Abstract

Indigoidine is a dark-blue natural pigment with application prospect and synthesized from glutamine (Gln) by series of indigoidine synthetases (IndCs). Indigoidine production can be improved by enhancing Gln pool via supplementing Gln directly or converting metabolism glutamate (Glu) to Gln by glutamine synthetase (GlnA). But, Gln is expensive, and excess Gln inhibits indigoidine production of the recombinant strain. Supplementing Glu instead of Gln may improve the productive and economic efficiency of indigoidine, but the local activities and positions of the indigoidine pathway enzymes GlnA, Sc-IndC, and the helper protein of Sc-IndC (IndB) should be well arranged. We identified the Streptomyces chromofuscus ATCC 49982 derived IndC (Sc-IndC) as an more efficient IndC compared to other IndCs applied for constructing indigoidine-producting strains, and designed series of protein scaffold complexes with architectures of PDZ, SH3, and GBD domains (PxSyG1) to arrange the pathway enzymes. The strain recruiting GlnA, Sc-IndC, and IndB on the PDZ, SH3, and GBD domains of scaffold P1S2G1, respectively, was the most efficient. In the strain, the GlnA supplied sufficient local Gln for Sc-IndC from Glu, and the generated Gln was immediately consumed by Sc-IndC to relieve cell growth inhibition caused by Gln. The optimum Glu concentration (6 g/L) for the strain was higher than those of the strains recruiting Sc-IndC on the GBD domain, which was away from the PDZ domain recruiting GlnA. The highest titer of indigoidine was 12 g/L, which was two folds of the control without scaffold (5.8 g/L). The titer is 5 g/L higher than the control without Glu supplemented (6.9 g/L), meaning that 97% of the supplemented Glu was transformed into indigoidine. The batch fermentation with the optimum strain in a 5-L reactor achieved an indigoidine titer of 14 g/L in 60 h. To our knowledge, this was the most efficient indigoidine productivity achieved so far. The optimization strategies by protein scaffold should be applicative to other pathways with complex substrate demands.

Key points

•Protein scaffold systems were designed to arrange the indigoidine synthetic pathway.

•The scaffold system improved supplement of Gln for indigoidine production from Glu.

•The inhibition caused by excess Gln was relieved by proper designed scaffold.

•The yield and titer of indigoidine was improved by arranging the pathway enzymes.

中文翻译：

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蛋白质支架优化了靛蓝定合成途径中组成酶的排列，以改善色素生成

摘要

靛蓝定是一种深蓝色的天然颜料，具有应用前景，是由谷氨酰胺（Gln）通过一系列靛蓝合成酶（IndCs）合成的。可通过直接补充Gln或通过谷氨酰胺合成酶（GlnA）将新陈代谢谷氨酸（Glu）转化为Gln来增强Inlnigo的产量。但是，Gln是昂贵的，并且过量的Gln抑制了重组菌株的靛蓝生产。补充Glu而不是Gln可以提高靛蓝定的生产效率和经济效率，但是应合理安排靛蓝定途径酶GlnA，Sc-IndC和Sc-IndC的辅助蛋白（IndB）的局部活性和位置。我们确定了链霉菌ATCC 49982衍生的IndC（Sc-IndC）与用于构建靛蓝生产菌株的其他IndC相比效率更高，并设计了一系列具有PDZ，SH3和GBD结构域（PxSyG1）结构的蛋白支架复合物酶。分别在支架P1S2G1的PDZ，SH3和GBD域上募集GlnA，Sc-IndC和IndB的菌株效率最高。在该菌株中，GlnA从Glu中为Sc-IndC提供了足够的局部Gln，生成的Gln立即被Sc-IndC消耗，从而减轻了由Gln引起的细胞生长抑制。该菌株的最佳Glu浓度（6 g / L）高于在GBD域募集Sc-IndC的菌株的最佳Glu浓度，该菌株远离PDZ域募集GlnA的菌株。靛蓝定的最高滴度为12 g / L，这是不含支架的对照的两倍（5.8 g / L）。滴度比不添加Glu的对照（6.9 g / L）高5 g / L，这意味着97％的添加的Glu被转化为茚满丁。在5升反应器中以最佳菌株进行分批发酵，在60小时内达到了14 g / L的茚满丁效价。据我们所知，这是迄今为止达到的最有效的茚满丁生产率。蛋白质支架的优化策略应适用于具有复杂底物需求的其他途径。这是迄今为止达到的最有效的靛蓝生产率。蛋白质支架的优化策略应适用于具有复杂底物需求的其他途径。这是迄今为止达到的最有效的靛蓝生产率。蛋白质支架的优化策略应适用于具有复杂底物需求的其他途径。

关键点

•设计了蛋白质支架系统，以安排茚满丁的合成途径。

•支架系统改善了从Glu生产靛蓝的Gln补充剂。

•适当设计的支架可减轻过量Gln引起的抑制作用。

•通过设置途径酶，可提高靛蓝定的收率和效价。