Construction of Engineered Escherichia coli and Optimization of Conditions for Carcinine Synthesis via Multi-Enzyme Cascade Catalysis

Construction and systematic engineering of Saccharomyces cerevisiae for efficient de novo biosynthesis of pentostatin.

Pentostatin is a potent adenosine deaminase inhibitor, yet its industrial application is hindered by low extraction yields and complex chemical synthesis. Here, we report an efficient de novo biosynthesis platform for pentostatin in Saccharomyces cerevisiae. Starting with the heterologous expression of cns3 from Cordyceps militaris, we optimized the cell factory via promoter engineering, multicopy integration, and AAH1 knockout. This integration strain achieved a maximum pentostatin titer of 16.28mg/L in shake-flask cultivation, representing a 19.38-fold improvement over our initial production. Separately, to alleviate severe product toxicity, we implemented flux balance analysis (FBA)-guided transporter engineering; the engineered strain expressing the episomal efflux pump Cns4 yielded a titer of 8.27mg/L while significantly accelerating the production process. Molecular docking revealed a distinct binding cavity where key residues (e.g., Asp296, Ala292) capture pentostatin via specific hydrogen bonds and hydrophobic interactions. Furthermore, transcriptomics demonstrated that Cns4 globally reprograms carbon and energy metabolism to boost precursor supply and cellular robustness. This work integrates structural insights with systems metabolic engineering, providing a generalizable paradigm for biosynthesizing toxic nucleoside natural products.

An expression framework for production of recombinant methionine aminopeptidase in Escherichia coli: a case study of upstream process.

A case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli, evaluating the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions without a side-by-side comparison with alternative expression systems.

Comprehensive derivatization of thiostrepton via heterologous expression.

Sustainable co-production of L-theanine via an integrated biocatalytic system exploiting atrazine degradation.

L-Theanine is a health-beneficial tea-specific amino acid. This amino acid is synthesized through the catalytic action of L-theanine synthase or its isoenzymes, utilizing ethylamine and L-glutamic acid as primary substrates. As the rate-limiting substance, traditional ethylamine synthesis relies on tea root metabolism, and exploring new synthetic pathways can boost L-theanine production and explore environmental application values. Self-developed Tongji_Software was used to predict a potential pathway for ethylamine synthesis. A cell-free protein synthesis (CFPS) system was adopted for verification, and genome engineering was performed to construct a recombinant strain. According to the software predictions, atrazine chlorohydrolase (AtzA) and hydroxyatrazine deethylaminase (AtzB) could convert the herbicide atrazine into ethylamine. CFPS based on Pseudomonas knackmussii (P. knackmussii) produced 88.5 μM L-theanine within a 24-h period, while the engineered strain P. knackmussii-AtzAB yielded 445.7 μM with the conversion rate of 44.6% over 84 h of fermentation. In summary, we revealed a novel pathway for ethylamine synthesis, which can promote L-theanine accumulation in tea trees and degrade environmental pollutants.

Antibiotic- and inducer-free biosynthesis of indican from crude glycerol using engineered probiotic Escherichia coli Nissle 1917

Chemical synthesis of indigo relies on aniline and formaldehyde feedstocks, inevitably generating carcinogenic byproducts and imposing severe environmental burdens, driving the urgent demand for sustainable bio-manufacturing alternatives. As a water-soluble glycoside precursor of indigo, indican circumvents the cytotoxicity and product accumulation issues caused by hydrophobic indigo precipitation, emerging as an ideal intermediate for green indigo production. Herein, we developed an antibiotic- and inducer-free biosynthesis platform using the probiotic Escherichia coli Nissle 1917 (EcN). By harnessing its native high-copy plasmids, constitutive expression of the flavin-containing monooxygenase FMOK223R and glycosyltransferase PtUGT1 was achieved to enable the de novo synthesis of indican. To redirect metabolic flux toward indican accumulation, competing pathways were blocked via inactivation of pheA, tyrA, pykA and ppc, while disruption of the trpR gene alleviated feedback inhibition to the tryptophan biosynthetic pathway. Combined overexpression of rate-limiting genes tnaA and the feedback-resistant trpES40F synergistically enhanced indican accumulation. Further semi-rational mutagenesis of PtUGT1(S112A), coupled with fusion expression optimization, substantially elevated indican titers to 449.3 mg/L in shake-flask cultivation, and crude glycerol-fed-batch fermentation reached a peak titer of 1923.2 mg/L at 32 h. This study demonstrates that the EcN endogenous plasmid engineering eliminates dependence on antibiotics and inducers, establishing a robust and eco-friendly alternative to traditional chemical synthesis.

An integrated engineering strategy enhances expression and activity of Pseudonocardia thermophila nitrile hydratase in Escherichia coli.

Nitrile hydratase is a key enzyme for nitrile-to-amide hydration under mild conditions, yet its application is limited by low heterologous expression, poor solubility, and suboptimal catalysis. Here, we present an integrated strategy to enhance expression, assembly, and function of Pseudonocardia thermophila NHase (PtNHase) in Escherichia coli, and dissect its mechanism via kinetic analysis. Codon optimization improved total whole-cell activity by 99% at 36 °C, likely through enhanced translational efficiency and co-translational folding at elevated temperatures. Replacing the native Shine-Dalgarno sequence with a strong RBS increased α-subunit expression 1.8-fold and boosted total whole-cell activity by 215%, outperforming solubilization tag and subunit fusion. Structure-guided saturation mutagenesis identified βTrp72 as a key specificity determinant. The βW72F variant increased apparent activity toward 3-cyanopyridine by 45% while retaining wild-type acrylonitrile activity. However, kinetic analysis revealed that βW72F has lower intrinsic catalytic efficiency (kcat/Km 10.8 vs. 30.9 mM-1·s-1), and its apparent gain arises from relieved substrate inhibition at high substrate concentrations (Kᵢ of βW72F increased from 306.1 to 688.4 mM), rather than from enhanced intrinsic rate. This mutation exhibits a trade-off: enhanced activity and tolerance toward 3-cyanopyridine but reduced thermostability and nicotinamide tolerance. Computational analyses suggested that βW72F expands the substrate tunnel entrance (bottleneck radius 0.92 → 1.32 Å), optimizes CoN coordination (5.8 → 4.9 Å), but compromises thermostability due to increased backbone flexibility (RMSD 2.78 → 3.42 Å). This work establishes a high-efficiency expression system for PtNHase and highlights that apparent activity gain does not necessarily equate to improved intrinsic catalytic efficiency, providing a framework for engineering complex metalloenzymes.