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Citrinin produced by Aspergillus, Penicillium, and Monascus species is a polyketide compound that has nephrotoxic activity in mammals and is bactericidal toward gram-positive bacteria. To avoid the risk of citrinin contamination in other fermentation products produced by Monascus purpureus, knowledge of the citrinin biosynthetic genes is needed so that citrinin-nonproducing strains can be generated. We cloned a polyketide synthase (PKS) gene from M. purpureus with degenerate primers designed to amplify the conserved region of a ketosynthase domain of a fungal PKS. A 13-kb genomic DNA fragment was identified that contained a full-length PKS gene (pksCT) of 7,838 bp with a single 56-bp intron. pksCT encodes a 2,593-amino-acid protein that contains putative domains for ketosynthase, acyltransferase, acyl carrier protein (ACP), and a rare methyltransferase. There was no obvious thioesterase domain, which usually is downstream of the ACP domain in multi-aromatic-ring PKSs. pksCT transcription was correlated with citrinin production, suggesting that the pksCT gene product was involved in citrinin biosynthesis. Homologous recombination between the wild-type allele and a truncated disruption construct resulted in a pksCT-disrupted strain of M. purpureus. The disruptant did not produce citrinin, but a pksCT revertant generated by successive endogenous recombination events in the pksCT disruptant restored citrinin production, indicating that pksCT encoded the PKS responsible for citrinin biosynthesis in M. purpureus.Fungi and actinomycetes are the primary producers of bioactive secondary metabolites (2

In a green alga, Haematococcus pluvialis, a morphological change of vegetative cells into cyst cells was rapidly induced by the addition of acetate or acetate plus Fe2+ to the vegetative growth phase. Accompanied by cyst formation, algal astaxanthin formation was more enhanced by the addition of acetate plus Fe2+ than by the addition of acetate alone. Encystment and enhanced carotenoid biosynthesis were inhibited by either actinomycin D or cycloheximide. However, after cyst formation was induced by the addition of acetate alone, carotenoid formation could be enhanced with the subsequent addition of Fe2+ even in the presence of the inhibitors. The Fe2+-enhanced carotenogenesis was inhibited by potassium iodide, a scavenger for hydroxyl radical, suggesting that hydroxyl radical formed by an iron-catalyzed Fenton reaction may be required for enhanced carotenoid biosynthesis. Moreover, it was demonstrated that four active oxygen species, singlet oxygen, superoxide anion radical, hydrogen peroxide, and peroxy radical, were capable of replacing Fe2+ in its role in the enhanced carotenoid formation in the acetate-induced cyst. From these results, it was concluded that oxidative stress is involved in the posttranslational activation of carotenoid biosynthesis in acetate-induced cyst cells.

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Inhibition of the fermentation of propionate to methane and carbon dioxide by hydrogen, acetate, and propionate was analyzed with a mesophilic propionate-acclimatized sludge that consisted of numerous flocs (size, 150 to 300 ,um). The acclimatized sludge could convert propionate to methane and carbon dioxide stoichiometrically without accumulating hydrogen and acetate in a propionate-minimal medium. Inhibition of propionate utilization by propionate could be analyzed by a second-order substrate inhibition model (shown below) given that the substrate saturation constant, K., was 15.9 ,uM; the substrate inhibition constant, Ki, was 0.79 mM; and the maximum specific rate of propionate utilization, q,, was 2.15 mmol/g of mixed-liquor volatile suspended solids (MLVSS) per day: q, = qmSI[Ks + S + (S2IKj)], where q., is the specific rate of propionate utilization and S is the initial concentration of undissociated propionic acid. For inhibition by hydrogen and acetate to propionate utilization, a noncompetitive product inhibition model was used: q, = qmI[l + (PIKp)-], where P is the initial concentration of hydrogen or undissociated acetic acid and Kp is the inhibition constant. Kinetic analysis gave, for hydrogen inhibition, KP(H2) = 0.11 atm (= 11.1 kPa, 71.5 ,iM), q,,, = 2.40 mmol/g of MLVSS per day, and n = 1.51 and, for acetate inhibition, KP(HAC) = 48.6 ,uM, qn = 1.85 mmol/g of MLVSS per day, and n = 0.96. It could be concluded that the increase in undissociated propionic acid concentration was a key factor in inhibition of propionate utilization and that hydrogen and acetate cooperatively inhibited propionate degradation, suggesting that hydrogenotrophic and acetoclastic methanogens might play an important role in enhancing propionate degradation to methane and carbon dioxide. Production of CH4 from propionate is an important intermediate step in the bioconversion of organic matter to CH4 and CO2. Propionate degradation requires obligate protonreducing acetogens that form acetate, H2, and CO2 from propionate and H2-and acetate-utilizing methanogens (2, 4, 13). The equations for the reactions of the respective bacterial groups are as follows: