基于全基因组测序和基因组尺度代谢网络分析的钝齿棒杆菌产精氨酸分子育种研究

Molecular breeding techniques have been intensively employed for modification of the metabolic and regulatory networks of corynebacterium, a normal arginine production strain, for improving arginine yield. However, classical strain engineering only focus on arginine biosynthetic pathway and has some limitations because of not considering systems metabolic network. In silico genome-scale metabolic network (GSMN) model is a new methods for analysis of characteristics of system metabolic pathway of organisms. Application of GSMN method in corynebacterium for improving arginine production has never been reported. In this proposal, Corynebacterium crenatum MT, a mutant strain from C. crenatum AS 1.542 with ability of arginine accumulation during fermentation process, will be used as targeted strain for analysis of GSMN. Optimal strategy for high yield and stable arginine-producing genetic engineered bacteria will be obtained. The genomes of C. crenatum will be sequenced and the relationship of genotype and phenotype will be established. The new GSMN model Cg_iKK446_arginie was obtained by modifying the Cg_iKK446 of C. glutamicum ATCC 13032 according to the genotype of C. crenatum MT. Flux distribution comparison analysis (FDCA) will be employed mainly to predict knocked-out and enhance expressed target genes. Markless knock-out technology and chemically induced chromosomal evolution (CIChE) technology will be used to knock out or strengthen the target genes. In short, we wish to comprehend the arginine metabolic network of C. crenatum systematically, and aim to improve arginine production and thus service industrialized production.

棒杆菌是发酵生产精氨酸的常用菌株，采用分子育种方法在提高精氨酸产量方面已取得了大量的成果，但大部分改造工作集中在精氨酸合成途径上，具有一定的局限性。基因组尺度代谢网络（GSMN）分析方法是从代谢网络全局进行分子改造的一种新方法，目前利用GSMN进行棒杆菌精氨酸分子育种的研究未见报道。本课题拟以一株钝齿棒杆菌精氨酸积累突变株为改造对象，探索GSMN分析方法应用于分子育种，提出构建稳定高产精氨酸的分子改造最佳策略。首先通过基因组测序建立突变株基因型与表型的关系；利用谷氨酸棒杆菌ATCC 13032的GSMN模型构建基于突变株扰动下的新模型Cg_iKK446_arginine；在新模型基础上采用通量分布比较分析方法预测关键敲除及强化表达位点。采用无痕敲除及化学诱导染色体进化表达技术构建精氨酸高产菌株,其目的为解析钝齿棒杆菌精氨酸代谢途径提供实验数据和理论支撑，最终为精氨酸的工业化生产服务。

为了提高C. crenatum分子育种效率，本课题对C. crenatum MT和C. crenatum AS1.542基因组进行测序，建立其基因型与表型的关系；在此基础上以已报道的C. glutamcium ATCC 13032的基因组尺度代谢网络模型Cg_iKK446为基础，结合C. crenatum MT的基因型改造获得Cg_iKK446_arginine新模型；采用FDCA算法预测新模型Cg_iKK446_arginine基因改造靶点。为充分体现改造靶点的效应，本课题组以C. crenatum MT为出发菌株，首先利用点突变解除减弱终产物精氨酸对精氨酸生物合成途径中关键酶的反馈抑制，即改造argB基因；然后根据预测结果筛选基因敲除靶点，采用无痕敲除技术逐一敲除并观测精氨酸产率变化。结果显示，通过点突变改造了argB，使其对精氨酸的半抑制率提高了3.7倍，且精氨酸产量提高了10.8%；利用新模型预测到了64个基因敲除位点、24个基因强化位点及7个基因弱化位点；其中筛选敲除了8个基因，包括proB、pta、proC、cgl2310、putP、Ncgl1221、ack、dtsR1，强化了lysE基因，导致精氨酸产量从7.8mg/mL升高到40.7mg/mL，提高了422%。同时，通过荧光定量PCR发现，dtsR1的敲除极大地下调了三羧酸循环中α-酮戊二酸脱氢酶复合体的编码基因的转录水平，上调了磷酸戊糖途径中zwf基因及精氨酸合成途径的基因argB的转录水平，这是导致精氨酸大幅提高的原因。