Akinori Ando

Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding.

ABSTRACT Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5− strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for ω3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/108 spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal ω3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.

Ricinoleic Acid and Castor Oil as Substrates for Conjugated Linoleic Acid Production by Washed Cells of Lactobacillus plantarum

Ricinoleic acid (12-hydroxy-cis-9-octadecaenoic acid) was an effective substrate for conjugated linoleic acid (CLA) production by washed cells of Lactobacillus plantarum AKU 1009a. The CLA produced was a mixture of cis-9,trans-11- and trans-9,trans-11-octadecadienoic acids. Addition of α-linolenic acid to the culture medium increased the CLA productivity of the washed cells. In the presence of lipase, castor oil, in which the main fatty acid component is ricinoleic acid, also was a substrate for CLA.

Structural Analysis of Conjugated Linoleic Acid Produced by Lactobacillus plantarum, and Factors Affecting Isomer Production

An isomer of the conjugated linoleic acid (CLA) produced from linoleic acid by Lactobacillus plantarum was identified as cis-9,trans-11-octadecadienoic acid by proton nuclear magnetic resonance spectroscopy. Together with earlier results, we concluded that the bacterium produces two CLA isomers, cis-9,trans-11- and trans-9,trans-11-octadecadienoic acid from linoleic acid. The addition of L-serine, glucose, AgNO3, or NaCl to the reaction mixture reduced production of the latter.

Microbial production of conjugated γ-linolenic acid from γ-linolenic acid by Lactobacillus plantarum AKU 1009a

Aims:  Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated.

Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria

The PUFAs include many bioactive lipids. The microbial metabolism of C18 PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C20 PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C20 PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C20 PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C18 and C20 PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.

Selection and characterization of promoters based on genomic approach for the molecular breeding of oleaginous fungus Mortierella alpina 1S-4.

To express a foreign gene effectively, a good expression system is required. In this study, we investigated various promoters as useful tools for gene manipulation in oleaginous fungus Mortierella alpina 1S-4. We selected and cloned the promoter regions of 28 genes in M. alpina 1S-4 on the basis of expression sequence tag abundance data. The activity of each promoter was evaluated using the β-glucuronidase (GUS) reporter gene. Eight of these promoters were shown to enhance GUS expression more efficiently than a histone promoter, which is conventionally used for the gene manipulation in M. alpina. Especially, the predicted protein 3 and the predicted protein 6 promoters demonstrated approximately fivefold higher activity than the histone promoter. The activity of some promoters changed along with the cultivation phase of M. alpina 1S-4. Seven promoters with constitutive or time-dependent, high-level expression activity were selected, and deletion analysis was carried out to determine the promoter regions required to retain activity. This is the first report of comprehensive promoter analysis based on a genomic approach for M. alpina. The promoters described here will be useful tools for gene manipulation in this strain.

Isolation and characterization of psychrotolerant endospore-forming Sporosarcina species associated with minced fish meat (surimi).

We studied the changes of resident microbiota in surimi-minced fish meat-during heat-treatment and subsequent cold-storage via the sequencing of partial 16S rRNA gene. Raw surimi made from Alaska pollock, pike conger, and white croaker was contaminated with 10(4) to 10(6)CFU/g of various non-endospore-forming bacteria. Immediately after heat-treatment, the bacterial counts were significantly reduced to less than 1CFU/g, and only endospore-forming bacteria, identified as Bacillus species were retrieved. Subsequently, the bacterial counts increased up to 10 to 10(5)CFU/g in the heated surimi after refrigerated storage at 5 °C for 2 weeks or at 10 °C for 1 week. Most of the isolates from the refrigerated surimi were identified as Sporosarcina species. The Sporosarcina isolates have an increased ability to grow at 10 °C than the isolates related to the other endospore-forming bacteria, such as Bacillus, Lysinibacillus, and Paenibacillus species. Endospores of the Sporosarcina isolates were able to germinate and proliferate in a fish-paste product model system stored at 10 °C within 8 days. In order to study the cold-adaptation mechanism of Sporosarcina species, the fatty acid composition of the isolates was analyzed. At the growth temperature of 10 °C, the proportions of unsaturated to saturated fatty acids and anteiso to iso fatty acids were higher than those at 28 °C. The alteration of the fatty acid composition suggests that Sporosarcina species adapt to cold by maintaining the fluidity of the cell membrane because unsaturated and anteiso fatty acids have lower melting points than saturated and iso fatty acids, respectively. We concluded that the endospores of Sporosarcina species are widely distributed in surimi, and that they can survive heat-treatment and proliferate during cold-storage in fish-paste products. Controlling Sporosarcina species would contribute to improving the quality of surimi product.

Gene targeting in the oil-producing fungus Mortierella alpina 1S-4 and construction of a strain producing a valuable polyunsaturated fatty acid

To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed ku80 gene-disrupted strains via single-crossover homologous recombination. The Δku80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the Δku80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the ku80 gene, but the construction of DGLA-producing strain by disruption of the Δ5-desaturase gene was succeeded using the Δku80 strain as a host strain. This report describes the first study on the identification and disruption of the ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4.

The rhizosphere microbial community in a multiple parallel mineralization system suppresses the pathogenic fungus Fusarium oxysporum

The rhizosphere microbial community in a hydroponics system with multiple parallel mineralization (MPM) can potentially suppress root‐borne diseases. This study focused on revealing the biological nature of the suppression against Fusarium wilt disease, which is caused by the fungus Fusarium oxysporum, and describing the factors that may influence the fungal pathogen in the MPM system. We demonstrated that the rhizosphere microbiota that developed in the MPM system could suppress Fusarium wilt disease under in vitro and greenhouse conditions. The microbiological characteristics of the MPM system were able to control the population dynamics of F. oxysporum, but did not eradicate the fungal pathogen. The roles of the microbiological agents underlying the disease suppression and the magnitude of the disease suppression in the MPM system appear to depend on the microbial density. F. oxysporum that survived in the MPM system formed chlamydospores when exposed to the rhizosphere microbiota. These results suggest that the microbiota suppresses proliferation of F. oxysporum by controlling the pathogens morphogenesis and by developing an ecosystem that permits coexistence with F. oxysporum.

Selective production of cis‐9,trans‐11 isomer of conjugated linoleic acid from trans‐vaccenic acid methyl ester by Delacroixia coronata

Aims:  Bio‐process development for isomer selective and efficient production of cis‐9,trans‐11‐octadecadienoic acid (CLA) from trans‐vaccenic acid (t‐VA, trans‐11‐octadecenoic acid) through microbial fatty acid Δ9‐desaturation reaction.