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.
Preliminary studies performed in our group indicate that such difficulties may constitute major obstacles for the genetic manipulation of the oleaginous yeast Lipomyces starkeyi DSM 70296, a promising candidate for biodiesel production. In the present work, we have employed an alternative strategy to achieve the genetic optimization of L. starkeyi: the random mutagenesis by UV irradiation associated to a cerulenin-based mutant screening assay.
Cerulenin, or 2,3-epoxy-4-oxo-10-dodecadienamide, is a natural product of ascomycete fungus Cephalosporium caerulens, being originally described by Hata and colleagues as presenting antifungal properties (Hata et al.
1960). Further studies revealed that cerulenin constitutes a potent inhibitor of lipid biosynthesis (
Satoshi 1976), and this response was demonstrate to be related to its inhibitory effects on the activity of the enzyme fatty acid synthase (Kawaguchi et al.
Since its characterization, cerulenin has been widely used as a valuable tool in biochemistry studies focusing on the synthesis and metabolism of fatty acids and sterols by microorganisms (
Altenbern 1977, Parrish et al.
1999, Parsons et al.
2011). Recent studies have employed this molecule as nutritional supplement for obtaining improvements on the native production of poly-unsaturated fatty acids by Moritella marina and Shewanella marinintestina (Morita et al.
2005), as well as a selective agent in the screening of high lipid production by Rhodotorula glutinis mutants (Wang et al.
2009) and high gluthatione production by Saccharomyces cereviseae and Candida utilis mutants (Nishiushi et al.
In the case of the selection of mutants presenting high lipid production, it sounds quite controversial the use of cerulenin in the screening steps since this molecule inhibits the metabolism of interest. The rationale of this strategy resides in the fact that the lipid biosynthesis is an essential metabolism for cell growth, thus its inhibition by cerulenin determines a marked reduction in the growth of the colonies on the culture plates. Therefore, it allows a simple assay to assess metabolic alterations introduced by mutagenesis: mutants displaying normal growth in the presence of cerulenin are good candidates to be high-lipid producers due to their ability to overcome this inhibition.
Accordingly, in the original work presenting this methodology, Wang and colleagues identified two R. glutinis mutants presenting an increase of approx. 60% in lipid content in comparison to the wild-type (Wang et al.
2009). In the study using S. cereviseae and C. utilis, authors obtained a > 100% increase in glutathione production when associating cerulenin to the screening of mutants (Nishiushi et al.
Thus, we employed a similar strategy in the screening of candidates for high lipid production among Lipomyces starkeyi DSM 70296 mutants. Our method of choice for mutagenesis was UV radiation due its good cost-benefit relation. In our experimental setting, the optimal UV exposure time for L. starkeyi was 40 min, corresponding to a 5% cell survival. We preferred to work with 5% survival rather than 1% to increase the population of viable cells in the mutant selection step.
With the cerulenin-based screening strategy, we isolated a total of 90 mutants presenting large colony size in comparison to wild-type strain (non-irradiated cells), from which the 6 largest were further studied by cultivation in liquid media. The mutant identified as A1 showed a significant increase of both biomass and lipid productivity rates, which were further confirmed by fermentation in bioreactor following the same process parameters used for optimization of L. starkeyi lipid production in our laboratory. Results confirmed the increase in both biomass (15.1%) and lipid productivity (30.7%) of A1 mutant.
The analysis of lipid profile shows that the fatty acid composition of this mutant did not vary significantly from wild-type, despite a small increase (approx. 7%) in the unsaturated fatty acid content. From a biotechnological perspective, these results are interesting since the saturation degree of fatty acids determines important physical-chemical properties of oils, like viscosity, oxidation susceptibility and melting point (
The alteration on unsaturated fatty acid content observed in A1 derived mostly on an increase in oleic acid (C18:1) accompanied by a proportional reduction on stearic acid (C18:0), indicating a possible involvement on gene encoding the enzyme delta-9 fatty acid desaturase (EC 126.96.36.199). This enzyme introduces a double bond in saturated fatty acyl-CoA substrates producing monounsaturated fatty acids (Martin et al.
2007); thus, an increase of its activity could result in a superior content of oleic acid in detriment of its precursor, stearic acid, which is in accordance to our results.
Moreover, these alterations may be secondary to the mutant screening by cerulenin, as reported by other studies on literature. Morita and colleagues obtained an increase in poly-unsaturated fatty acids after cerulenin treatment in marine bacteria (Morita et al.
2005); Hauvermale and colleagues investigated the influence of cerulenin on the synthesis of fatty acids in Schizochytrium sp. and their results revealed a significant reduction in the synthesis of saturated fatty acids after cerulenin supplementation (Hauvermale et al.
2006). Same response was observed by Chaung and colleagues in Aurantiochytrium mangrovei (Chaung et al.
In these studies, authors suggest the involvement of polyketide pathway to overcome FAS inhibition by cerulenin, resulting in these alterations on fatty acid composition. Despite these works are referring to prokaryotes and the effects reported therein were triggered in the presence of cerulenin, the effects observed by our work may indicate a similar response even if we consider that we are focusing on eukaryotes and in the absence of cerulenin (fatty acid composition was determined from oils produced during fermentation on a culture media with no trace of cerulenin). The reason would be that, since we employed cerulenin during mutant screening step, we may have selected those individuals presenting mutations on genes present in the same metabolic pathways regulated in the studies above mentioned.
However, the genetic characterization on A1 mutant was not performed yet therefore we are not able to investigate this hypothesis, neither the possible involvement on delta-9 fatty acid desaturase in the differences of oleic-to-stearic fatty acid proportion in A1.
In conclusion, our results constitute an important step on the genetic optimization of non-domesticated oleaginous species for industrial purposes, despite the increase on fermentative performance of mutants obtained from UV-irradiated L. starkeyi DSM 70296 was not so evident than those reported in these other studies (Wang et al.
2009; Nishiushi et al.
2012). Further studies should be performed to adapt this strain to the conditions of fermentation of hemicelluloses hydrolysates of sugarcane bagasse, as well as to characterize metabolic changes using the tools of genomics in order to identify which genes/enzymes were modified during mutagenesis procedures. Finally, such a mutant may be employed for improving its desirable characteristics through evolutionary engineering methodologies in order to speed the fixation and breeding of the genetic traits with industrial interest.