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  • Title: Protein scaffold optimizes arrangement of constituent enzymes in indigoidine synthetic pathway to improve the pigment production.
    Author: Wang L, Sun Y, Lv D, Liu B, Guan Y, Yu D.
    Journal: Appl Microbiol Biotechnol; 2020 Dec; 104(24):10493-10502. PubMed ID: 33151367.
    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. Graphical abstract.
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