林果特异功能性脂肪酸的高效制备:培育还是培养?

Lipid is one of the three major nutrients that can perform important physiological functions in the human body,which is vital to human health.To adopt the concept of"all-encompassing approach to food",a diversified lipid supply system can be built based on animal,herb seeds,forest fruit and oleaginous yeasts.Lipids from different sources in nature provide different nutritional features depending on the composition of their functional fatty acids.As a very long-chain fatty acid(VLCFA),NA has attracted considerable attention due to its specific biological functions in cognitive improvement,brain development,and therapies for brain diseases.NA was traditionally extracted from sharks in the early years,but shark hunting destroyed marine ecosystems and has been banned in many regions by relevant policies and regulations.Furthermore,the natural NA-producing woody plants such as Malania oleifera,Acer truncatum and Xanthoceras sorbifolium suffer from harsh growing conditions and low lipid-yield.Applying food synthetic biology to produce high-level of NA in other organisms is thus a research hotspot.Currently,the heterologous biosynthesis of NA was achieved by both plant and microbial metabolic engineering,which were expected to lead to the sustainable production of NA by breeding transgenic crops or cultivating engineered strains.The species of 3-ketoacyl-CoA synthases(KCS),seed-specific promoters,transgene copy numbers,optimal genome integration sites,and sufficient specific substrates are critical factors for increasing NA content in plant metabolic engineering.The average percentage of NA in total fatty acid from transgenic Brassica carinata was up to 42.2%,which could produce about 27～32 Kg of NA per acre.In contrast to metabolic engineering of plants,microbial cell factory possesses the significant advantages of rapid growth rate,high productivity,strong controllability,no seasonal requirements,and not occupying arable land.Oleaginous yeasts are excellent chassis to produce functional fatty acids and their derivatives by metabolic engineering,owing to its characteristics of high lipid content,strong robustness and high cell-density cultivation.However,the following problems still exist in the high NA yield of oleaginous yeast modified by metabolic engineering:(1)During the elongation of C18:1 to C24:1,whether the synergistic effect of multiple 3-ketoacyl-CoA synthases(KCSs)with different substrate specificity is required,so as to improve the efficiency of fatty acid elongation(FAE)module;(2)the dynamic relationship between NA and lipid accumulation,and the genes coding enzymes that may lead to insufficient NA biosynthesis during the lipogenic stage have not been explored;(3)lack of effective promoter elements to drive the expression of key genes during lipid synthesis;(4)lack of fermentation process matching with engineered yeast producing high-yield NA.Therefore,Rhodosporidium toruloides with high-yield lipid was used as the chassis in this work.Firstly,the effects of individual and combined expression of KCSs genes coding enzymes preferencing different substrate on the synthesis of VLCFAs were investigated,so as to preliminarily construct engineered strains capable of synthesizing NA.Secondly,the dynamic relationship between NA synthesis and lipid synthesis was revealed through the analysis of fermentation process,and the transcriptome changes between cell proliferation phase and lipogenic phase were compared through transcriptomic techniques,so as to establish the relationship between lipid accumulation,NA synthesis and gene transcription level,and reveal the key target genes of NA synthetic pathway.Then,according to the transcriptome data of cell proliferation phase and lipogenic phase,the activated promoter of lipogenic phase was further mined,and the key steps of NA and lipid synthesis were regulated,and the desaturation pathway for NA synthesis was constructed.Finally,the high yield of NA was achieved at the fermenter level through fermentation optimization and process control.Through fed-batch fermentation by the final strain,the NA percentage of TFA,NA titer and NA productivity reached 46.3%,44.2 g/L and 0.230 g/L/h,markedly exceeding the highest current reports of metabolically engineered plants and oleaginous yeasts.