Enhancing The Biolipid Production Of Rhodotorula Sp. U13N3 By Metabolic Regulation

The development of renewable energy to replace fossil fuel to meet the needs of energy,environmental protection,sustainable development has become the consensus of the whole society.As an important raw material of the third-generation biofuel,microbial lipid synthesized by fermentation which has high calorific value and less pollutant emission.Therefore,the research has attracted more and more attention which aimed at the large-scale production of microbial lipids.Among them,the screening of microorganisms with low-cost substrates for high lipid production is the key.In this study,Rhodotorula sp.U13N3,a color mutant,was used as the starting strain.Firstly,the metabolomics method combined with the transcription level of key genes was used to construct the metabolic network of lipid production using glycerol as the carbon source,and the variation trends of intracellular metabolites content and lipid metabolism cell growth and lipid synthesis were analyzed respectively.Then Triacylglycerol lipase ATG15 gene and peroxidase AOX2 gene deletion engineering bacteria were constructed by CRISPR/Cas9.Subsequently,the lipid fermentation performance of engineering strain was determined and a better engineering strain was screened.The results were as follows:(1)The ~1H NMR metabolomics technology was used to determine the changes of intracellular metabolites in the growth phase(30 h)and lipid synthesis phase(96 h)of Rhodotorula sp.U13N3 and the original strain when they were fermented with glycerol as the carbon source,and the substrate and lipid metabolism networks were constructed combined with the gene transcription level.A total of 33 metabolites were identified by NMR technology.Compared with the original strain,there were 13 metabolites with significantly increased content in the growth phase of U13N3;There are nine declines.Only two metabolites in U13N3 increased and 17 metabolites decreased during lipid synthesis.After comprehensive analysis of the metabolites level,it was found that the intracellular carbon flow of U13N3 changed significantly,and the TCA cycle was strengthened during the growth period of U13N3 cells,showing a higher cell growth rate.In the later period,the carbon flow moved to the direction of lipid synthesis,showing a higher lipid accumulation rate.Further analysis showed that a series of gene transcription levels of U13N3 in fatty acid?-oxidation pathway were up-regulated,which was not conducive to lipid accumulation.(2)ATG15 and AOX2 gene deletion engineering bacteria were constructed by CRISPR/Cas9 gene editing system.The above analysis showed that the high activities of ATG15 and AOX2 might promote the decomposition and metabolism of triacylglycerol,which was not conducive to lipid accumulation.Therefore,the corresponding gRNA sequences of ATG15and AOX2 were designed and the recombinant plasmids were constructed.The recombinant plasmids and Cas9 plasmids were successively introduced into Rhodotorula U13N3 by Agrobacterium transformation.A total of 24 positive clones with ATG15 and AOX2 gene defects were screened and their fermentation performance was determined.Subsequently,the related sequences of four mutants(?AOX2-2,?AOX2-8,?ATG15-8 and?ATG15-12)with good fermentation performance were analyzed.The results showed that there were two repair modes of base deletion and partial increase near the target site.The number of base deletion was 15-50 bp,and the gene sequences of AOX2 and ATG15 were edited successfully.(3)Using glycerol as carbon source,the lipid production performance of four engineering strains was determined by batch fermentation method,and?ATG15-8 with good performance was screened.Firstly,according to the different optimal pH for biomass accumulation and lipid production in the growth of Rhodotorula,a two-stage pH control strategy was established and tested using?AOX2-2.The biomass of?AOX2-2 was 14.77g/L,and the lipid yield was 10.62 g/L.The biomass and lipid yield increased by 49.7%and80.26%,respectively,compared with the results of natural pH batch fermentation.Then,the fermentation performance of Rhodotorula was determined at constant high pH(pH 7.0).The results showed that high pH was helpful to further improve cell biomass and lipid yield.Among them,the intracellular lipid content of four engineering strains(?ATG15-12,?ATG15-8,?AOX2-2 and?AOX2-8)increased by 12.24%,6.88%,4.36%and 3.45%,respectively,compared with U13N3,indicating that ATG15 or AOX2 deficiency contributes to intracellular lipid accumulation.At the end of batch fermentation,the lipid yield,glycerol yield,glycerol yield and lipid production intensity of the engineered strain?ATG15-8reached 12.57 g/L,0.373 g/L,0.274 g/L and 0.114 g/L/h,respectively,which were 10.4%,1.6%,12.3%and 9.6%higher than those of U13N3,respectively,showing good application potential.