase by 1.5–2.4 times and that of glucanase by three times.     

Citric acid fermentation by the mutant strain of theAspergillus niger resistant to manganese ions inhibition

Summary A new mutant strain,Aspergillus niger GS-III, showing resistance to manganese ions inhibition of citric acid fermentation on a sugarcane molasses containing medium was induced fromAspergillus niger KCU 520, a high citric acid-yielding strain. In submerged, surface or continuous cultures in the presence of manganese ions concentration upto 1.5 ppm the mutant strain yielded citric acid about 90 KgM^–3 . The citric acid yield was comparable to that obtained with the parental strain KCU 520 in the absence of manganese ions, but it was atleast 3-fold higher than that obtained by the latter in the presence of manganese ions. The mutant strain immobilized in calcium alginate beads was used in combination with surface-stabilized cultures for about 36-days in a continuous flow horizontal fermenter without any apparent loss in citric acid productivity. These results indicate that the manganese-resistant mutant is stable and may be used in the presence of sufficient manganese ions concentration (1.5 ppm) in the fermentation medium. This capability of the mutant strainA. niger GS-III has been correlated with greatly reduced levels (about one-thirds) of the NADP⁺ -isocitric dehydrogenase, one of the control points for citric acid accumulation.     

A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid)

A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl₄) transformation ability of wild-type KC. Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl₄ dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spe I fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt⁻) phenotype. One cosmid (pT31) complemented the Pdt⁻ phenotype of CTN1 and conferred CCl₄ transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25 746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.     

Effect of some broad-spectrum antibiotics on the high-production strainClaviceps fusiformis W1

Summary The effect of chloramphenicol, streptomycin, oxytetracycline and tetracycline on growth intensity and production of clavine alkaloids in submerged cultures ofClaviceps fusiformis W1 was studied. Chloramphenicol (70–275 g/mL) is suitable for prevention of bacterial contaminations of saprophyticClaviceps cultures and has no negative effect on the magnitude and spectrum of alkaloid production.     

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate.     

Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii

A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-β-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-β-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.     

Effect of aeration efficiency and carbon source on the production of anthracyclines inStreptomyces galilaeus

Biosynthesis of anthracyclines inStreptomyces galilaeus during submerged cultivation is considerably influenced by aeration and by the concentration of glucose in the medium. At higher values of oxygen absorption rate both the production of ε-pyrromycinone glycosides in the wild strain JA 3043 and its production mutant G-167 and accumulation of free ε-pyrromycinone in the blocked mutant G-162 were found to be higher; the production of 7-deoxyaglycones was lower in all strains. The studied strains differed in the rate of glucose consumption and in the ability to utilize starch for the biosynthesis of anthracyclines. A two-fold concentration of glucose in the medium resulted in the G-162 strain in an increase of the yield of ε-pyrromycinone by 120 %. The production of glycosides in strain G-167 increased even after exhaustion of glucose from the medium and the amount of 7-deoxyaglycones simultaneously decreased.