Secondary metabolism is commonly associated with morphological development in microorganisms, including fungi. We found that veA, a gene previously shown to control the Aspergillus nidulans sexual/asexual developmental ratio in response to light, also controls secondary metabolism. Specifically, veA regulates the expression of genes implicated in the synthesis of the mycotoxin sterigmatocystin and the antibiotic penicillin. veA is necessary for the expression of the transcription factor aflR, which activates the gene cluster that leads to the production of sterigmatocystin. veA is also necessary for penicillin production. Our results indicated that although veA represses the transcription of the isopenicillin synthetase gene ipnA, it is necessary for the expression of acvA, the key gene in the first step of penicillin biosynthesis, encoding the delta-(L-alphaaminoadipyl)-L-cysteinyl-D-valine synthetase. With respect to the mechanism of veA in directing morphological development, veA has little effect on the expression of the known sexual transcription factors nsdD and steA. However, we found that veA regulates the expression of the asexual transcription factor brlA by modulating the ␣/␤ transcript ratio that controls conidiation.

Fumitremorgin C, a diketopiperazine mycotoxin produced by Aspergillus fumigatus, is a potent and specific inhibitor of breast cancer resistance protein (BCRP). Elucidation of the fumitremorgin C biosynthetic pathway provides a strategy for new drug design. A structure-activity relationship study based on metabolites related to the ftm gene cluster revealed that the process most crucial for inhibitory activity against BCRP was cyclization to form fumitremorgin C. To determine the gene involved in the cyclization reaction, targeted gene inactivation was performed with candidate genes in the ftm cluster. Analysis of the gene disruptants allowed us to identify ftmE, one of the cytochrome P450 genes in the cluster, as the gene responsible for the key step in fumitremorgin biosynthesis. Additionally, we demonstrated that the other two cytochrome P450 genes, ftmC and ftmG, were involved in hydroxylation of the indole ring and successive hydroxylation of fumitremorgin C, respectively.

No abstract

Enzyme-catalyzed [4+2] cycloaddition has been proposed to be a key transformation process in various natural product biosynthetic pathways. Recently Fsa2 was found to be involved in stereospecific trans-decalin formation during the biosynthesis of equisetin, a potent HIV-1 integrase inhibitor. To understand the mechanisms by which fsa2 determines the stereochemistry of reaction products, we sought an fsa2 homologue that is involved in trans-decalin formation in the biosynthetic pathway of an enantiomerically opposite analogue, and we found phm7, which is involved in the biosynthesis of phomasetin. A decalin skeleton with an unnatural configuration was successfully constructed by gene replacement of phm7 with fsa2, thus demonstrating enzymatic control of all stereochemistry in the [4+2] cycloaddition. Our findings highlight enzyme-catalyzed [4+2] cycloaddition as a stereochemically divergent step in natural product biosynthetic pathways and open new avenues for generating derivatives with different stereochemistry.

Asexual spores (conidia) are reproductive structures that play a crucial role in fungal distribution and survival. As fungal conidia are, in most cases, etiological agents of plant diseases and fungal lung disease, their stress resistance and interaction with their hosts have drawn increasing attention. In the present study, we investigated whether environmental temperature during conidiation affects the stress tolerance of the conidia of the human pathogenic fungus Aspergillus fumigatus. Conidia from a 25°C culture showed a lower tolerance to heat (60°C) and oxidative (H2O2) stresses and a marked resistance to ultraviolet radiation exposure, compared with those produced at 37 and 45°C. The accumulation of trehalose was lower in the conidia from the 25°C culture. Furthermore, the conidia from the 25°C culture showed darker pigmentation and increased transcripts of dihydroxynaphthalene (DHN)-melanin biosynthesis-related genes (i.e., pksP, arp1, and arp2). An RNA-sequencing analysis revealed that the transcription level of the trypacidin (tpc) gene cluster, which contains 13 genes, was sharply and coordinately activated in the conidia from the 25°C culture. Accordingly, trypacidin was abundant in the conidia from the 25°C culture, whereas there was little trypacidin in the conidia from the 37°C culture. Taken together, these data show that the environmental temperature during conidiation affects conidial properties such as stress tolerance, pigmentation, and mycotoxin accumulation. To enhance our knowledge, we further explored the temperature-dependent production of DHN-melanin and trypacidin in clinical A. fumigatus isolates. Some of the isolates showed temperature-independent production of DHN-melanin and/or trypacidin, indicating that the conidia-associated secondary metabolisms differed among the isolates.

No abstract

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full‐length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3‐hydroxylase (F3H), flavonoid 3′,5′‐hydroxylase (F3′5′H), dihydroflavonol 4‐reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3′5′H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.

Eucalyptus is a diverse genus of flowering trees with more than 700 genotypic species which are mostly native to Australia. We selected 19 wild provenances of Eucalyptus camaldulensis grown in Australia, compared their growth rate and drought tolerance and determined the protein levels of plasma membrane aquaporins (PIPs). There was a positive relationship between the drought tolerance and PIP content. PIPs are divided into two subgroups, PIP1 and PIP2. Most members of the PIP2 subgroup, but not PIP1 subgroup, exhibit water channel activity. We introduced two radish (Raphanus sativus L.) PIPs, RsPIP1;1 and RsPIP2;1, into a hybrid clone of Eucalyptus grandis and Eucalyptus urophylla to examine the effect of their overexpression. Expression of these genes was confirmed by real-time polymerase chain reaction (PCR) and the protein accumulation of RsPIP2;1 by immunoblotting. Drought tolerance was not enhanced in transgenic lines of either gene. However, one transgenic line expressing RsPIP2;1 showed high photosynthesis activity and growth rate under normal growth conditions. For RsPIP1;1-transformed lines, the RsPIP1;1 protein did not accumulate, and the abundance of endogenous PIP1 and PIP2 was decreased. The endogenous PIP1 and PIP2 genes were suppressed in these lines. Therefore, the decreased levels of PIP1 and PIP2 protein may be due to co-suppression of the PIP genes and/or high turnover of PIP proteins. RsPIP1;1-expressing lines gave low values of photosynthesis and growth compared with the control. These results suggest that down-regulation of PIP1 and PIP2 causes serious damage and that up-regulation of PIP2 improves the photosynthetic activity and growth of Eucalyptus trees.