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Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides

  • Applied genetics and molecular biotechnology
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Abstract

Commercial interest in microbial lipids is increasing due to their potential use as feedstock for biodiesel production. The supply of NADPH generated by malic enzyme (ME; NADP+-dependent; EC 1.1.1.40) has been postulated as being the rate-limiting step for fatty acid biosynthesis in oleaginous fungi, based mainly on data from the zygomycete Mucor circinelloides studies. This fungus contains five genes that code for six different ME isoforms. One of these genes, malA, codes for the isoforms III and IV, which have previously been associated with lipid accumulation. Following a strategy of targeted integration of an engineered malA gene, a stable strain overexpressing malA and showing high ME activity has been obtained, demonstrating the feasibility of this strategy to overexpress genes of biotechnological interest in M. circinelloides. This is the first report showing the integration and overexpression of a gene in Zygomycetes. Unexpectedly, the genetically modified strain showed a lipid content similar to that of a prototrophic non-overexpressing control strain, suggesting that another limiting step in the fatty acid synthesis pathway may have been revealed as a consequence of the elimination of malic enzyme-based bottleneck. Otherwise, the fact that prototrophic strains showed at least a 2.5-fold increase in lipid accumulation in comparison with leucine auxotrophic strains suggests that a wild-type leucine biosynthetic pathway is required for lipid accumulation. Moreover, increasing concentrations of leucine in culture medium increased growth of auxotrophs but failed to increase lipid content, suggesting that the leucine synthesized by the fungus is the only leucine available for lipid biosynthesis. These results support previous data postulating leucine metabolism as one of the pathways involved in the generation of the acetyl-CoA required for fatty acid biosynthesis.

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Acknowledgment

We thank J. A. Madrid for technical assistance. This work was funded by the Dirección General de Investigación y Política Científica (Comunidad Autónoma de la Región de Murcia, Spain), Project BIO-BMC 07/01-0005. The authors declare that they have no conflict of interest.

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  1. Rosa Amarilis Rodríguez-Frómeta

    Present address: Departamento de Ciencia de Alimentos, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Avda. Agustín Escardino, 7, 46980, Paterna, Valencia, Spain

Authors and Affiliations

  1. Departamento de Genética y Microbiología (Unidad asociada al IQFR-CSIC), Facultad de Biología, Universidad de Murcia, 30071, Murcia, Spain

    Rosa Amarilis Rodríguez-Frómeta, Adrián Gutiérrez, Santiago Torres-Martínez & Victoriano Garre

  2. Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, 30071, Murcia, Spain

    Victoriano Garre

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  1. Rosa Amarilis Rodríguez-Frómeta
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  2. Adrián Gutiérrez
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  3. Santiago Torres-Martínez
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  4. Victoriano Garre
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Correspondence to Victoriano Garre.

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Rodríguez-Frómeta, R.A., Gutiérrez, A., Torres-Martínez, S. et al. Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides . Appl Microbiol Biotechnol 97, 3063–3072 (2013). https://doi.org/10.1007/s00253-012-4432-2

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  • DOI: https://doi.org/10.1007/s00253-012-4432-2

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