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2.2.1 Genetic and molecular aspects of aflatoxin biosynthesis

After the discovery that the AF biosynthesis was regulated by a gene cluster (Trail et al., 1995; Yu et al., 1995; Brown et al., 1996), the biosynthetic pathway of AFs has been extensively studied, and most of the enzymes and corresponding genes involved have been identified. Also, most of their functions have been elucidated (e.g. Trail et al., 1994; Yu et al., 1998, 2000, 2004a, 2004b; Ehrlich et al., 2004; Yabe & Nakajima, 2004; Ehrlich et al., 2005; Wen et al., 2005; Cary & Ehrlich, 2006), with possible alternative pathways (Detroy et al., 1973). AF biosynthesis requires at least 25 enzymes and two regulatory proteins encoded by contiguous genes in an 80-kb cluster (reviewed in Yu et al., 2004b). Clustered biosynthetic genes for fungal secondary metabolism are not only regulated by specific transcription factors, as a global epigenetic control mechanism may be conducted by genes, beyond the biosynthetic cluster, which are able to regulate multiple physiological processes and the response to environmental and nutritional factors such as temperature, pH, light, carbon and nitrogen sources (reviewed by Georgianna & Payne, 2009).

The genes involved in the major convertion steps from early precursors to AFs and their funtions are discussed in Yu et al. (2004b). These authors have proposed the use of a three-letter code “afl” to represent AF pathway genes. A capital letter in alphabetical order from “A” to “Y” represents each individual gene confirmed to be (or potentially be) involved in AF biosynthesis, e.g. aflA to aflY for all of the 25 genes (Figure 2.4). Those genes whose pathway involvement has already been characterised and confirmed are designated aflA to aflQ from the initial conversion of fatty acids to the final products, AFs.

aflR and aflS (formerly aflJ) are named for transcription regulators. Those genes whose pathway involvements are ambiguous or remain unclear are designated aflT, aflU (= cypA), aflV (= cypX), aflW (= moxY), aflX (= ordB), and aflY (= hypA).

Figure 2.4 Clustered genes and the AF biosynthetic pathway.

The gene names proposed by Yu et al. (2004b) are given on the left of the vertical line and the old gene names are given on the right. Arrows along the vertical line indicate the direction of gene transcription. The ruler at far left indicates the relative sizes of these genes in kilobases. Arrows indicate the connections from the genes to the enzymes they encode, from the enzymes to the bioconversion steps they are involved in, and from the intermediates to the products in the AF bioconversion steps.

Abbreviations: NOR, norsolorinic acid; AVN, averantin; HAVN, 5-hydroxyaverantin;

OAVN, oxoaverantin; AVNN, averufanin; AVF, averufin; VHA, versiconal hemiacetal acetate; VAL, versiconal; VERB, versicolorin B; VERA, versicolorin A;

DMST, demethylsterigmatocystin; DHDMST, dihydrodemethylsterigmatocystin; ST, sterigmatocystin; DHST, dihydrosterigmatocystin; OMST, O-methylsterigmatocystin;

DHOMST, dihydro-O-methylsterigmatocystin (Adapted from Yu et al., 2004b).

Generally, the AF biosynthesis genes of A. flavus, A. parasiticus and A. nomius are highly homologous, the order of the genes within the cluster being the same (Yu et al., 1995; Ehrlich et al., 2005; Chang et al., 2007). Also, AF genes and gene organisation in A.

sojae are most similar to those of A. parasiticus (identity 98-99%). A significant proportion, but not all, of non-aflatoxigenic A. flavus isolates have been found to contain various deletions in the AF gene cluster (Prieto et al., 1996; Ehrlich & Cotty, 2004; Ehrlich et al., 2004; Chang et al., 2005, 2006) which are common to some strains of A. oryzae (Chang et al., 2005, 2006). Also, additional enzymes are required for AFGs formation in A.

parasiticus. The loss of the ability to produce AFGs in A. flavus seems to result from a deletion in the terminal region of the cluster corresponding to genes aflF (= norB) and aflU (= cypA) (Ehrlich et al., 2004). Several studies confirmed that separate pathways lead to the formation of AFBs and AFGs (Henderberg et al., 1988; Bhatnagar et al., 1991; Yabe et al., 1999; Ehrlich et al., 2004).

2.2.2 Molecular differentiation of aflatoxigenic and non-aflatoxigenic strains

Molecular techniques have been widely applied in the attempt to distinguish aflatoxigenic and non-aflatoxigenic strains of A. flavus and related species, through the correlation of presence/absence of one or several genes involved in the AF biosynthetic pathway with the ability/inability to produce AFs. Some studies have been able to distinguish these species from other foodborne fungi and, in some cases, they were capable of distinguishing aflatoxigenic from non-aflatoxigenic strains.

The studies by Geisen (1996) and Shapira et al. (1996) can be regarded as the starting point for PCR-based diagnosis of aflatoxigenic and non-aflatoxigenic fungi.

Geisen (1996) used multiplex PCR with three sets of primers specific for three structural genes of the AF biosynthetic pathway aflD, aflM and aflO, and was able to differentiate A.

flavus and A. parasiticus from other food-borne fungi, but not aflatoxigenic and non-aflatoxigenic strains of the same species. Shapira et al. (1996) used non-aflatoxigenic strains and carried out monomeric PCRs with three different sets of primers for aflR, aflO and aflM genes, but they could only discriminate aflatoxigenic strains from other moulds.

Färber et al. (1997) detected aflatoxigenic strains of A. flavus in contaminated figs by performing a monomeric PCR with the same sets of primer used by Geisen (1996). Other

multiplex PCR with the AF pathway genes aflR, aflD, aflM and aflO did not produce a clear pattern that would allow to accurately differenciate aflatoxigenic from non-aflatoxigenic strains (Criseo et al., 2001). Lee et al. (2006) detected the differences in the aflR gene of A. flavus/A. oryzae and A. parasiticus/A. sojae, but they were not able to clearly differentiate the species. Baird et al. (2006) tested a different methodology based on DNA fingerprinting with two consecutive amplifications with arbitrary primers, with which the majority, but not all, of the aflatoxigenic isolates was differentiated from the non-aflatoxigenic.

AF production ability and aflatoxigenic strains differentiation have also been assessed by monitoring AF genes expression in the A. flavus group, using the reverse transcription PCR (RT-PCR) methodology. RT-PCR allows the detection of mRNAs transcribed by specific genes by PCR amplification of cDNA intermediates synthesised by reverse transcription. Such systems have been applied to monitor AF production and AF gene expression based on various regulatory and structural AF pathway genes in A. parasiticus and/or A. flavus (Sweeney et al., 2000; Mayer et al., 2003a, 2003b; Sherm et al., 2005; Degola et al., 2007), and were found to be very rapid and sensitive. Scherm et al.

(2005) studied 13 strains of both species and found consistency of 3 genes (aflD, aflO and aflP) in detecting AF production ability, further indicating them as potential markers.

But, as said, AF biosynthesis is based on a highly complex pathway. It is thus not surprising that genetic protocols that can fully differentiate between AF producers and non-producers have not yet been successfully established. Furthermore, one has to be aware that some genes are not exclusive of the AF biosynthetic pathway, which could create false-positives from sterigmatocystin producing fungi (Paterson, 2006). As an example, A.

nidulans harbours the complete AF biosynthesis pathway except for the final step that converts sterigmatocystin to AF (Brown et al., 1996).