The rare sugar D-allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises D-allulose 3-epimerase (DAEase) or D-psicose 3-epimerase (DPEase) to convert D-fructose into D-allulose. Additionally, the process can also utilise D-tagatose 3-epimerase (DTEase). However, the process is not efficient due to the poor thermotolerance of the enzymes and low conversion rates between the sugars. This review describes three newly identified DAEases that possess desirable properties for the industrial-scale manufacturing of D-allulose. Other methods used to enhance process efficiency include the engineering of DAEases for improved thermotolerance or acid resistance, the utilization of Bacillus subtilis for the biosynthesis of D-allulose, and the immobilization of DAEases to enhance its activity, half-life, and stability. All these research advancements improve the yield of D-allulose, hence closing the gap between the small-scale production and industrial-scale manufacturing of D-allulose.

Background: Two-armed FabscFv-Fc is a favoured bispecific antibody (BsAb) format due to its advantages of the conventional IgG structure. Production of FabscFv-Fc requires expression of three polypeptide chains, one light chain (LC), one heavy chain (HC) and a scFv fused to the Fc (scFvFc) at optimal ratios. Methods: We designed a set of internal ribosome entry site (IRES)-mediated multi-cistronic vectors tailoring to various expression ratios of the three polypeptides to study how the chain ratios affect the FabscFv-Fc production. Results: Expression of HC and scFvFc chains at 1:1 ratio and excess LC gave the highest yield of correctly assembled product. Compared to the use of IRES and multiple promoters, using 2A peptides for co-expression of the three polypeptides gave the highest titre and correctly assembled product. Conclusion: The results obtained in this work provide insights to the impacts of hetero-chain ratios on the BsAb production.

Halogenation of pyrrole requires strong electrophilic reagents and often leads to undesired polyhalogenated products. Biocatalytic halogenation is a highly attractive approach given its chemoselectivity and benign reaction conditions. While there are several reports of enzymatic phenol and indole halogenation in organic synthesis, corresponding reports on enzymatic pyrrole halogenation have been lacking. Here we describe the in vitro functional and structural characterization of PrnC, a flavin-dependent halogenase that can act on free-standing pyrroles. Computational modeling and site mutagenesis studies identified three key residues in the catalytic pocket. A moderate resolution map using single-particle cryogenic electron microscopy reveals PrnC to be a dimer. This native PrnC can halogenate a library of structurally diverse pyrrolic heterocycles in a site-selective manner and be applied in the chemoenzymatic synthesis of a chlorinated analog of the agrochemical fungicide Fludioxonil.

Objective: We developed a targeted integration-based CHO cell platform for simultaneous antibody display and secretion, enabling a streamlined transition from antibody library screening to production without requiring the re-cloning of antibody genes. Methods: The platform consists of a CHO master cell line with a single-copy landing pad, a helper vector expressing FLPe recombinase, and bi-functional targeting vectors. Recombinase-mediated cassette exchange was utilized to integrate targeting vectors into the landing pad. Bi-functional vectors were designed by incorporating a minimal furin cleavage sequence (mFCS), RRKR, and various 2A peptides between the heavy chain (HC) and a membrane anchor. Results: Incomplete cleavage at the mFCS and 2A sites facilitated the expression of both membrane-bound and secreted antibodies, while mutations in the 2A peptide produced a range of display-to-secretion ratios. However, a fraction of secreted antibodies retained 2A residues attached to the HC polypeptides. Further analysis demonstrated that modifying the first five amino acids of the 2A peptide significantly influenced furin cleavage efficiency, resulting in different display-to-secretion ratios for targeting vectors containing mFCS-2A variant combinations. To overcome this, we designed nine-amino-acid FCS variants that, when placed between the HC and membrane anchor, provided a range of display-to-secretion ratios and eliminated the issue of attached 2A residues in the secreted antibodies. Vectors with lower display levels proved more effective at distinguishing cells expressing high-affinity antibodies with closely matched binding affinities. The platform also demonstrated high sensitivity in isolating high-affinity antibody-expressing cells and supported robust antibody production. Conclusion: This targeted integration-based CHO platform enables efficient, in-format screening and production of antibodies with tunable display-to-secretion profiles. It provides a powerful and scalable tool for accelerating the development of functional, manufacturable therapeutic antibodies.