Effects Of Matps Gene And Mapepck Gene On Lipid Accumulation In Mortierella Alpin A

Mortierella alpina is a promising oil producing filamentous fungus with excellent PUFAs synthesis ability.In order to adapt to the industrial production of PUFAs,it is necessary to further improve its lipid accumulation ability.The overall carbon metabolic flux of central carbon metabolism in the direction of glucose-6-phosphate(G-6-P)to pyruvate(Pyr)provides more precursors for fatty acid synthesis,which promotes the accumulation of carbon source in form of storage lipid.On the contrary,the metabolic pathway of carbon flux shunt in the direction of G-6-P to Pyr results in the reduction of final fatty acid synthesis.Trehalose can also serve as storage carbon source and it also helps microorganisms to resist to the environmental stress.Besides lipid,trehalose synthesis is another major carbon storage mode in M.alpina.By diverting G-6-P,carbon flux in lipid accumulation pathway is weakened,while gluconeogenesis pathway will cause the diversion of Pyr and lead carbon flux into carbohydrate synthesis.In order to improve the redistribution of carbon flux from carbohydrate to lipid synthesis,RNA interference was performed on the trehalose-6-phosphate synthase(Matps)and phosphoenolpyruvate carboxykinase(Mapepck),the key genes in trehalose synthesis and gluconeogenesis in M.alpina,respectively.The gene transcription level,growth status,glucose consumption and lipid metabolism of the recombinant strains were analyzed,and the effects of Matps and Mapepck on the synthesis of carbohydrate and lipids in M.alpina were investigated.In addition,the response to environmental stress in Matps RNA interference strain and the difference between carbohydrate and lipid accumulation in Mapepck RNA interference strain were studied in a 7.5 L fermentator.This paper provides a theoretical basis for analyzing the effect of mattps and Mapepck in M.alpina on the distribution of carbon flux in the process of lipid accumulation and improving the ability of lipid accumulation of M.alpina.1.The extraction and analysis methods of sugars in M.alpina were established.Three kinds of heating extraction and three kinds of circulating freeze-thaw extraction methods were compared.The total sugars were determined by hydrochloric acid heating extraction combined with phenol sulfuric acid method,and the sugars were analyzed by high performance liquid chromatography combined with evaporative light scattering detector after acetonitrile water circulating freeze-thaw extraction The results showed that M.alpina accumulated high levels of sugars in the process of lipid accumulation.The highest content of total sugars was 26% of the dry weight of M.alpina,and trehalose was about 20% of the total sugars.Carbohydrate synthesis is the main competitive pathway of lipid synthesis.Trehalose with the highest content may be an effective target to interfere with carbohydrate accumulation.In addition,gluconeogenesis,which provides carbon source for total sugar accumulation,may be another effective target.2.The key enzymes in trehalose biosynthesis(Matps)and gluconeogenesis(Mapepck)in M.alpina were selected by bioinformatics analysis.The recombinant strains of Matps and Mapepck were constructed by Agrobacterium tumefaciens mediated transformation and RNA interference.The transcriptional level of Matps and Mapepck were decreased about by 60% and41%,respectively,indicating that RNA interference successfully reduced the expression of related pathways at gene and protein levels.3.In the later stage of fermentation,the content of trehalose was decreased by 37%,and the content of fatty acids increased by 20%.There was no significant effect on fatty acid composition,but the yield of ARA increased by 30% in the later stage.The results showed that under the condition of nitrogen limitation,down-regulation of Matps expression by RNA interference could reduce trehalose accumulation,and distribute part of carbon flux to fatty acid synthesis;under mild stress,Matps mainly played a role in controlling carbon flux diversion,while under severe stress,the ability of trehalose in stress resistence could not be ignored,and the normal expression of Matps had a significant effect on stress state It plays an important role in maintaining the activity of the strain.4.In the process of lipid accumulation,the RNA interference of Mapepck reduced the content of intracellular total carbohydrates by 17%?28%,while the proportion of trehalose in total carbohydrates remained unchanged,and the lipid content was increased by 17% ? 26%and the yield of ARA increased by 30% in the later stage.Unlike Matps,Mapepck changed the distribution of carbon metabolic flux between intracellular carbohydrates and lipid.However,it did not affect the proportion of trehalose in total carbohydrates,indicating that repressed Mapepck expression reduced the level of gluconeogenesis and led to the decreased carbohydrates synthesis level,thus promoting lipid accumulation.Metabolite analysis of the recombinant strain showed that down-regulated Mapepck reduced the carbon flux into gluconeogenesis,but also accelerated the pyruvate-citric acid cycle and promoted the production of fatty acid precursors.Overall,Mapepck RNAi could not only increase the production of fatty acids,but also advance the time to reach the maximum accumulation of lipid.The analysis of carbohydrates and lipid accumulation is helpful to optimize the distribution of carbon metabolism flow in M.alpina,and provides a theoretical basis for promoting lipid synthesis by regulating sugar synthesis in M.alpina.