微生物合成植物源神经酸的代谢途径组装与调控研究

Nervonic acid is a kind of very long chain monounsaturated fatty acid with special function of repairing the human brain nerve cells and fibers. Therefore, it is regarded as the essential nutrient for brain health. Nowadays, the nervonic acid is generally extracted from some rare plants. However, this method takes a long period, and usually has a high cost. Therefore, it cannot meet the gradually expanded needs of people. In order to solve the problem, in this proposal, based on the principle of synthetic biology, we are going to develop a ‘push and pull’ strategy to construct and optimize the metabolic pathway in Yarrowia lipolytica for accumulating nervonic acid. Firstly, we will try to strengthen the supply of precursor (C18:1, oleinic acid) for nervonic acid synthesis by knocking out the delta12-desateurase catalyzing the reaction from C18:1 to C18:2 (linoleic acid) using the CRISPR/Cas9 genome editing technology. Secondly, we will try to assemble different genes encoding β-ketoacyl-CoA synthases of fatty acid elongase complex with different substrate specificity of fatty acid to construct the artificial fatty acid elongation pathway in Y. lipolytica. The final object is to extend the C18:1 in the yeast cells to C24:1 (nervonic acid) with the help of the artificial pathway. We will use the DNA-guided spatial optimization strategy to assemble the artificial elongation pathway for precisely controlling the chain length of the object fatty acid. Thirdly, we will try to alter the intracellular nervonic acid storage form by introducing a plant derived acyl transfer reaction pathway with substrate specificity towards nervonic acid. Through the above operations, the three modules, i.e., nervonic acid precursor supply, synthesis, and accumulation modules would be balanced dynamically. Thus the nervonic acid heterologous biosynthesis efficiency would be improved, and the adaptation mechanism of synthetic nervonic acid metabolic pathway and microbial cell would be revealed. The method developed in the present proposal, i.e., optimizing the heterologous metabolic pathway through ‘push and pull’ modular construction and spatial optimization strategy, aiming at maximizing the metabolic flux to the heterologous metabolites, will provide a powerful toolbox for the bio-manufacturing plant derived natural products.

神经酸是一种对神经细胞和纤维有修复功能的超长链单不饱和脂肪酸，是脑健康必须的营养素；目前采用从稀有植物中提取获得，该法周期长成本高，不能满足人们的需求。针对上述问题，基于合成生物学原理，拟采用“推拉式”模块化的研究思路，在不能合成神经酸的解脂耶氏酵母体内，构建并优化神经酸合成的代谢途径。通过CRISPR/Cas9基因编辑技术，强化神经酸合成前体的供应；通过DNA介导的支架结构区域化调控不同底物特异性脂肪酸延长酶，精准控制目标脂肪酸的链长；通过引入对神经酸具有底物特异性转酰基反应途径，改变其胞内储存形式。通过上述操作，实现神经酸前体供应模块、合成模块与积累模块的动态平衡，提高神经酸异源生物合成效率的同时，剖析人工构建的神经酸合成途径和微生物细胞的适配机制。发展一种通过模块化构建、区域化调控优化异源代谢途径，实现代谢流最大化地导向异源代谢产物的方法，为植物源天然产物的生物制造提供新的技术手段。

神经酸（C24:1）是一种对神经细胞和纤维有修复功能的超长链单不饱和脂肪酸，是脑健康必须的营养素。目前采用从稀有植物中提取获得，该法周期长成本高，不能满足人们的需求。针对上述问题，拟在产油脂酵母解脂耶氏酵母中再造神经酸的合成代谢途径，以实现神经酸的高效生产。根据解脂耶氏酵母脂肪酸与油脂合成途径将拟构建的神经酸代谢通路划分为四个模块：前体模块，合成模块，积累模块，降解模块。分别对四个模块进行优化，以“推-拉-阻”（Push-Pull-Block）策略实现四个模块的动态平衡。在此过程中，针对前体模块与合成模块，发现效仿植物中以油酸（C18:1）为前体的延长-去饱和途径在解脂耶氏酵母中并不奏效，分析其原因是解脂耶氏酵母中油脂积累的三个关键酶：甘油-3-磷酸酰基转移酶(GPAT)，溶血磷脂酸酰基转移酶(LPAAT)，二脂酰甘油酰基转移酶(DGAT)，天然对C18:1有较强的特异性，对C24:1特异性较弱，C18:1还未经过β-酮脂酰辅酶A合成酶（KCS）催化延伸便被积累到甘油三酯（TAG）中了。为此，尝试构建了一条自然界不存在的去饱和-延长途径，使用胞内硬脂酸（C18:0）作为前体合成神经酸；通过筛选不同植物和微生物来源的关键基因元件，再造了一条全新的神经酸合成通路（组合表达银扇草来源的β-酮脂酰辅酶A合成酶LaKCS3，拟南芥来源的β-酮脂酰辅酶A合成酶AtKCS18以及高山被孢霉来源的Δ15去饱和酶Maω9），经过初步优化，并结合降解模块的改造，神经酸占总脂肪酸的比例达到了10%，对积累模块的优化正在进行中。采用该人工再造的神经酸合成途径有望实现解脂耶氏酵母中神经酸产量的进一步提高。此外，在不同神经酸代谢途径的构建与评价过程中，解析了人工合成代谢途径与宿主解脂耶氏酵母底盘细胞的适配机制。