Tylosin

Tylosin is a bulky molecule expected to induce the size-exclusion effect upon adsorption onto microporous adsorbents. Ji et al. (2010a) proposed that the adsorption of bulky tylosin is lower onto activated carbons with more micropores than onto synthesized carbons with more mesopores because of the size-exclusion effect. Fu et al. (2011) noted that commercial microporous-activated carbon treated with potassium hydroxide activation markedly increases the specific surface area and enlarges the pore sizes of the mesopores. The adsorption of tylosin is greatly increased by more than one order of magnitude due to the enhanced size-exclusion effect. Obvious size-exclusion effects were also found in the adsorption of other bulky organic chemicals. Ji et al. (2009) observed that the adsorbent-to-solution distribution coef-ficient (Kd, in liter/kilogram) of tetracycline is in the order of 10₄ to 10₆ L/kg for single-wall nanotube (SWNT), 10₃ to 10₄ L/kg for multiwall nanotube (MWNT), 10₃ to 10₄ L/kg for activated carbon (AC), and 10₃ to 10₅ L/kg for graphite. Upon normalization of the adsorbent surface area, the adsorption affinity of tetracycline decreases in the following order: graphite/SWNT > MWNT > AC. The weaker adsorption of tetracycline onto AC indicates that for bulky adsorbates, adsorption affinity is caused by the molecular sieving effect. Liu et al. (2006) found that the maximum adsorption amount of nonylphenol ethoxylates (NPE) over activated carbon is approximately 350 mg g⁻¹, which was markedly lower than that of mesoporous carbon CMK-3.

Complementation of tylM-KO strains with tylM DNA None of the three tylM-KO strains accumulated macrolide material when fermented in tylosin production medium (Figures 3a, 4a and 5a) although, as expected, each could produce tylosin when complemented with a block of wild type DNA containing tyl[MIII–MII–MI ] under control of the strong, constitutive promoter, ermEp* ( see Figures 3b and 5b). Note, however, that wild type levels of tylosin production were not achieved by the complemented strains (compare Figure 3b and d), since the C31 attB site, used for integration of complementing DNA, is not a neutral site in the context of tylosin production. Thus, integration of the ‘‘empty’’ vector, pLST9828, into that site reduces tylosin yields by up to 50% (unpublished data; this laboratory). Note also that it was not necessary to include the ccr gene (Figure 1), downstream of and codirectional with the tylM genes, in the blocks of complementing DNA, since strains lacking ccr can still produce tylosin.

As yet, relatively little is known about the regulation of macrolide biosynthesis nor is it yet obvious whether a common pattern of control might emerge. For example, the intensively studied erythromycin-biosynthetic ( ery ) gene cluster of Saccha-ropolyspora erythraea contains no regulatory genes and none that control erythromycin production has yet been identified elsewhere in the Sacc. erythraea genome. In contrast, the tylosin-biosynthetic ( tyl ) gene cluster of Streptomyces fradiae contains at least five candidate regulatory genes. Although the precise functions of the latter remain to be established, a current model proposes that tylosin production is controlled in pathway-specific fashion by regulatory protein( s ) of the SARP family and/or by the product of tylR and also by one or more gamma-butyrolactone signalling factors. The TylR protein appears to mediate global control of tylosin production, since both polyketide and deoxysugar metabolism were abolished in a tylR-disrupted strain of S. fradiae. As an added complication, glycosylated precursors of tylosin are required to stimulate production of the aglycone, tylactone. In this respect, glycosylated macrolides act catalytically ( in an uncharacterized manner ) to turn a trickle of polyketide metabolism into a flood. However, none of this implies that the polyketide aglycone and the three tylosin sugars are necessarily produced in stoichiometrically equivalent amounts. The primary purpose of the present work was to address this point and to determine whether tylosin production in S. fradiae is limited by availability of the aglycone or of the sugars. It was also of interest to ascertain whether such limitation has changed significantly during the empirical selection of enhanced production strains.