A microarray analysis was performed to study the effect of varying combinations of water activity and temperature on the activation of aflatoxin biosynthesis genes in Aspergillusflavus grown on YES medium. Generally A. flavus showed expression of the aflatoxin biosynthetic genes at all parameter combinations tested. Certain combinations of a(w) and temperature, especially combinations which imposed stress on the fungus resulted in a significant reduction of the growth rate. At these conditions induction of the whole aflatoxin biosynthesis gene cluster occurred, however the produced aflatoxin B(1) was low. At all other combinations (25 degrees C/0.95 and 0.99; 30 degrees C/0.95 and 0.99; 35 degrees C/0.95 and 0.99) a reduced basal level of cluster gene expression occurred. At these combinations a high growth rate was obtained as well as high aflatoxin production. When single genes were compared, two groups with different expression profiles in relation to water activity/temperature combinations occurred. These two groups were co-ordinately localized within the aflatoxin gene cluster. The ratio of aflR/aflJ expression was correlated with increased aflatoxin biosynthesis.

Systematic expression analysis of mycotoxin biosynthesis genes by real-time PCR and microarray was carried out to examine the relationship between growth and general expression patterns in relation to single environmental factors such as temperature, water activity (a(w)) and pH and water activity x temperature interactions. For single parameters, one major peak of expression occurred close to optimum growth conditions. However, a second minor peak was observed under suboptimal growth conditions, when intermediate environmental stress was imposed on Aspergillus parasiticus (afl genes), Penicillium verrucosum (ota genes) and Fusarium culmorum (tri genes). This expression profile pattern was more pronounced in relation to changes in temperature and a(w) than to pH. In a two-factorial experimental design with temperature xa(w) regimes, again two peaks of expression were observed for cluster genes after microarray analysis, one close to those giving optimal growth and one under imposed stress conditions. Interestingly, when the activity of single genes of the microarray data were plotted in relation to the two parameters, again a two-peak expression profile became obvious independently for both parameters. Expression of the mycotoxin biosynthesis genes was followed exactly by phenotypic mycotoxin production. This expression profile appears to be generic across the mycotoxigenic fungi examined.

A microarray analysis was used to examine the effect of combinations of water activity (a w , 0.995-0.90) and temperature (20 -428C) on the activation of aflatoxin biosynthetic genes (30 genes) in Aspergillus flavus grown on a conducive YES (20 g yeast extract, 150 g sucrose, 1 g MgSO 4 . 7H 2 O) medium. The relative expression of 10 key genes (aflF, aflD, aflE, aflM, aflO, aflP, aflQ, aflX, aflR and aflS) in the biosynthetic pathway was examined in relation to different environmental factors and phenotypic aflatoxin B 1 (AFB 1 ) production. These data, plus data on relative growth rates and AFB 1 production under different a w  temperature conditions were used to develop a mixed-growth-associated product formation model. The gene expression data were normalized and then used as a linear combination of the data for all 10 genes and combined with the physical model. This was used to relate gene expression to a w and temperature conditions to predict AFB 1 production. The relationship between the observed AFB 1 production provided a good linear regression fit to the predicted production based in the model. The model was then validated by examining datasets outside the model fitting conditions used (378C, 408C and different a w levels). The relationship between structural genes (aflD, aflM) in the biosynthetic pathway and the regulatory genes (aflS, aflJ) was examined in relation to a w and temperature by developing ternary diagrams of relative expression. These findings are important in developing a more integrated systems approach by combining gene expression, ecophysiological influences and growth data to predict mycotoxin production. This could help in developing a more targeted approach to develop prevention strategies to control such carcinogenic natural metabolites that are prevalent in many staple food products. The model could also be used to predict the impact of climate change on toxin production.

Summary The filamentous fungus Alternaria alternata is a potent producer of many toxic secondary metabolites, which contaminate food and feed. The most prominent one is the polyketide‐derived alternariol (AOH) and its derivative alternariol monomethyl ether (AME). Here, we identified the gene cluster for the biosynthesis of AOH and AME by CRISPR/Cas9‐mediated gene inactivation of several biosynthesis genes in A. alternata and heterologous expression of the gene cluster in Aspergillus oryzae. The 15 kb‐spanning gene cluster consists of a polyketide synthase gene, pksI, an O‐methyltransferase, omtI, a FAD‐dependent monooxygenase, moxI, a short chain dehydrogenase, sdrI, a putative extradiol dioxygenase, doxI and a transcription factor gene, aohR. Heterologous expression of PksI in A. oryzae was sufficient for AOH biosynthesis. Co‐expression of PksI with different tailoring enzymes resulted in AME, 4‐hydroxy‐alternariol monomethyl ether (4‐OH‐AME), altenusin (ALN) and altenuene (ALT). Hence, the AOH cluster is responsible for the production of at least five different compounds. Deletion of the transcription factor gene aohR led to reduced expression of pksI and delayed AOH production, while overexpression led to increased expression of pksI and production of AOH. The pksI‐deletion strain displayed reduced virulence on tomato, citrus and apple suggesting AOH and the derivatives as virulence and colonization factors.

The influence of varying combinations of water activity (aw) and temperature on growth, aflatoxin biosynthesis and aflR/aflS expression of Aspergillus parasiticus was analysed in the ranges 17-42°C and 0.90-0.99 aw. Optimum growth was at 35°C. At each temperature studied, growth increased from 0.90 to 0.99 aw. Temperatures of 17 and 42°C only supported marginal growth. The external conditions had a differential effect on aflatoxin B1 or G1 biosynthesis. The temperature optima of aflatoxin B1 and G1 were not at the temperature which supported optimal growth (35°C) but either below (aflatoxin G1, 20-30°C) or above (aflatoxin B1, 37°C). Interestingly, the expression of the two regulatory genes aflR and aflS showed an expression profile which corresponded to the biosynthesis profile of either B1 (aflR) or G1 (aflS). The ratios of the expression data between aflS:aflR were calculated. High ratios at a range between 17 and 30°C corresponded with the production profile of aflatoxin G1 biosynthesis. A low ratio was observed at >30°C, which was related to aflatoxin B1 biosynthesis. The results revealed that the temperature was the key parameter for aflatoxin B1, whereas it was water activity for G1 biosynthesis. These differences in regulation may be attributed to variable conditions of the ecological niche in which these species occur.