Metabolic pathway engineering using the central signal processor PII.
Metabolic pathway engineering using the central signal processor PII.
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DOI：
10.1186/s12934-015-0384-4
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发表时间：
2015-11-25
影响因子：
6.4
通讯作者：
Forchhammer K
中科院分区：
文献类型：
--
作者：
Watzer B;Engelbrecht A;Hauf W;Stahl M;Maldener I;Forchhammer K
PII信号处理器蛋白广泛分布于原核生物和植物中，它们控制着大量的合成代谢反应。有效地过量生产代谢物需要放松紧密的细胞控制回路。在这里，我们证明了蓝藻聚胞藻PII信号蛋白的单点突变。PCC6803足以解锁导致生物聚合物蓝藻素(多L-精氨基-聚-L-天冬氨酸)过度积累的精氨酸途径。作为氨基酸和聚天冬氨酸的来源，该产品具有生物技术价值。这项工作展示了一种新的途径工程方法，通过设计定制的PII信号蛋白。在这里，工程聚球藻sp.具有PII-I86N突变的PCC6803菌株通过关键酶N-乙酰谷氨酸激酶(NAGK)的结构性激活而过度积累精氨酸。在工程菌株BW86中，体内NAGK活性显著增加，导致精氨酸含量比野生型高10倍以上。结果表明，在本试验条件下，菌株BW86的单位细胞干物质中蓝藻素的积累量达到57%，是迄今为止报道的最高的蓝藻素产量。菌株BW86产生的蓝藻素的分子质量范围为25到100 kDa；野生型产生的聚合物的分子质量范围为30到100 kDa。菌株BW86生产的蓝藻素产量高、相对分子质量高，同时蓝藻对营养的要求较低，是一种很有前途的生物技术生产蓝藻素的方法。这项研究进一步证明了利用PII信号蛋白进行代谢途径工程的可行性，PII信号蛋白存在于许多细菌物种中。本文的在线版本(doi：10.1186/s12934-0150384-4)包含补充材料，授权用户可以使用。
PII signal processor proteins are wide spread in prokaryotes and plants where they control a multitude of anabolic reactions. Efficient overproduction of metabolites requires relaxing the tight cellular control circuits. Here we demonstrate that a single point mutation in the PII signaling protein from the cyanobacterium Synechocystis sp. PCC 6803 is sufficient to unlock the arginine pathway causing over accumulation of the biopolymer cyanophycin (multi-l-arginyl-poly-l-aspartate). This product is of biotechnological interest as a source of amino acids and polyaspartic acid. This work exemplifies a novel approach of pathway engineering by designing custom-tailored PII signaling proteins. Here, the engineered Synechocystis sp. PCC6803 strain with a PII-I86N mutation over-accumulated arginine through constitutive activation of the key enzyme N-acetylglutamate kinase (NAGK). In the engineered strain BW86, in vivo NAGK activity was strongly increased and led to a more than tenfold higher arginine content than in the wild-type. As a consequence, strain BW86 accumulated up to 57 % cyanophycin per cell dry mass under the tested conditions, which is the highest yield of cyanophycin reported to date. Strain BW86 produced cyanophycin in a molecular mass range of 25 to >100 kDa; the wild-type produced the polymer in a range of 30 to >100 kDa. The high yield and high molecular mass of cyanophycin produced by strain BW86 along with the low nutrient requirements of cyanobacteria make it a promising means for the biotechnological production of cyanophycin. This study furthermore demonstrates the feasibility of metabolic pathway engineering using the PII signaling protein, which occurs in numerous bacterial species. The online version of this article (doi:10.1186/s12934-015-0384-4) contains supplementary material, which is available to authorized users.