Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803.
Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803.
复制标题
通过Synechocystis sp。探索用于乳酸光合作用的代谢工程设计原理。 PCC6803。
DOI：
10.1186/1754-6834-7-99
复制
发表时间：
2014
影响因子：
6.3
通讯作者：
Hellingwerf KJ
中科院分区：
文献类型：
--
作者：
Angermayr SA;van der Woude AD;Correddu D;Vreugdenhil A;Verrone V;Hellingwerf KJ
蓝藻中间生理学的分子工程对于“设计微生物”利用二氧化碳和阳光可持续生产生物燃料和商品化合物变得非常重要。化学商品L-乳酸可以由这些生物体的一种关键中间代谢物丙酮酸在乳酸脱氢酶的催化下一步合成。制造“设计微生物”的合成生物工程包括引入和过度表达形成产物的生化途径。为了进一步优化产物形成，必须对中间代谢的周围生化网络进行修改。为了提高光驱动的 CO2 产生 L-乳酸的能力，我们使用先前设计的集胞藻 (Synechocystis sp.) 产生 L-乳酸的突变株，探索了几种代谢工程设计原理。以PCC6803为基准。这些策略包括：（i）提高相关产物形成酶、乳酸脱氢酶（LDH）的表达水平，例如通过复制质粒的表达； (ii) 异源丙酮酸激酶的共表达以增加丙酮酸的通量； (iii) 磷酸烯醇丙酮酸羧化酶的敲低，以减少通过竞争途径的通量（从磷酸烯醇丙酮酸到草酰乙酸）。此外，我们测试了选定的乳酸脱氢酶，其中一些通过定点诱变进一步优化，以提高酶对辅因子烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 的亲和力。通过菌株优化，生物质和乳酸之间的碳分配从约 5% 增加到 50% 以上。高效的光合微生物细胞工厂将底物 (CO2) 转化为产物（此处：L-乳酸）的速率和程度很高。在文献中描述的现有的基于二氧化碳的蓝藻细胞工厂中，到目前为止，对产物形成的大部分控制都存在于基因引入的发酵途径中。在这里，我们表明，强启动子与增加的基因表达相结合，可以取消对二氧化碳生产乳酸这一步骤的重要控制。在这些前提下，调节细胞内前体丙酮酸可以显着提高生产率。此外，通过蛋白质工程提高异源表达的 LDH 的辅因子特异性，从而实现产量增强。
Molecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by “designer microbes.” The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make “designer microbes” includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made. To improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme’s affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization. An efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH.
登录
查看更多内容
影响因子：
4.1
作者：
KLECZKOWSKI, LA;RANDALL, DD
通讯作者：
RANDALL, DD
影响因子：
6.4
作者：
Li H;Liao JC
通讯作者：
Liao JC
影响因子：
4.3
作者：
Knoop H;Gründel M;Zilliges Y;Lehmann R;Hoffmann S;Lockau W;Steuer R
通讯作者：
Steuer R
影响因子：
2.9
作者：
Brown, WM;Yowell, CA;Jagt, DLV
通讯作者：
Jagt, DLV
影响因子：
14.9
作者：
Huang HH;Camsund D;Lindblad P;Heidorn T
通讯作者：
Heidorn T