Koichi Tamano

Identification and characterization of genes responsible for biosynthesis of kojic acid, an industrially important compound from Aspergillus oryzae

Kojic acid is produced in large amounts by Aspergillus oryzae as a secondary metabolite and is widely used in the cosmetic industry. Glucose can be converted to kojic acid, perhaps by only a few steps, but no genes for the conversion have thus far been revealed. Using a DNA microarray, gene expression profiles under three pairs of conditions significantly affecting kojic acid production were compared. All genes were ranked using an index parameter reflecting both high amounts of transcription and a high induction ratio under producing conditions. After disruption of nine candidate genes selected from the top of the list, two genes of unknown function were found to be responsible for kojic acid biosynthesis, one having an oxidoreductase motif and the other a transporter motif. These two genes are closely associated in the genome, showing typical characteristics of genes involved in secondary metabolism.

Characterization of the biosynthetic gene cluster for the ribosomally synthesized cyclic peptide ustiloxin B in Aspergillus flavus

Ustiloxin B is a secondary metabolite known to be produced by Ustilaginoidea virens. In our previous paper, we observed the production of this compound by Aspergillus flavus, and identified two A. flavus genes responsible for ustiloxin B biosynthesis (Umemura et al., 2013). The compound is a cyclic tetrapeptide of Tyr-Ala-Ile-Gly, whose tyrosine is modified with a non-protein coding amino acid, norvaline. Although its chemical structure strongly suggested that ustiloxin B is biosynthesized by a non-ribosomal peptide synthetase, in the present study, we observed its synthesis through a ribosomal peptide synthetic (RiPS) pathway by precise sequence analyses after experimental validation of the cluster. The cluster possessed a gene (AFLA_094980), termed ustA, whose translated product, UstA, contains a 16-fold repeated peptide embedding a tetrapeptide, Tyr-Ala-Ile-Gly, that is converted into the cyclic moiety of ustiloxin B. This result strongly suggests that ustiloxin B is biosynthesized through a RiPS pathway and that UstA provides the precursor peptide of the compound. The present work is the first characterization of RiPS in Ascomycetes and the entire RiPS gene cluster in fungi. Based on the sequence analyses, we also proposed a biosynthetic mechanism involving the entire gene cluster. Our finding indicates the possibility that a number of unidentified RiPSs exist in Ascomycetes as the biosynthetic genes of secondary metabolites, and that the feature of a highly repeated peptide sequence in UstA will greatly contribute to the discovery of additional RiPS.

Increased production of fatty acids and triglycerides in Aspergillus oryzae by enhancing expressions of fatty acid synthesis-related genes

Microbial production of fats and oils is being developed as a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillus oryzae. Examination of the A. oryzae genome demonstrates that it contains two fatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhanced the expression of fatty acid synthesis-related genes by replacing their promoters with the promoter from the constitutively highly expressed gene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthase genes we successfully increased the production of fatty acids and triglycerides by more than two-fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesterase increased productivity to a lesser extent. Increasing expression of acetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored using quantitative real-time reverse transcription polymerase chain reaction. Our data demonstrate that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

The β-1,3-Exoglucanase Gene exgA (exg1) of Aspergillus oryzae Is Required to Catabolize Extracellular Glucan, and Is Induced in Growth on a Solid Surface

The biological role of ExgA (Exg1), a secretory β-1,3-exoglucanase of Aspergillus oryzae, and the expression pattern of the exgA (exg1) gene were analyzed. The exgA disruptant and the exgA-overexpressing mutant were constructed, and phenotypes of both mutants were compared. Higher mycelial growth rate and conidiation efficiency were observed for the exgA-overexpressing mutant than for the exgA disruptant when β-1,3-glucan was supplied as sole carbon source. On the other hand, no difference in phenotype was observed between them in the presence or absence of the inhibitors of cell wall β-glucan remodeling when grown with glucose. exgA Expression was induced in growth on solid surfaces such as filter membrane and onion inner skin. A combination of poor nutrition and mycelial attachment to a hydrophobic solid surface appears to be an inducing factor for exgA expression. These data suggest that ExgA plays a role in β-glucan utilization, but is not much involved in cell wall β-glucan remodeling.

Comparative Genome Analysis Between Aspergillus oryzae Strains Reveals Close Relationship Between Sites of Mutation Localization and Regions of Highly Divergent Genes among Aspergillus Species

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome.

Genomics of industrial Aspergilli and comparison with toxigenic relatives

Aspergillus oryzae has been used in Japanese fermentation industries for more than a thousand years. The species produces large amounts of various hydrolytic enzymes and has been successfully applied to modern biotechnology. The size of the A. oryzae genome (37.5 Mb) is very close to that of A. flavus and A. niger, and 20–30% larger than that of either A. nidulans or A. fumigatus. A. oryzae and A. flavus have exactly the same number of aspartic proteinase genes, of which each orthologous pair shares highly conserved amino acid sequences. Synteny analysis with A. fumigatus and A. nidulans showed that the A. oryzae genome has a mosaic structure consisting of syntenic and non-syntenic blocks. In the microorganisms to be compared, the density of the genes having homologs was obviously higher on the syntenic than on the non-syntenic blocks. Expression analysis by the DNA microarray supported the significantly lower expression of genes on the non-syntenic than on the syntenic blocks.

Enhancing microbial metabolite and enzyme production: current strategies and challenges.

The metabolites and enzymes synthesizedby microorganisms have been widely usedas food (Mitsuhashi, 2014; Wendisch,2014), pharmaceuticals (Elander, 2003;Endo, 2010), biofuels (Geddes et al.,2011), pesticides (Waldron et al., 2001;Yoon et al., 2004), and detergents(Shaligram and Singhal, 2010), as wellas in the manufacturing process of theseindustrial products (Kirk et al., 2002;Merino and Cherry, 2007). They playimportant roles in our daily lives. Theproduction methods used for usefulmetabolites and enzymes have improvedsince the time their importance was firstestablished.If the genes involved in the synthe-sis of a metabolite or enzyme of inter-est are unknown, the production yield isenhanced by introducing random muta-tions into the chromosomes of the syn-thesizing microbe by ultraviolet (UV)irradiation or treatment with mutagens(Adrio and Demain, 2006). In addition,culture conditions have been adaptedto further enhance production (Demain,2000; Mukherjee et al., 2006). On theother hand, if the genes involved areknown, their expression is also enhancedby metabolic engineering strategies suchas gene disruption and overexpressionusing genetic modification techniques(Stephanopoulos et al., 1998; Adrio andDemain, 2010). When genetic modifi-cation of the producing microorganismis not possible because of difficulties intransformation, heterologous expressionof the product of interest in other micro-bial species in which genetic modifica-tion can be more easily achieved hasalso been utilized for mass production(Stephanopoulos et al., 1998; Keasling,2012).Primary metabolites essential for thenormalgrowthoforganismsareconservedbetween closely related microbial species,and their metabolic pathways includinggenetic components are almost fully elu-cidated. Therefore, metabolic engineer-ing has been the chosen strategy usedfor increasing the microbial productionof primary metabolites (Stafford andStephanopoulos, 2001; Kern et al., 2007).About microbial enzymes, the codinggenes are highly likely to be identifiedif both N-terminal amino acid sequencesand molecular weights are not only iden-tified by using highly purified samples butthegenomicdataoftheproducermicroor-ganisms are also available. Searching agene from the genomic data, on the basisof the N-terminal amino acid sequenceand molecular weight, will help us iden-tifyanenzyme-codinggene.Oncethegenehas been identified, inducing overexpres-sion of this gene in the original produceror another microbial host is one of thestrategies adopted to increase the produc-tionoftheenzyme(DemainandVaishnav,2009).Four strategies are considered to beeffective in enhancing the productionof primary metabolites. The first strat-egy is enhancing the expression of genesinvolved in metabolite synthesis. Thisstrategy should be the most commonlyusedandreliableapproach,butitdoesnotalwayscontributetoelevatedproduc-tion. In fact, we enhanced the expres-sion of four enzyme genes, individually,that were involved in palmitic acid [C16-fatty acid] synthesis, aiming to increasethe production of free fatty acids (pri-mary metabolites) in

Genome Sequence of the Mucoromycotina Fungus Umbelopsis isabellina, an Effective Producer of Lipids

ABSTRACT Umbelopsis isabellina is a fungus in the subdivision Mucoromycotina, many members of which have been shown to be oleaginous and have become important organisms for producing oil because of their high level of intracellular lipid accumulation from various feedstocks. The genome sequence of U. isabellina NBRC 7884 was determined and annotated, and this information might provide insights into the oleaginous properties of this fungus.

Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes

Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

High-efficiency extracellular release of free fatty acids from Aspergillus oryzae using non-ionic surfactants

Free fatty acids (FFAs) are useful for generating biofuel compounds and functional lipids. Microbes are increasingly exploited to produce FFAs via metabolic engineering. However, in many microorganisms, FFAs accumulate in the cytosol, and disrupting cells to extract them is energy intensive. Thus, a simple cost-effective extraction technique must be developed to remove this drawback. We found that FFAs were released from cells of the filamentous fungus Aspergillus oryzae with high efficiency when they were cultured or incubated with non-ionic surfactants such as Triton X-100. The surfactants did not reduce hyphal growth, even at 5% (w/v). When the faaA disruptant was cultured with 1% Triton X-100, more than 80% of the FFAs synthesized de novo were released. When the disruptant cells grown without surfactants were incubated for 1h in 1% Triton X-100 solution, more than 50% of the FFAs synthesized de novo were also released. Other non-ionic surfactants in the same ether series, such as Brij 58, IGEPAL CA-630, and Tergitol NP-40, elicited a similar FFA release. The dry cell weight of total hyphae decreased when grown with 1% Triton X-100. The decrement was 4.9-fold greater than the weight of the released FFAs, implying release of other intracellular compounds. Analysis of the culture supernatant showed that intracellular lactate dehydrogenase was also released, suggesting that FFAs are not released by a specific transporter. Therefore, ether-type non-ionic surfactants probably cause non-specific release of FFAs and other intracellular compounds by increasing cell membrane permeability.