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We constructed dihomo-γ-linolenic acid (DGLA)-producing strains with disruption of the Δ5-desaturase (Δ5ds) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Δlig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the Δlig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Δlig4 strain was constructed, and then transformed for disruption of Δ5ds. The isolation of nine Δ5ds-disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Δ5ds-disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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 oil-producing zygomycete Mortierella alpina 1S-4 is known to accumulate beneficial polyunsaturated fatty acids. We identified the lig4 gene that encodes for a DNA ligase 4 homolog, which functions to repair double strand breaks by non-homologous end joining. We disrupted the lig4 gene to improve the gene targeting efficiency in M. alpina. The M. alpina 1S-4 Δlig4 strains showed no defect in vegetative growth, formation of spores, and fatty acid production, but exhibited high sensitivity to methyl methansulfonate, an agent that causes DNA double-strand breaks. Importantly, gene replacement of ura5 marker by CBXB marker occurred in 67% of Δlig4 strains and the gene targeting efficiency was 21-fold greater than that observed in disruption of the lig4 gene in the M. alpina 1S-4 host strain. Further metabolic engineering of the Δlig4 strains is expected to result in strains that produce higher levels of rare and beneficial polyunsaturated fatty acids and contribute to basic research on the zygomycete., (Copyright © 2015 Elsevier B.V. All rights reserved.)