Biotechnology Journal最新文献

Enzymatic biofuel cells face substrate limitations due to enzyme specificity of the electrode. A universal electrode was designed by immobilizing ferredoxin-NADP⁺ oxidoreductase (FNR) with bacterial cellulose (BC), carbon nanotubes (CNTs), and silver nanowires (AgNWs). The electrode coupled with NADPH-dependent malic enzyme or glucose dehydrogenase generated electricity using malic acid and glucose, respectively. The open-circuit voltage reached 79.36 and 75.8 mV, respectively, and the accumulation of pyruvate and gluconate reached 0.30 and 0.25 mM, respectively, after 12 h. This strategy enables electron transfer from diverse substrates via NADPH.

Caffeic acid, a high-value natural phenolic compound synthesized through plant metabolism, plays a critical role in producing phenylpropanoid derivatives and serves as a direct precursor to several key phenolic acids. As a food additive and medicine, caffeic acid has garnered significant attention for its potential in various applications. Recent advances in synthetic biology and metabolic engineering have enabled its biosynthesis via microbial cell factories. This review summarizes five strategies for optimizing caffeic acid production: caffeic acid biosynthetic pathway, modification of metabolic pathway, systems biology and synthetic biology, cofactor engineering, and modular co-culture. However, caffeic acid production via microbial chassis faces bottlenecks such as limited precursor availability for biosynthesis, toxicity from metabolic intermediates, inefficient cofactor utilization, and over-reliance on conventional host microorganisms. Breaking through these bottlenecks by integrating the five strategies outlined is expected to further increase caffeic acid production.

Biomass productivities in shake flasks are often not reproduced in bioreactors for plant cell cultures due to change in hydrodynamics. Considering shake flask biomass productivity as benchmark, this study employs shake flask geometries as a model system to understand hydrodynamic changes with volume and identify suitable scale-up criteria for plant cell cultivations, with minimal cost and time, given their slow growth time, using computational fluid dynamics (CFD) and experiments. Cultivation of Viola odorata cells in increasing flask volumes (100–3000 mL) revealed no significant change in biomass productivity. CFD analysis indicated that volumetric oxygen mass transfer coefficient (k_La), increased up to 1000 mL and then decreased, due to saturation of energy dissipation rates (k_L is a function of energy dissipation rates) and decreasing interfacial area. The unaffected biomass concentration, despite decreased k_La, suggests that k_La may not be a significant scale-up parameter. Instead, maintaining a constant shear environment, indicated by power per unit volume saturation at higher volumes, was proposed as a suitable scale-up parameter for V. odorata cell cultivation in bioreactors. Moreover, the decrease in velocity difference between fluid layers with increased flask volume, indicated that minimizing velocity gradients in bioreactors could help achieve shake flask biomass productivity.

Glucosamine (GlcN), a high-value nutraceutical, is currently produced via environmentally detrimental chitin hydrolysis or inefficient microbial fermentation. While acidic hydrolysis of crustacean chitin raises environmental and allergen concerns, microbial fermentation faces challenges in strain engineering and byproduct formation. One-pot production of GlcN from maltodextrin by an in vitro synthetic enzymatic biosystem (ivSEB) containing glucosamine 6-phosphate phosphatase, which dephosphorylates glucosamine 6-phosphate (GlcN6P) to GlcN, was developed recently. In this study, we identified a thermostable haloacid dehalogenase (HAD) phosphatase, TmHAD, from Thermophilibacter mediterraneus through database mining. Biochemical characterization revealed its remarkable dephosphorylation specificity for GlcN6P, exhibiting 27.6- and 138.0-fold higher activity toward GlcN6P compared to glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), respectively. The enzyme demonstrated Mg²⁺-dependent activity and moderate thermal stability with a half-life of 6.6 h at 45°C. When incorporated into an ivSEB (phosphorylation, isomerization, amination, and dephosphorylation), TmHAD enabled GlcN production from maltodextrin with a molar yield of 44.5%. This biosystem represented an effective complement to current GlcN production methods, with the exceptional substrate specificity and thermal stability of TmHAD making it particularly promising for industrial-scale GlcN manufacturing in vitro.

Gibberellic acid 3 (GA₃), a diterpenoid phytohormone industrially biosynthesized by Fusarium fujikuroi, serves as a pivotal plant growth regulator with extensive agricultural applications. Currently, industrial GA₃ production predominantly relies on prolonged submerged microbial fermentation with F. fujikuroi as the main production strain, valued for its native biosynthetic capacity. Nevertheless, large-scale industrialization of GA₃ remains constrained by low production yields. In this study, a systematic multimodular metabolic engineering framework was implemented to enhance GA₃ biosynthesis in F. fujikuroi. The engineering strategy encompassed four synergistic modules: reinforcement of fatty acid biosynthesis, augmentation of acetyl-CoA metabolic flux, optimization of redox cofactor homeostasis, and overexpression of the positive transcriptional regulator. This integrated approach yielded the engineered strain OE: Lae1-AGP3 demonstrating a 2.58 g/L GA₃ titer in shake-flask fermentation. Subsequent bioprocess optimization through exogenous fatty acid supplementation further elevated GA₃ production to 2.86 g/L, representing a 10.9% increase. This study demonstrates the feasibility of coordinated metabolic modifications for improving GA₃ biosynthesis in F. fujikuroi, offering practical insights for overcoming productivity limitations in fungal secondary metabolite fermentation processes.

DNA cloning methods are fundamental tools in molecular biology, synthetic biology, and genetic engineering that enable precise DNA manipulation for various scientific and biotechnological applications. This review systematically summarizes the major restriction-free overlapping sequence cloning (RFOSC) techniques currently used in synthetic biology and examines their development, efficiency, practicality, and specific applications. In vitro methods, including Gibson Assembly, Circular Polymerase Extension Cloning (CPEC), Polymerase Incomplete Primer Extension (PIPE), Overlap Extension Cloning (OEC), Uracil DNA Glycosylase-based Cloning (UDG-Cloning), and commercially available techniques such as In-Fusion, have been discussed alongside hybrid approaches such as Ligation-Independent Cloning (LIC), Sequence-Independent Cloning (SLIC), and T5 Exonuclease-Dependent Assembly (TEDA). Additionally, in vivo methods leveraging host recombination machinery, including Yeast Homologous Recombination (YHR), In Vivo Assembly (IVA), Transformation-Associated Recombination (TAR), and innovative approaches such as Phage Enzyme-Assisted Direct Assembly (PEDA), are critically evaluated. The review highlights that method selection should consider individual research projects’ scale, complexity, and specific needs, noting that no single technique is universally optimal. Future trends suggest the increased integration of enzymatic efficiency, host versatility, and automation, broadening the accessibility and capabilities of DNA assembly technologies.

As the biopharmaceuticals industry becomes increasingly competitive, improving speed-to-market and achieving “Right First Time” are key factors that propel a company to success. Building upon our foundational work on the Automatic Assay Preparation Platform (A2P2) initially introduced to enhance process analytical technology (PAT), this publication describes its innovative application for real-time sample preparation, acquisition, and monitoring of critical quality attributes (CQAs) for biotherapeutic production. Determination of CQA in cell culture is typically supported by analytical laboratories. Titer is determined before product purification by affinity capture column using a liquid handler. Purified product concentration is then measured before subjecting to a subsequent product quality assay. This process takes up to four separate instruments and several analysts to complete. The A2P2 system, embodying an end-to-end automated and autonomous PAT solution, is configured to streamline titer measurement, product purification, purified product concentration measurement, and CQA assay within a single run sequence using a modified ultrahigh performance liquid chromatography (UHPLC) instrument. By utilizing Chromeleon's System Suitability Tests (SST) and Intelligence Run Control (IRC) feature and the Agilent injector program, A2P2 significantly reduces analysts’ hands-on time and shortens result turnaround time for improved efficiency. This evolution of the A2P2 system not only reduces laboratory footprint but also positions it as an ideal solution for real-time bioprocess monitoring of CQA, further advancing our commitment to accelerating the delivery of safe and effective biotherapeutics.