Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases
Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases
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DOI：
10.1016/j.ymben.2011.07.001
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发表时间：
2011-09-01
影响因子：
8.4
通讯作者：
Chhabra, Swapnil R.
中科院分区：
文献类型：
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作者：
Ma, Suzanne M.;Garcia, David E.;Chhabra, Swapnil R.
外源途径的表达通常由于非预期因素（如引入毒性代谢物、辅因子失衡或途径组分表达不良）而导致次优性能。在这项研究中，我们报道了类异戊二烯衍生的倍半萜烯紫穗槐二烯的生产提高了120%，该倍半萜烯是由大肠杆菌的工程菌株生产的，该工程菌株被开发用于表达来自酿酒酵母的天然七基因甲羟戊酸途径（Martin等人，2003）。这种实质性的改善是通过仅改变途径的单一组分（HMG-CoA还原酶）和随后的宿主优化来改善辅因子可用性而实现的。我们的特点和测试五种变体HMG-CoA还原酶从公开的基因组数据库获得不同的动力学特性和辅因子的要求。这些酶的体外和体内分析结果表明，甲羟戊酸激酶的底物抑制是优化工程化甲羟戊酸途径的重要因素。因此，来自Delftia acidovorans的NADH依赖性HMG-CoA还原酶似乎具有最佳动力学参数，以平衡HMG-CoA水平低于E.大肠杆菌和低于甲羟戊酸激酶抑制浓度的甲羟戊酸的那些，被鉴定为紫穗槐二烯的最佳生产者（比天然途径酶提高54%，48小时后产生2.5 mM或520 mg/L紫穗槐二烯）。进一步提高了含D.通过使用来自博伊丁假丝酵母的NAD（+）依赖性甲酸脱氢酶以及沿着甲酸补充物增加其优选的辅因子（NADH）的细胞内水平来抑制嗜酸菌HMG-CoA还原酶。这导致系统的总体改善120%，导致在发酵48小时后产生3.5mM或700 mg/L紫穗槐二烯。这项综合研究纳入了代谢设计的几个关键参数的分析，如变体酶的体外和体内动力学性能、蛋白质表达的细胞内水平、途径内底物抑制和辅因子管理，以实现观察到的改善。这些指标可应用于广泛的异源途径，以改善生物衍生化合物的生产。(C)2011 Elsevier Inc. All rights reserved.
Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5 mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD(+)-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5 mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds. (C) 2011 Elsevier Inc. All rights reserved.