Protein scaffold optimizes arrangement of constituent enzymes in indigoidine synthetic pathway to improve the pigment production
Protein scaffold optimizes arrangement of constituent enzymes in indigoidine synthetic pathway to improve the pigment production
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蛋白质支架优化靛蓝苷合成途径中组成酶的排列，以提高色素产量
DOI：
10.1007/s00253-020-10990-5
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发表时间：
2020-11
影响因子：
5
通讯作者：
Yu Dayu
中科院分区：
文献类型：
--
作者：
Wang Lei;Sun Yue;Lv Di;Liu Bin;Guan Yuekai;Yu Dayu
靛蓝是一种具有应用前景的深蓝色天然色素，由谷氨酰胺经靛蓝合成酶系列（IndCs）合成。通过直接补充谷氨酰胺或通过谷氨酰胺合成酶（GlnA）将代谢谷氨酸（Glu）转化为谷氨酰胺（Gln）来增加谷氨酰胺库，可以提高靛蓝的产量。但是，谷氨酰胺是昂贵的，过量的谷氨酰胺抑制靛蓝生产的重组菌株。补充Glu可提高靛蓝的生产效率和经济效益，但应合理安排靛蓝途径酶GlnA、Sc-IndC和Sc-IndC辅助蛋白（IndB）的活性和位置。本研究将产靛蓝的链霉菌ATCC 49982来源的IndC（Sc-IndC）与其它IndC进行比较，确定其为更有效的IndC，并设计了一系列具有PDZ、SH 3和GBD结构域（PxSyG 1）的蛋白质支架复合物，以排列途径酶。分别在支架P1 S2 G1的PDZ、SH 3和GBD结构域上募集GlnA、Sc-IndC和IndB的菌株是最有效的。在该菌株中，GlnA从Glu为Sc-IndC提供足够的局部Gln，并且所产生的Gln立即被Sc-IndC消耗以减轻Gln引起的细胞生长抑制。该菌株的最适Glu浓度（6 g/L）高于在GBD结构域上募集Sc-IndC的菌株，其远离募集GlnA的PDZ结构域。靛蓝的最高滴度为12 g/L，是无支架对照（5.8 g/L）的2倍。滴度比没有补充Glu的对照（6.9g/L）高5g/L，这意味着97%的补充的Glu被转化成靛红。在5L反应器中分批发酵60 h，靛蓝素效价可达14 g/L。据我们所知，这是迄今为止实现的最有效的靛蓝生产率。蛋白质支架的优化策略也适用于其他底物需求复杂的合成途径。关键点·设计了蛋白质支架系统来安排靛蓝的合成途径。该支架系统改善了Gln的补充以用于从Glu生产靛蓝。通过适当的支架设计，可以缓解过量Gln引起的抑制作用。通过对途径酶的排列，提高了靛蓝的产率和效价。图形摘要
AbstractIndigoidine 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 theStreptomyces chromofuscusATCC 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.Graphical abstract
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影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
8.4
作者：
Moon, Tae Seok;Dueber, John E.;Prather, Kristala L. Jones
通讯作者：
Prather, Kristala L. Jones
影响因子：
4.1
作者：
Brachmann, Alexander O.;Kirchner, Ferdinand;Bode, Helge B.
通讯作者：
Bode, Helge B.
影响因子：
4.5
作者：
Ç. Yumuşak;A. Prochazkova;D. Apaydin;Hathaichanok Seelajaroen;N. S. Sariçiftçi;M. Weiter;J. Krajcovic;Yong Qin;Wei Zhang;J. Zhan;A. Kovalenko
通讯作者：
Ç. Yumuşak;A. Prochazkova;D. Apaydin;Hathaichanok Seelajaroen;N. S. Sariçiftçi;M. Weiter;J. Krajcovic;Yong Qin;Wei Zhang;J. Zhan;A. Kovalenko
影响因子：
5.2
作者：
Gromek SM;Suria AM;Fullmer MS;Garcia JL;Gogarten JP;Nyholm SV;Balunas MJ
通讯作者：
Balunas MJ