RESUMO
Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.
Assuntos
Proteínas de Algas/genética , Meios de Cultura/farmacologia , Ácidos Docosa-Hexaenoicos/biossíntese , Ácido Eicosapentaenoico/biossíntese , Proteoma/genética , Estramenópilas/efeitos dos fármacos , Proteínas de Algas/classificação , Proteínas de Algas/isolamento & purificação , Biomassa , Carotenoides/biossíntese , Carotenoides/isolamento & purificação , Meios de Cultura/química , Ácidos Docosa-Hexaenoicos/isolamento & purificação , Ácido Eicosapentaenoico/isolamento & purificação , Expressão Gênica , Ontologia Genética , Glucose/metabolismo , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Maltose/metabolismo , Maltose/farmacologia , Anotação de Sequência Molecular , Proteoma/classificação , Proteoma/isolamento & purificação , RNA Ribossômico 18S/genética , Água do Mar/química , Análise de Sequência de DNA , Amido/metabolismo , Amido/farmacologia , Estramenópilas/genética , Estramenópilas/crescimento & desenvolvimento , Estramenópilas/metabolismoRESUMO
Background: Mutation breeding is one of the most important routes to achieving high docosahexaenoic acid (DHA) productivity using Schizochytrium. However, few selection strategies have been reported that aim to generate a high DHA content in Schizochytrium lipids. Results: First, culture temperature altered the butanol tolerance of Schizochytrium limacinum B4D1. Second, S. limacinum E8 was obtained by selecting mutants with high butanol tolerance. This mutant exhibited a 17.97% lower proportion of DHA than the parent strain S. limacinum B4D1. Third, a negative selection strategy was designed in which S. limacinum F6, a mutant with poor butanol tolerance, was obtained. The proportion of DHA in S. limacinum F6 was 11.22% higher than that of parent strain S. limacinum B4D1. Finally, the performances of S. limacinum B4D1, E8 and F6 were compared. These three strains had different fatty acid profiles, but there was no statistical difference in their biomasses and lipid yields. Conclusion: It was feasible to identified the relative DHA content of S. limacinum mutants based on their butanol tolerance.