Improvement of industry-applied rifamycin B-producing strain,Amycolatopsis mediterranei,by rational screening

An industrially applied rifamycin B-producing strain, Amycolatopsis mediterranei XC 1-02, was used for further screening. A special mutation and screening procedure was adopted to select a strain, which can alleviate the inhibition caused by both aromatic amino acid and p-hydroxybenzoic acid in the pathway of rifamycin B biosynthesis as well as enhance the production of propionate, one of the precursors of rifamycin B biosynthesis. By the above methods, a strain A. mediterranei XC 9-25 was obtained, and its rifamycin B productivity in shaking flask reaches 10 g/L, which is 2.38 times higher than that of the ancestral strain XC 1-02. The productivity of rifamycin B fed-batch fermentation in 60000 L fermentor with A. mediterranei XC 9-25 reached 19.11 g/L.     

Rifamycin B oxidase from Monocillium spp., a new type of diphenol oxidase

It was found that enzyme from a microbial strain, Monocillium spp. ATCC 20621, catalyzed the oxidative reaction of rifamycin B to form rifamycin O. The identification of the reaction products suggested that the reaction proceeded by the oxidative cyclization of rifamycin B to give rifamycin O, which spontaneously hydrolyzed to rifamycin S in neutral aqueous milieu. The characteristic of the enzyme was different as compared with that of other polyphenol oxidases such as laccase. It is proposed that this new type of enzyme be classified into a subgroup EC 1.10.3.6 with a trivial name rifamycin B oxidase.     

Development of balanced medium composition for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3

Aim:  To develop optimum fermentation environment for enhanced rifamycin B production by isolated Amycolatopsis sp. RSP-3.
Methods and Results:  The impact of different fermentation parameters on rifamycin B production by isolated Amycolatopsis sp. RSP-3 was investigated using Taguchi methodology. Controlling fermentation factors were selected based on one variable at a time methodology. The isolated strain revealed more than 25% higher production compared to literature reports. Five different nutritional components (soyabean meal, glucose, potassium nitrate, calcium carbonate and barbital) and inoculum concentration showed impact on rifamycin B production at individual and interactive level. At optimized environment, 65% contribution was observed from selected fermentation parameters.
Conclusions:  Soyabean meal and calcium carbonate were the most significant factors among the selected factors followed by barbital and potassium nitrate. Glucose, however, showed the least significance on rifamycin B production with this strain. A maximum of 5·12 g l⁻¹ rifamycin B production was achieved with optimized medium containing (g l⁻¹) soyabean meal, 27; glucose, 100; potassium nitrate, 4; calcium carbonate, 3 and barbital, 1·2.
Significance and Impact of the Study:  The present study signifies identification of balanced medium component concentrations for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3. This strain requires organic and inorganic nitrogen sources for effective product yield. Yet at individual level, organic nitrogen source has c. nine-fold higher influence compared to inorganic one.     

Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.     

Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.     

Improvement of rifamycin B production using mutant strains of Amycolatopsis mediterranei

Two mutant strains of Amycolatopsis mediterranei VA17 and VA18 were isolated using physical (UV) and chemical (NTG) mutagens gave high rifamycin B than the parent type when grown in the same fermentation medium with a pH of 7.2, temperature 32v°C for a period of 12 days. The cultural conditions of both mutant strains are similar to the parent strain except temperature which was higher by 4v°C. By this mutation and selection study, rifamycin B production was improved from 1400 mg/l to 2450 mg/l.     

Oxidation of biphenyl by a Beijerinckia species

A species of Beijerinckia that utilizes biphenyl as sole source of carbon for growth was isolated by enrichment culture. A mutant strain, Beijerinckia B8/36, oxidizes biphenyl to cis-2, 3-dihydroxy-1-phenylcyclohexa-4, 6-diene. Cell extracts, prepared from the parent organism, oxidize cis-2, 3-dihydroxy-1-phenylcyclohexa-4, 6-diene to 2, 3-dihydroxy-biphenyl. The physical properties of both metabolites are described.     

Effect of low molecular weight regulators on the biosynthesis of rifamycin B by Amycolatopsis mediterranei strains

Formation of differentiation regulators of the A-factor group in representatives of Nocardia and Nocardia-like actinomyces: N. asteroides, N. brasiliensis, Amycolatopsis mediterranei and "Streptomyces listeri" was observed. The effect of the regulators of different nature (barbital, A-factor and B-factor) on biosynthesis of rifamycin B by A. mediterranei strains was studied. It was shown that the A-factor stimulated rifamycin B production in the adifferentiated low active variant isolated from a natural population of the active strain VNIIA 1713 of the rifamycin B-producing culture. B-Factor insignificantly inhibited biosynthesis of rifamycin B in the studied strains of A. mediterranei.     

Enzymatic conversion of rifamycin B in a rotating packed disk reactor

The biological conversion of rifamycin B to rifamycin S was attempted using immobilized rifamycin B oxidase in a rotating packed disk reactor (RPDR). Humicola sp. (ATCC 20620) was treated with acetone and the cell powder was immobilized with cellulose acetate. The three phase reaction involving oxygen, substrate, and the immobilized enzyme beads was done in the RPDR.The physical strength of the immobilized enzyme beads was very good and was suitable as packing material in RPDR. The optimum submergence in the RPDR was 0.5 and the rotating speed of the disk did not affect the conversion very much. In continuous operation as the residence time increased, the conversion of rifamycin B increased, but the productivity decreased.     

The properties of immobilized whole cell ofHumicola spp. with rifamycin oxidase activity

Summary The whole cell ofHumicola spp. ATCC 20620 with rifamycin oxidase activity was immobilized by copolymerization with acrylamide. The whole cell was defatted by treatment with acetone to reduce the diffusional resistance through the cell membrane. The recovery of enzyme activity after the immobilization step was about 50%. The acetone-defatted cell showed the maximum activity at pH 7.5 for both free and the immobilized forms. No appreciable activity loss could be detected when stored at 4 °C and pH 7.8 for one month, while the half life at 40 °C and pH 8 was decreased to about 8 days. The apparent Km values of rifamycin oxidase for the free and immobilized acetonedefatted cells were 0.3mM and 0.6mM, respectively. The enzyme demonstrated substrate inhibition, but the degree of substrate inhibition was different between two forms of the enzyme preparation. A complete substrate inhibition was observed for the immobilized cell, whereas the enzyme activity was partially inhibited at high substrate concentration in the acetone-defatted cells.     

Partial characterization of anEscherichia coll strain harboring a xylanase encoding plasmid

Summary The plasmid pRH271, harboring a xylanase gene cioned fromBacilius subtilis, has been transferred into a mutant ofE. coli SK2284 which allowed the release of part of the xylanase in the culture supernatant. Kinetic parameters of this recombinantE. coll strain were determined in microscale batch culture with and without the selective pressure of antibiotics. No significant difference in µ_max was observed for the nontransformedE. coli strain when compared to the recombinant strain. However, K₅ values for glucose were two times higher in the case of the recombinant strain. Preliminary study of xylanase production in a large batch farmenter was also described.     

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved.     

Solid state cultivation of Curvularia lunata for transformation of rifamycin B to S

Biotransformation of rifamycin B to rifamycin S using two strains of C. lunata namely NCIM 716 and NMU grown on various solid substrates viz., grass, paper, jowar/wheat straw, bran and bagasse was studied. Almost complete biotransformation efficiency of rifamycin B at 0. 06 mM concentration was observed within 24 hr. Among these two strains, C. lunata NMU showed 90% of biotransformation and higher rate of cellulose utilization on solid substrates vis-à-vis reference strain. Cellulase activity of both strains was also studied for exoglucanase, endoglucanase and beta-glucosidase. Column bioreactor studies with bagasse revealed further improvement in biotransformation efficiency of C. lunata NMU.     

Biological conversion of rifamycin B by live Humicola sp. cells immobilized in a dual hollow fiber bioreactor

The biological transformation from rifamycin B to rifamycin S was carried out with the live whole cells of Humicola sp., ATCC 20620, immobilized in a dual hollow fiber bioreactor (DHFBR). Humicola sp., inoculated in the DHFBR, proliferated successfully to a high density cell mass within the space between an outer silicone tubing and three inner polypropylene hollow fiber membranes. In order to control the cell growth a nitrogen deficient medium was fed. Conversion of rifamycin B continued for more than 30 d, whereas that of immobilized rifamycin B oxidase lasted only for 3 d in comparable conditions.In the DHFBR the volumetric productivity of rifamycin S was 0.65–1.03 mmol/(dm³ · h) with 60% conversion, while that in the rotating packed disk reactor was 0.27 mmol/(dm³ · h) with 40% conversion at a residence time of 0.5–1.5 h.     

The stability of the yeast plasmid pJDB248 depends on growth rate of the culture

Summary The stability of the plasmid pJDB 248 has been measured in theS. cerevisiae strain S150-2B growing in a chemostat under conditions of glucose limitation. It was found that reducing the growth rate of the culture led to a more rapid loss of the plasmid from the cells.     

Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens

Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and axenic cultures were established. Octopine or nopaline, respective of the strain type used for inoculation, was detected in tumorous cultures. Southern blot analyses demonstrated T-DNA integration by hybridization of DNA from tumors with tmr and nos gene probes. One clone of B. papyrifera produced tumors with a morphogenic character, unusual in calli of this species, generating viable shoots which did not synthesize opine.Abbreviations Cb Carbenicillin - Cf Cefotaxime - 2,4-D 2,4-Dichloro-phenoxyacetic acid     

2,3-Butanediol production from xylose and other hemicellulosic components by Bacillus polymyxa

In the xylose fermentation of Bacillus polymyxa strain 9035, best 2,3-butanediol yields were obtained with 1.0 % yeast extract, 4–6 % xylose, shaking at 125 rpm and incubation at 30°C. Under these conditions, mannose, galactose, L-arabinose, cellobiose, starch and glucose were readily metabolized and yielded significant amounts of diol. Diol production from xylan was also demonstrated. In addition, the screening of a number of B. polymyxa strains on xylose revealed that only strains 9031-1 and 9035 used xylose extensively and produced significant amounts of diol. The latter strain proved best under scaled-up conditions.NRCC #22775     

Isolation and characterization of a novel bacterium, <Emphasis Type="Italic">Sphingomonas bisphenolicum</Emphasis> strain AO1, that degrades bisphenol A

Bisphenol A (2,2-bis(4-hydroxyphenyl) propane, BPA), which is used as a synthetic resin material or a plasticizer, is a pollutant that␣possesses endocrine-disrupting activity. Bioremediation of BPA is used to decrease its polluting effects, and here we report a novel bacterial strain AO1, which is able to degrade BPA. This strain was isolated using enrichment cultivation from a soil sample from a vegetable-growing field; the sample was one of 500 soil samples collected across Japan. Strain AO1 degraded 100 mg/l BPA to an undetectable level within 6 h in MYPG medium (containing malt extract, yeast extract, peptone, and glucose) and within 48 h in minimum medium containing 1% glucose at 30°C. Strain AO1 can utilize BPA as a sole source of carbon and as an energy source under aerobic conditions. The estrogenic activity of BPA in MYPG medium was ultimately reduced by strain AO1, although the activity initially increased. Taxonomical analysis showed that strain␣AO1 is closely related to Sphingomonas chlorophenolicum and S. herbicidovorans, neither of which have a capacity for BPA degradation. DNA–DNA hybridization showed that strain AO1 is a novel species of the Sphingomonas genus, and we designated AO1 as S. bisphenolicum.     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum