Date Published: December 5, 2012
Author(s): Eulalia Tapia V, Andréia Anschau, Alessandro LV Coradini, Telma T Franco, Ana Carolina Deckmann.
In this work we performed assays for the genetic improvement of the oleaginous yeast Lipomyces starkeyi DSM 70296 focusing on its utilization for lipid biosynthesis from renewable sources. The genetic optimization was carried out by random mutagenesis by ultraviolet irradiation and mutant selection by cerulenin, a compound displaying inhibitory effects on lipid biosynthesis. Mutants demonstrating normal growth in presence of cerulenin were considered as good candidates for further studies. Using this strategy, we selected 6 mutants for further studies, in which their productivities were evaluated by fermentation in shaken flasks and bioreactor. The evaluation of the fermentative performance of mutants was carried out using xylose as sole carbon source; the fermentation of wild-type strain was used as reference. Using this strategy it was possible to identify one mutant (termed A1) presenting a significant increase in the productivity rates of both biomass and lipid in comparison to wild-type strain. A1 mutant was further studied in bioreactor using the same fermentation parameters optimized for L. starkeyi lipid production from a mixed carbon source (xylose:glucose), as previously determined by other studies in our laboratory. A1 presented a productivity increase of 15.1% in biomass and 30.7% in lipid productivity when compared to the wild-type strain with a similar fatty acid composition, despite a slight increase (approx. 7%) on the unsaturated fraction. Our work demonstrates the feasibility of the random mutagenesis strategy coupled with mutant selection based on cerulenin screening for the genetic improvement of the oleaginous yeast L. starkeyi.
Microorganisms constitute a promising alternative for the production of second generation biofuels and other valuable biochemicals. The major advantage of using microorganisms to obtain products of industrial interest is the fact that they do not require large cultivation areas, being usually cultured in fermentation vats and therefore not competing for agricultural land (Vicente et al.
2004, Meng et al.,
2009). Another important characteristic of microorganisms is the ability to use several complex materials such as lignocellulosic wastes as source of nutrients (Meng et al.
Prior the mutagenesis experiments, a dose/response assay was performed to determine the optimal UV-exposition period required to obtain the highest accumulation of DNA mutations of L. starkeyi cells, as indicated by lower survival rates (Kava-Cordeiro et al.
1995). After 40 min irradiation, the colony number reduced to approx. 5% of the total colonies present in control plates (Figure
Traditional tools of genetic engineering often run into technical difficulties when used in non-domesticated species, which usually display protective mechanisms preventing the manipulation of their DNA. This constitutes a major obstacle to the industrial utilization of microorganisms in the biosynthesis of second generation biofuels and other bioproducts (Ageitos et al.
The authors declare that they have no competing interests.