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
Abstract Regulation of gene transcription based on clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool for constructing synthetic gene circuits in Saccharomyces cerevisiae. The current CRISPR-based regulatory approaches primarily focus on inhibiting the binding of dCas9 protein to single guide RNA (sgRNA) or blocking target site recognition. However, these regulation strategies are often at a single level, and their sensitivity still needs to be improved. In this study, 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. This strategy was used to regulate the transcription levels of fluorescent proteins, resulting in up to 2.58-fold increase in the fluorescence intensity of mCherry compared to that without regulation. This CRISPR-based multi-level optogenetic system should be extremely helpful in understanding gene regulatory networks and in designing robust genetic circuits for synthetic biology.
Yaokun Liang, Xianghui Qi, Song Gao et al.· Nucleic Acids Research· 0 citations