Qiuling Luo

DCEO Biotechnology: Tools To Design, Construct, Evaluate, and Optimize the Metabolic Pathway for Biosynthesis of Chemicals

Chemical synthesis is a well established route for producing many chemicals on a large scale, but some drawbacks still exist in this process, such as unstable intermediates, multistep reactions, complex process control, etc. Biobased production provides an attractive alternative to these challenges, but how to make cells into efficient factories is challenging. As a key enabling technology to develop efficient cell factories, design-construction-evaluation-optimization (DCEO) biotechnology, which incorporates the concepts and techniques of pathway design, pathway construction, pathway evaluation, and pathway optimization at the systems level, offers a conceptual and technological framework to exploit potential pathways, modify existing pathways and create new pathways for the optimal production of desired chemicals. Here, we summarize recent progress of DCEO biotechnology and examples of its application, and provide insights as to when, what and how different strategies should be taken. In addition, we highlight future perspectives of DCEO biotechnology for the successful establishment of biorefineries.

Enzymatic Production of Ascorbic Acid-2-phosphate by Recombinant Acid Phosphatase

In this study, an environmentally friendly and efficient enzymatic method for the synthesis of l-ascorbic acid-2-phosphate (AsA-2P) from l-ascorbic acid (AsA) was developed. The Pseudomonas aeruginosa acid phosphatase (PaAPase) was expressed in Escherichia coli BL21. The optimal temperature, optimal pH, Km, kcat, and catalytic efficiency of recombinant PaAPase were 50 °C, 5.0, 93 mM, 4.2 s-1, and 2.7 mM-1 min-1, respectively. The maximal dry cell weight and PaAPase phosphorylating activity reached 8.5 g/L and 1127.7 U/L, respectively. The highest AsA-2P concentration (50.0 g/L) and the maximal conversion (39.2%) were obtained by incubating 75 g/L intact cells with 88 g/L AsA and 160 g/L sodium pyrophosphate under optimal conditions (0.1 mM Ca2+, pH 4.0, 30 °C) for 10 h; the average AsA-2P production rate was 5.0 g/L/h, and the AsA-2P production system was successfully scaled up to a 7.5 L fermenter. Therefore, the enzymatic process showed great potential for production of AsA-2P in industry.

Fumarate Production by Torulopsis glabrata: Engineering Heterologous Fumarase Expression and Improving Acid Tolerance

Fumarate is a well-known biomass building block compound. However, the poor catalytic efficiency of fumarase is one of the major factors preventing its widespread production. To address this issue, we selected residues 159HPND162 of fumarase from Rhizopus oryzae as targets for site-directed mutagenesis based on molecular docking analysis. Twelve mutants were generated and characterized in detail. Kinetic studies showed that the Km values of the P160A, P160T, P160H, N161E, and D162W mutants were decreased, whereas Km values of H159Y, H159V, H159S, N161R, N161F, D162K, and D162M mutants were increased. In addition, all mutants displayed decreased catalytic efficiency except for the P160A mutant, whose kcat/Km was increased by 33.2%. Moreover, by overexpressing the P160A mutant, the engineered strain T.G-PMS-P160A was able to produce 5.2 g/L fumarate. To further enhance fumarate production, the acid tolerance of T.G-PMS-P160A was improved by deleting ade12, a component of the purine nucleotide cycle, and the resulting strain T.G(△ade12)-PMS-P160A produced 9.2 g/L fumarate. The strategy generated in this study opens up new avenues for pathway optimization and efficient production of natural products.

A selective and sensitive nanosensor for fluorescent detection of specific IgEs to purified allergens in human serum

Food allergies are increasingly recognized as a major healthcare concern. In order to sensitively and specifically detect allergies from blood samples of at-risk allergic patients, an effective magnetic fluorescence sensing platform (EMFP) was constructed. The EMFP incorporated hollow mesoporous silica nanospheres (HMNs) to amplify signal from the target IgE in addition to magnetic nanoparticles (MNPs) to capture and separate the target IgE. The application of EMFP to immunoassays indicated a detection limit of 0.0159 ng mL−1 for low concentration specific immunoglobulin E (sIgE) against purified shellfish Metapenaeus ensis (Meta. E.) allergens, which is 15 fold more sensitive than the commercially available Food and Drug Administration-approved analyzers. Notably, EMFP was specific for the targeted sIgE even with interference by other sIgEs. In addition, the detection time is only 75 min, considerably faster than current commercial ELISA kits for IgE assays. Together, these results demonstrated that EMFP has excellent sensitivity and selectivity for the rapid detection of sIgE. The method thus exhibits potential toward the rapid monitoring of sIgE against Meta. E. allergens in clinical application.

Production of β‐Alanine from Fumaric Acid Using a Dual‐Enzyme Cascade

The aim of this study was to develop an environmentally safe and efficient method for β‐alanine production using a dual‐enzyme cascade route with L‐aspartase (AspA) from E. coli and L‐aspartate‐α‐decarboxylase (PanD) from Corynebacterium glutamicum. Poor cooperativity in this system due to the divergent catalysis efficiencies of AspA and PanD led to an imbalance between the two reactions. To address this issue, we employed ribosome binding site regulation and gene duplication to coordinate the expression levels of AspA and PanD. Finally, we achieved β‐alanine production of 80.4±1.6 g L−1 with a conversion rate of 95.3±1.6 % in a 5‐L bioreactor. The dual‐enzyme cascade reported herein represents a promising strategy to meet industrial requirements for large‐scale β‐alanine production in the future.