| Oleaginous microorganisms can accumulate a large amount of fatty acids in the cell and store them in the form of triacylglycerol, which is an important alternative to traditional animal and vegetable oils. Furthermore, the accumulated lipid of some microbial has important application value and commercial value as it is rich in polyunsaturated fatty acids. Lipid accumulation in oleaginous microorganisms is species- and strain-dependent. Amounts of cellular lipid vary between 20% cell dry weight and more than 80%. It is difficult to decipher the molecular mechanism of the different capacity of lipid accumulation among different microbial species because of their diverse physiological and biochemical characteristics. However, the research of different strains, whose capacity of lipid accumulation have significant difference, within the same species can be a good solution for the problem. This study systematically investigated the regulation mechanism between high lipid-producing strain WJ11 and low lipid-producing strain CBS 277.49 in Mucor circinelloides by genomics, proteomics and key enzyme activity analysis. This study also found some key genes regulating lipid biosynthesis through transcription analysis and the lipid content was increased when these genes were overexpressed in M. circinelloides. In addition, the effect of amino acid nitrogen sources on γ-linolenic(GLA) production was investigated. The main research contents and results are as follows.The lipid analysis showed strain WJ11 can produce lipid up to 36%, while the value of CBS 277 is no more than 15%. The genome of M. circinelloides WJ11 was sequenced and the genome assembly size was 35.4 Mb. The genome of strain WJ11 consisted of 827 scaffolds and 2530 sequence contigs with a G+C content of 39.7%. The numbers of predicted protein-coding genes and t RNAs were 10973 and 177, respectively. The general genome features of WJ11(sequenced in this study) and CBS 277.49(sequenced by Joint Genome Institute) were similar. Whole genome alignments with MAUVE revealed the presence of numerous blocks of homologous regions in WJ11 and CBS 277.49, and MUMmer analysis showed that the genomes of these two strains were mostly co-linear. The central carbon and lipid metabolism pathways of these two strains were reconstructed. These mapped the utilization of glucose, the biosynthesis of fatty acids, triacylglycerols, phospholipids, steroids and carotenoids, and β-oxidation. The different numbers of genes encoding the enzymes in these pathways between WJ11 and CBS 277.49 may have an effect on the lipid accumulation. Some unique genes were identified for each strain using Ortho MCL clustering procedure, which may play a role in different cell growth and lipid accumulation in these two strains.Comparative proteomic analysis between the lipid accumulation stage, under nitrogen(N) deficiency, and the balanced phase of growth in strain WJ11 demonstrated that N depletion increased the expression of proteins involved in nitrogen assimilation but decreased the metabolism of amino acids. Upon N deficiency, many proteins involved in glycolytic pathway were up-regulated while proteins involved in the tricarboxylic acid cycle were down-regulated, indicating this activity was retarded thereby leading to a greater flux of carbon into fatty acid biosynthesis. Moreover, the pentose phosphate pathway was up-regulated, leading to the increased production of NADPH, the reducing power for fatty acid biosynthesis. Furthermore, protein and nucleic acid metabolism were down-regulated and some proteins involved in energy metabolism, signal transduction, molecular chaperone and redox homeostasis were up-regulated upon N depletion, which may be the cellular response to the stress produced by the onset of N deficiency. On the other hand, the comparative proteomic analysis between strains WJ11 and CBS 277.49 at the fast lipid accumulation stage indicated the expression of proteins related to branched chain amino acid metabolism were significantly increased in WJ11, this may provide more sources of substrates for fatty acid synthesis. The expression of proteins involved glycolytic pathway were up-regulated while that involved in tricarboxylic acid cycle were down-regulated in WJ11 compared with CBS 277.49, which resulted in a greater flux of carbon into fatty acid biosynthesis. In addition, some proteins taking part in pentose phosphate pathway were up-regulated, leading to more production of NADPH in WJ11 than CBS 277.49.Comparison of biochemical activities between WJ11 and CBS 277.49 showed the activity of phosphofructokinase(PFK) was lower in WJ11, while activities of glucose-6-phosphate dehydrogenase(G6PDH) and 6-phosphogluconate dehydrogenase(6PGDH), involved in pentose phosphate pathway, in strain WJ11 were greater than that in CBS 277.49. This suggested that the glucose flux to pentose phosphate pathway was up-regulated in strain WJ11 and increased amounts of NADPH for fatty acid biosynthesis. The activity of malic enzyme was only slightly higher in WJ11 than in CBS 277.49 during the initial part of the lipid accumulation phase. The activity of isocitrate dehydrogenase(ICDH) was lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase(ACL) to produce acetyl-Co A. Also, the activity of ACL and fatty acid synthase(FAS) in strain WJ11 were greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains. The key enzymes from comparison of biochemical activities and key proteins identified from proteomic research were analyzed at transcription level by RT-q PCR. The results of transcription analysis were in accordance with the data from biochemical activities and comparative proteomics. Interestingly, the expressions of g6pdh1 and g6pdh2 in WJ11 were increased drastically at the lipid accumulation stage than the balanced phase of growth. In addition, comparing to the balanced phase of growth, transcription level of some genes(e.g., leu B) involved in branched chain amino acid metabolism at lipid accumulation stage in WJ11 were decreased much less than that in CBS 277.49, these genes may also take part in regulation of lipid biosynthesis.The results of genomics, proteomics, enzyme activities and transcription analysis suggested that g6pdh1, g6pdh2 and leu B from WJ11 might play a critical role in regulation of lipid accumulation. The recombination strains, overexpressed g6pdh1, g6pdh2 and leu B from WJ11, all increased the lipid accumulation but had no effect on cell growth. Among them, the lipid content of recombination strains overexpressed g6pdh1 were enhanced by 23~28% and that overexpressed g6pdh2 were enhanced by 41~47%. This indicated that both g6pdh1 and g6pdh2 can regulate the lipid accumulation, and g6pdh2 is more effective. Furthermore, the lipid content of recombination strains overexpressed leu B was increased by 67~73%, suggesting that leu B plays an important role in lipid accumulation.In order to investigate the regulation of amino acids on lipid accumulation, the cell growth, total fatty acids production and GLA yield were analyzed when 20 standard amino acids were used as single nitrogen source. The comparison of three strains(WJ11, CBS 108.16 and CBS 277.49) in this laboratory on the capacity of producing GLA showed CBS 108.16 had the highest capacity of accumulating GLA. The strain WJ11 had the highest total fatty acids production but the GLA yield was much lower than CBS 108.16 as its low GLA content in total fatty acids. Therefore, strain CBS 108.16 was used as further research and then the results showed that different amino acids affected the cell growth, fatty acids production, and yield of GLA in M. circinelloides. Tyrosine appeared to be the most favorable amino acid nitrogen source for the cell growth and total fatty acids production, which lead to the highest yield of GLA(0.81 g/L) among the 20 amino acids. The analysis of lipogenic enzyme activities of the fungus during lipid accumulation phase indicated that tyrosine enhanced the activities of G6 PDH and 6PGDH, providing more NADPH for fatty acids biosynthesis, and increased the activity of ACL, providing more acetyl-Co A for fatty acid biosynthesis. Also, the activity of ICDH was down-regulated by tyrosine, which might result in a more carbon flux to fatty acid biosynthesis rather than tricarboxylic acid cycle. This is in according with the results of proteomic analysis and enzyme activities related to lipid biosynthesis between strains WJ11 and CBS 277.49. |
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