Ergothioneine (EGT) is a high-value antioxidant for food, pharmaceutical, nutraceutical, and cosmetic applications. Microbial production from renewable feedstocks is promising, but efficient biosynthesis requires coordinated precursor supply. Precursor-supplementation experiments indicated that multiple amino acids may jointly influence EGT production. Based on pathway biochemistry and previous engineering evidence, endogenous l-cysteine supply was selected as one mechanistically relevant engineering target. Here, an Escherichia coli platform for glucose-derived EGT production was developed by combining biosensor-driven evolution and transcriptome-guided chassis engineering. A dual-output l-cysteine-responsive biosensor linked kanamycin resistance and fluorescence, enabling sequential growth-based enrichment and fluorescence-based prioritization. Mutagenesis, microdroplet-assisted adaptive evolution, and single-cell sorting generated an evolved chassis with 47.10% higher extracellular l-cysteine accumulation and 64.49% higher EGT production. Transcriptome-guided bsmA activation further improved production, and the final strain produced 1.20 g/L EGT in a 3 L fed-batch fermentation without additional l-cysteine supplementation.
Su-Yu Wang, H. Zabed, Guoyan Zhang et al.· Journal of Agricultural and...· 0 citations
d-Allulose is a promising low-calorie rare sweetener; however, its industrial production is limited by the insufficient catalytic activity and thermostability of d-allulose 3-epimerases (DAEases). In this study, a combinatorial engineering strategy integrating lid-domain modulation and computational design was developed to improve enzyme performance. Lid engineering identified a key mutation (N120D) associated with enhanced catalytic turnover, while computational screening identified beneficial mutations (T69S, D71E, and S128A). The best mutant, T69S/D71E/N120D/S128A, exhibited a 1.9-fold increase in specific activity, a 2.2-fold improvement in catalytic efficiency, and a 34.1% longer half-life at 60 °C. It produced 152 g/L d-allulose from 500 g/L d-fructose within 3 h, achieving a 30.4% conversion yield under mildly acidic and high-temperature conditions. Mechanistic analyses suggested that improved performance resulted from the combinatorial effects of enhanced structural rigidity and altered lid-domain dynamics, consistent with improved catalytic turnover while maintaining thermostability. This work provides an efficient biocatalyst and a generalizable protein engineering strategy.
Qi-Chen Liu, Xinrui Tang, Wenjing Sun et al.· Journal of Agricultural and...· 0 citations
Caffeic acid (CA) is a valuable phenylpropanoid with applications in food, pharmaceutical, and chemical industries. Microbial production of CA is often limited by the terminal hydroxylation catalyzed by 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, we constructed a de novo CA biosynthetic pathway in Escherichia coli and enhanced production through systematic metabolic and spatial engineering. Optimization of l-tyrosine supply and HpaBC expression increased CA production to 61.0 mg/L. However, further enhancement of glucose uptake and precursor supply was insufficient, indicating that hydroxylation remained a major limitation. To address this, HpaBC was relocated to the periplasm via the Tat pathway, increasing CA production 4.9-fold to 299.1 mg/L with reduced byproduct formation. Further lpp+14 mediated periplasmic remodeling and fermentation optimization increased shake-flask production to 463.7 mg/L. Finally, fed-batch fermentation achieved 5.1 g/L CA in a 3 L bioreactor. This study highlights periplasmic engineering as an effective strategy for improving oxidation-dependent phenylpropanoid biosynthesis.
Shangyi Wang, Yuqi Zhuo, Jamila A Tuly et al.· Journal of Agricultural and...· 0 citations
Simultaneously enhancing the thermostability and catalytic activity of acetylxylan esterases (AxEs) remains a significant challenge due to the inherent stability-activity trade-off. Here, a previously uncharacterized CE7 family acetylxylan esterase (TsAxE) from Thermoclostridium stercorarium was identified and engineered through a parallel multidimensional engineering strategy integrating consensus motif engineering, molecular docking, and interface engineering. The best-performing mutant BCF (D218L/D245P/G183Y) exhibited a 44.10 ± 0.44% increase in activity toward p-nitrophenyl acetate (pNPA). Notably, the thermostability of BCF was substantially improved, with the half-life (t1/2) at 60 °C extended from 0.63 ± 0.04 h to 43.82 ± 3.90 h. In addition, BCF showed improved catalytic efficiency toward p-nitrophenyl butyrate (pNPB). Molecular docking and molecular dynamics analyses suggested that these mutations may reshape substrate-binding pocket and improve structural stability. This study demonstrates the effectiveness of integrating complementary engineering strategies for the rational improvement of AxEs activity and thermostability.
Yujiao Tao, Xinrui Tang, Mei Zhao et al.· Journal of Agricultural and...· 0 citations
The gene regulatory approaches at the translational and post-translational levels were integrated with optogenetic control patterns to attain very sensitive multi-level precision regulation of the dCas9 protein, thereby facilitating flexible regulation of transcription levels of target genes.
Yaokun Liang, Xianghui Qi, Song Gao et al.· Nucleic Acids Research· 0 citations
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