细胞膜合成途径模块化调控与形态改造提高大肠杆菌β-胡萝卜素的积累与产量

Improving β-carotene production in Escherichia coli by modularized regulation of the membrane synthetic pathway and morphology engineering

β-胡萝卜素是类胡萝卜素家族中的典型代表,属于疏水性较强的化合物,前期研究表明,改变细胞膜形态以及增加3-磷酸甘油二酯的供给,均可容纳更多的β-胡萝卜素,从而提高其产量。然而在之前的研究中,没有对细胞膜的磷脂中主要组分磷脂酰乙醇胺的合成途径对β-胡萝卜素积累的影响进行系统的讨论。本研究将磷脂酰乙醇胺的合成途径分为上中下游3个模块,对它们的多种表达组合策略进行比较。首先过表达了上游模块1,菌株CAR016的β-胡萝卜素的产量与单位细胞的β-胡萝卜素产量均有显著提高,分别可达到44 mg/L以及13.7 mg/g DCW。与对照菌株相比,分别提高30.5%与35.6%。过表达磷脂酰乙醇胺合成的中游模块,β-胡萝卜素的产量以及单位细胞的β-胡萝卜素的产量分别为103.5 mg/L DCW与19.8 mg/g DCW。与对照菌株CAR016(pACYC184-M)相比,分别提高1.4倍与53.5%。将上游模块1与中游模块2共表达,菌株CAR016(pModule1,pModule2)单位细胞的β-胡萝卜素产量为22.3 mg/g DCW。与CAR016(pModule2)相比,单位细胞产量提高18%,与出发菌株CAR016(pTrc99A-M,pACYC184-M)相比,单位细胞的β-胡萝卜素产量提高122%。本研究找到了磷脂酰乙醇胺合成途径表达的最优组合策略,可以产生更大量的细胞膜,为储存β-胡萝卜素提供了更多的空间,从而进一步提高β-胡萝卜素的产量。细胞膜形态和合成途径的模块化改造,是今后提高类胡萝卜素产量的新方向。

Carotenoids are a class of terpenes of commercial interest and exert important biological functions. Engineering morphological and biosynthetic aspects of Escherichia coli cell membrane could improve its storage capacity for β-carotene. However, how the synthesis of phosphatidylethanolamine, the major component of the cell membrane, was not discussed in detail. In this work, the synthesis of phosphatidylethanolamine was divided into three modules to discuss their synergetic effect, by expressing in different combinations. Overexpressing the upstream module 1 in CAR016 caused a 30.5% increase of β-carotene specific production（from 10.1 mg/g to 13.7 mg/g DCW）; combined overexpressing module 1 and module 2 in CAR016 led to a 122% increase of β-carotene specific production（from 10.5 mg/g to 22.3 mg/g DCW）. The optimal expression combination of the phosphatidylethanolamine synthetic pathway was obtained, which further increased the content of the cell membrane for β-carotene storage, and improved its production. The membrane engineering strategy opens up a new direction for engineering microbial producers for a large spectrum of hydrophobic molecules.