Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.

A unique highly reducing polyketide synthase (HR-PKS) with a reductase domain was identified in a betaenone biosynthetic gene cluster. Successful heterologous expression and characterization of the HR-PKS and trans-acting enoyl reductase (ER) provide insights into the core structure formation with a decalin scaffold and allow reconstitution of the betaenone biosynthetic machinery.

The current information on the polymorphism variation and haplotype structure of the domestic dog leukocyte antigen (DLA) genes is limited in comparison to other experimental animals. In this paper, to better elucidate the degree and types of polymorphisms and genetic differences for DLA-88, DLA-12 and DLA-64, we genotyped four families of 38 beagles and another 404 unrelated dogs representing 49 breeds by RT-PCR based Sanger sequencing. We also sequenced and analyzed the genomic organization of the DLA-88 and DLA-12 gene segments to better define these two-gene DLA haplotypes more precisely. We identified 45 alleles for DLA-88, 15 for DLA-12 and six for DLA-64, of which 20, 14 and six, respectively, were newly described alleles. Therefore, this study shows that the DLA-12 and DLA-64 loci are far more polymorphic than previously reported. Phylogenetic analysis strongly supported that the DLA-88, DLA-12 and DLA-64 alleles were independently generated after the original divergence of the DLA-79 alleles. Two distinct DLA-88 and DLA-12 haplotype structures, tentatively named DLA-88-DLA-12 and DLA-88-DLA-88L, were identified, and the novel haplotype DLA-88-DLA-88L contributed to 32.7% of the unrelated dogs. Quantitative real-time PCR analysis showed that the gene expression levels of DLA-88L and DLA-88 were similar, and that the gene expression level of DLA-12 was significantly lower. In addition, haplotype frequency estimations using frequently occurring alleles revealed 45 different DLA-class I haplotypes (88-88L/12-64) overall, and 22 different DLA-class I haplotypes in homozygous dogs for 18 breeds and mongrels.

We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae, using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3 rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization in A. oryzae results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.The filamentous fungus Aspergillus oryzae has been used in the production of fermented foods, such as sake, soy sauce, and miso (soybean paste), in Japan for over a thousand years. In addition, A. oryzae has the ability to secrete large amounts of proteins and has recently become a favorable host for recombinant protein production (5). By use of a series of classical random mutagenesis and screening procedures, hypersecretion mutants of heterologous proteins in aspergilli have been obtained (9, 49). However, the secretion yields of heterologous proteins are low compared to those of homologous proteins or proteins from closely related fungal species and generally do not exceed tens of milligrams per liter (16). In order to improve the expression levels of heterologous proteins, several trials have been conducted, and some strategies have been reported to be effective in increasing the level of heterologous protein production (39). These results provide information on how to increase the expression levels of heterologous genes; however, there is little information on the mechanisms hampering heterologous gene expression. In this study, we investigate the effect of codon optimization on heterologous gene expression. Such optimization has been effective in improving secretion levels of heterologous proteins in several hosts (19). Studies of heterologous gene expression through codon optimization have reported improved heterologous protein production in filamentous fungal species, such as Aspergillus awamori (2,15,35), Aspergillus niger (27, 35), Neurospora crassa (13, 35), and Trichoderma reesei (47). Based on studies of ...

The production of amylolytic enzymes in Aspergillus oryzae is induced in the presence of starch or maltose, and two Zn2Cys6-type transcription factors, AmyR and MalR, are involved in this regulation. AmyR directly regulates the expression of amylase genes, and MalR controls the expression of maltose-utilizing (MAL) cluster genes. Deletion of malR gene resulted in poor growth on starch medium and reduction in α-amylase production level. To elucidate the activation mechanisms of these two transcription factors in amylase production, the expression profiles of amylases and MAL cluster genes under carbon catabolite derepression condition and subcellular localization of these transcription factors fused with a green fluorescent protein (GFP) were examined. Glucose, maltose, and isomaltose induced the expression of amylase genes, and GFP-AmyR was translocated from the cytoplasm to nucleus after the addition of these sugars. Rapid induction of amylase gene expression and nuclear localization of GFP-AmyR by isomaltose suggested that this sugar was the strongest inducer for AmyR activation. In contrast, GFP-MalR was constitutively localized in the nucleus and the expression of MAL cluster genes was induced by maltose, but not by glucose or isomaltose. In the presence of maltose, the expression of amylase genes was preceded by MAL cluster gene expression. Furthermore, deletion of the malR gene resulted in a significant decrease in the α-amylase activity induced by maltose, but had apparently no effect on the expression of α-amylase genes in the presence of isomaltose. These results suggested that activation of AmyR and MalR is regulated in a different manner, and the preceding activation of MalR is essential for the utilization of maltose as an inducer for AmyR activation.