Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of ...fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20–25 %).
Aspergillus oryzae is used extensively for the production of the traditional Japanese fermented foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste). In recent years, recombinant DNA ...technology has been used to enhance industrial enzyme production by A. oryzae. Recently completed genomic studies using expressed sequence tag (EST) analyses and whole-genome sequencing are quickly expanding the industrial potential of the fungus in biotechnology. Genes that have been newly discovered through genome research can be used for the production of novel valuable enzymes and chemicals, and are important for designing new industrial processes. This article describes recent progress of A . oryzae genomics and its impact on industrial production of enzymes, metabolites, and bioprocesses.
Genomics of Aspergillus oryzae Kobayashi, T.(Nagoya Univ. (Japan)); Abe, K; Asai, K ...
Bioscience, biotechnology, and biochemistry,
03/2007, Letnik:
71, Številka:
3
Journal Article
Recenzirano
The genome sequence of Aspergillus oryzae, a fungus used in the production of the traditional Japanese fermentation foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste), has revealed ...prominent features in its gene composition as compared to those of Saccharomyces cerevisiae and Neurospora crassa. The A. oryzae genome is extremely enriched with genes involved in biomass degradation, primary and secondary metabolism, transcriptional regulation, and cell signaling. Even compared to the related species A. nidulans and A. fumigatus, an abundance of metabolic genes is apparent, with acquisition of more than 6 Mb of sequence in the A. oryzae lineage, interspersed throughout the A. oryzae genome. Besides the various already established merits of A. oryzae for industrial uses, the genome sequence and the abundance of metabolic genes should significantly accelerate the biotechnological use of A. oryzae in industry.
We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The ...approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70⁺ strain due to the low frequency of homologous recombination were efficiently obtained from Δku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.
Aspergillus oryzae penicillin biosynthetic genes were clustered. The penicillin production was positively regulated by VeA, a global gene regulator required for transcriptional expression of the ...penicillin biosynthetic genes. Overexpression of the biosynthetic genes by a strong promoter yielded a greater than 100-fold increase in penicillin production.
Fungal strain 14919 was originally isolated from a soil sample collected at Mt. Kiyosumi, Chiba Prefecture, Japan. It produces FR901512, a potent and strong 3-hydroxy-3-methylglutaryl-coenzyme A ...(HMG-CoA) reductase inhibitor. The genome sequence of fungal strain 14919 was determined and annotated to improve the productivity of FR901512.
Ustiloxin B, originally isolated from the fungus
Ustilaginoidea virens
, is a known inhibitor of microtubule assembly. Ustiloxin B is also produced by
Aspergillus flavus
and is synthesized through ...the ribosomal peptide synthesis pathway. In
A. flavus
, the gene cluster associated with ustiloxin B production contains 15 genes including those encoding a fungal C6-type transcription factor and ustiloxin B precursor. Although the koji mold
Aspergillus oryzae
, which is genetically close to
A. flavus
, has the corresponding gene cluster, it does not produce ustiloxin B, which may be explained by the fact that the gene encoding the transcription factor UstR is not expressed. Here, to investigate whether ustiloxin B can be produced by expressing
ustR
in
A. oryzae
, we constructed
ustR
expression (
ustR
EX
) strains and analyzed ustiloxin B production. In the
ustR
EX
strains, all genes in the cluster were up-regulated, in line with expression of
ustR
, and ustiloxin B produced. To elucidate whether the KexB protease is involved in the processing of the ustiloxin B precursor protein UstA, which has repeats of basic amino acid doublets resembling KexB target sites, we also constructed a
ustR
EX
strain with the ∆
kexB
genotype. Although
ustR
was expressed in this strain, ustiloxin B was barely detectable. This finding strongly suggests that KexB is required for ustiloxin B production.
Ustilaginoidea virens is a rice pathogenic fungus that causes false smut disease, a disease that seriously damages the yield and quality of the grain. Analysis of the U. virens IPU010 33.6-Mb genome ...sequence will aid in the understanding of the pathogenicity of the strain, particularly in regard to effector proteins and secondary metabolic genes.
Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins ...from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (~7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution.
We have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function.
This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.
PDF
