Meijuan Xu

Indigoidine is a sustainable natural blue pigment with broad industrial prospects, while its microbial production is hampered by growth inhibition of the producing strain, enzyme-host incompatibility, and insufficient industrial-scale titer. In this study, de novo biosynthesis of indigoidine was achieved in Corynebacterium glutamicum. Efflux pump engineering was employed to alleviate growth inhibition of the production strain, and semi-rational design of the key synthetase BpsA generated the optimal mutant Y433Q with enhanced catalytic activity. Strain CGI23, which integrated the two aforementioned strategies, produced 29.8 g/L of indigoidine at a mass yield of 0.248 g/g glucose in shake flasks; when further scaled up in a 5 L fed-batch fermenter, the titer reached 60.7 g/L with a volumetric productivity of 0.723 g/L/h. This work establishes an efficient microbial platform for industrial indigoidine production, providing a reference for integrated engineering of high-performance microbial cell factories.

L-Tryptophan, an essential amino acid, serves as a critical precursor for the biosynthesis of numerous bioactive compounds. In this study, we engineered tryptophan synthase (TrpS) to develop an efficient cell factory for l-tryptophan production. Initially, EcTrpS from Escherichia coli K-12 was expressed in E. coli BL21 (DE3) to construct strain BS, which produced 836.43 mg/L of l-tryptophan. To alleviate substrate inhibition, we constructed a mutant library of 10⁴–10⁵ variants through two rounds of error-prone PCR and utilized a biosensor employing a green fluorescent protein to report l-serine consumption for screening TrpS mutants. Mutant C10 showed a 375% increase in enzyme activity compared with strain BS. Computational analysis was used to explore an improved catalytic mechanism. Finally, through systematic process optimization and catalytic strategy, strain C10 produced 94.2 g/L of l-tryptophan, the highest titer via whole-cell transformation. This study provides new perspectives and strategies for TrpS engineering and the industrial production of l-tryptophan.

Nicotinamide mononucleotide (NMN) has received widespread attention as a supplement of NAD⁺ in cells. In this study, a dual-module reaction system was constructed to synthesize NR using uridine and nicotinamide, and further to efficiently synthesize NMN. First, module 1 was constructed, which catalyzed the synthesis of NMN from NR using an efficient NRK and ATP regeneration system. Then module 2 was constructed by introducing pyrimidine nucleoside phosphorylase (PyNP) to synthesize NMN from uridine and NAM under the synergistic catalysis of NRK. Based on the fact that NRK has both phosphorylation and group transfer functions in the dual-module system, the mutant KlmNRK_M4 with nearly 4-fold increased stability was obtained through predicted structure and evolutionary conservation analysis. At the same time, the pncC, deoD, ushA, nadR and deoB genes encoding endogenous degradative enzymes in Escherichia coli affect substrate and intermediate conversion were knocked out. Finally, by optimizing the reaction conditions of the dual-module recombination system, a high NMN conversion rate of 81.1% was achieved using 300 mM uridine and nicotinamide as substrates. This study provides a novel and efficient pathway for the biosynthesis of NMN.

D-tagatose is an ideal sucrose substitute with potential applications in food and healthcare. The combined catalysis of polyphosphate kinase (PPK), fructose kinase (FRK), D-tagatose-6-phosphate 3-differential anisomerase (FbaA) and phytase provides a low-cost and convenient pathway for the biosynthesis of D-tagatose from D-fructose; however, there is still a problem of low catalytic efficiency that needs to be solved urgently. Therefore, this study enhanced the biosynthesis of D-tagatose by optimizing the expression levels of PPK, FRK and FbaA in a polycistronic system and knocking out the gene pfka of Escherichia coli. With 30 g/L D-fructose as a substrate, the conversion rate increased to 52%, which was the highest after 24 h. In addition, by constructing a multienzyme self-assembly system with SpyTag and SpyCatcher to improve the whole-cell catalytic ability, the conversion rate was further increased to 75%. Finally, through the fed-batch strategy, the optimal strain Ec-7 produced 68.1 g/L D-tagatose from 100 g/L D-fructose. The multienzyme cascade route reported herein provides an efficient and elegant innovative solution for the generation of D-tagatose.