RESUMO
Long-chain (C20-24) polyunsaturated fatty acids (LC-PUFA) are physiologically important nutrients for vertebrates including fish. Previous studies have addressed the metabolism of LC-PUFA in the Amazonian teleost tambaqui (Colossoma macropomum), an emerging species in Brazilian aquaculture, showing that all the desaturase and elongase activities required to convert C18 polyunsaturated fatty acids (PUFA) into LC-PUFA are present in tambaqui. Yet, elongation of very long-chain fatty acid 4 (Elovl4) proteins, which participate in the biosynthesis of very long-chain (>C24) saturated fatty acids (VLC-SFA) and very long-chain polyunsaturated fatty acids (VLC-PUFA), had not been characterized in this species. Here, we investigate the repertoire and function of two Elovl4 in tambaqui. Furthermore, we present the first draft genome assembly from tambaqui, and demonstrated the usefulness of this resource in nutritional physiology studies by isolating one of the tambaqui elovl4 genes. Our results showed that, similarly to other teleost species, two elovl4 gene paralogs termed as elovl4a and elovl4b, are present in tambaqui. Tambaqui elovl4a and elovl4b have open reading frames (ORF) of 948 and 912 base pairs, encoding putative proteins of 315 and 303 amino acids, respectively. Functional characterization in yeast showed that both Elovl4 enzymes have activity toward all the PUFA substrates assayed (18:3n-3, 18:2n-6, 18:4n-3, 18:3n-6, 20:5n-3, 20:4n-6, 22:5n-3, 22:4n-6 and 22:6n-3), producing elongated products of up to C36. Moreover, both Elovl4 were able to elongate 22:5n-3 to 24:5n-3, a key elongation step required for the synthesis of docosahexaenoic acid via the Sprecher pathway.
Assuntos
Elongases de Ácidos Graxos/genética , Ácidos Graxos Insaturados/metabolismo , Proteínas de Peixes/genética , Peixes/metabolismo , Sequência de Aminoácidos , Animais , Brasil , Clonagem Molecular , Ácidos Docosa-Hexaenoicos/biossíntese , Elongases de Ácidos Graxos/isolamento & purificação , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/genética , Proteínas de Peixes/metabolismo , Genoma , Fases de Leitura Aberta , Filogenia , Alinhamento de SequênciaRESUMO
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
Alanine mother liquor, a type of industrial waste from alanine fermentation, was used as a nitrogen source to produce docosahexaenoic acid (DHA) by Schizochytrium sp. B4D1. The results indicated that yeast extract could trigger the utilization of the alanine mother liquor. Additionally, the alanine can be quenched during the culture, which aids in DHA accumulation. The medium components were optimized via response surface methodology as follows: 99.98-g/L glucose, 0.05-g/L yeast extract and a 183.17 dilution factor of the alanine mother liquid (v/v, with an alanine content of 0.72 g/L) and 17.98% inoculum concentration (v/v). Finally, in a 50-mL shake-flask fermentation, the DHA yield was 2.29 g/L.
Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Alanina/metabolismo , Estramenópilas/metabolismo , Leveduras , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Alanina/análise , Fermentação , Glucose , Resíduos IndustriaisRESUMO
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.
Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Butanóis/metabolismo , Estramenópilas/genética , Estramenópilas/metabolismo , Seleção Genética , Temperatura , Ácido Eicosapentaenoico/metabolismo , Biomassa , Butanóis/toxicidade , Ácidos Graxos/metabolismo , Ácidos Graxos/química , Estramenópilas/efeitos dos fármacos , Fermentação , MutaçãoRESUMO
The high costs and environmental concerns associated with using marine resources as sources of oils rich in polyunsaturated fatty acids have prompted searches for alternative sources of such oils. Some microorganisms, among them members of the genus Aurantiochytrium, can synthesize large amounts of these biocompounds. However, various parameters that affect the polyunsaturated fatty acids production of these organisms, such as the carbon and nitrogen sources supplied during their cultivation, require further elucidation. The objective of this investigation was to study the effect of different concentrations of carbon and total nitrogen on the production of polyunsaturated fatty acids, particularly docosahexaenoic acid, by Aurantiochytrium sp. ATCC PRA-276. We performed batch system experiments using an initial glucose concentration of 30 g/L and three different concentrations of total nitrogen, including 3.0, 0.44, and 0.22 g/L, and fed-batch system experiments in which 0.14 g/L of glucose and 0.0014 g/L of total nitrogen were supplied hourly. To assess the effects of these different treatments, we determined the biomass, glucose, total nitrogen and polyunsaturated fatty acids concentration. The maximum cell concentration (23.9 g/L) was obtained after 96 h of cultivation in the batch system using initial concentrations of 0.22 g/L total nitrogen and 30 g/L glucose. Under these conditions, we observed the highest level of polyunsaturated fatty acids production (3.6 g/L), with docosahexaenoic acid and docosapentaenoic acid 6 concentrations reaching 2.54 and 0.80 g/L, respectively.(AU)