High titer production of gastrodin enabled by systematic refactoring of yeast genome and an antisense-transcriptional regulation toolkit.

Gastrodin, a phenolic glycoside, is a prominent component of Gastrodia elata, which is renowned for its sedative, hypnotic, anticonvulsant, and neuroprotective activities. Engineering heterologous production of plant natural products in microbial host represents a safe, cost-effective, and scalable alternative to plant extraction. Here, we present the construction of an engineered Yarrowia lipolytica yeast that achieves a high-titer production of gastrodin. We systematically refactored the yeast genome by enhancing the flux of the shikimate pathway and optimizing the glucosyl transfer system. We introduced more than five dozen of genetic modifications onto the yeast genome, including enzyme screening, alleviation of rate-limiting steps, promoter selection, genomic integration site optimization, downregulation of competing pathways, and elimination of gastrodin degradation. Meanwhile, we developed a Copper-induced Antisense-Transcriptional Regulation (CATR) tool. The developed CATR toolkit achieved dynamic repression and activation of violacein synthesis through the addition of copper in Y. lipolytica. This strategy was further used to dynamically regulate the pyruvate kinase node to effectively redirect glycolytic flux towards the shikimate pathway while maintaining cell growth at proper rate. Taken together, these efforts resulted in 9477.1 mg/L of gastrodin in shaking flaks and 13.4 g/L of gastrodin with a yield of 0.149 g/g glucose in a 5-L bioreactor, highlighting the potential for large-scale and sustainable production of gastrodin from microbial fermentation.

Finite Element Analysis of Split Sleeve Cold Expansion Process on Multiple Hole Aluminum Alloy.

Multiple cold expansion holes are widely used in connection areas of aircraft structures, in order to achieve uniform load transfer of the skin or connection parts. Split sleeve cold expansion (SSCE) is widely used to enhance the fatigue life of fastener holes by applying compressive residual stresses around the holes. In this study, the finite element method (FEM) was used to research the distribution and variation of residual stresses along the hole edges of 7075AA single-hole and multi-hole cold expansion (CE) specimens. Full-field strain measurements of single-hole and multi-hole specimens were performed using two-dimensional digital image correlation (DIC), and the residual stress and strain at the hole edge of the specimens measured by FEM and DIC were compared. FEM results shows that the maximum circumferential and radial residual stresses of three-hole specimens with three-hole spacing are increased by 5.37% and 31.53% compared with single-hole specimens. The maximum circumferential residual stress of three-hole specimens with four-hole spacing increases by 7.25% compared with a single hole, but the radial residual stress decreases by 12.98%. In addition, for three-hole specimens with hole spacing three times the hole diameter, the strengthening effect of SSCE in the order of middle hole, then left hole, and, finally, right hole is better than that of SSCE in the order of left to right hole. FEM and DIC full-field strain results are basically consistent.

Recent developments in promiscuous enzymatic reactions for carbon-nitrogen bond formation.

Biocatalytic promiscuity is a new field of enzyme application in biochemistry, which has received much attention and has developed rapidly in recent years. The promiscuous biocatalysis has been promoted as a useful supplement to traditional strategy for the formation of C-heteroatom bonds. The generation of carbon-nitrogen (CN) bonds is an important issue in synthetic chemistry and is indispensable for the manufacturing of various pharmaceuticals and agrochemicals. Therefore, numerous efficient and reliable synthetic methods for the formation of CN bonds have been developed in recent years. Enzymatic CN bond forming reactions catalyzed by lipases, cytochrome P450 monooxygenases, glycosyltransferases, amine dehydrogenases, proteases, acylases, amylases and halohydrin dehalogenases are well established for synthetic purposes. This review introduces the recent progress in the construction of CN bonds using promiscuous enzymes.

Biocatalytic approaches for the synthesis of optically pure vic-halohydrins.

Enantiopure vicinal halohydrins (vic-halohydrins) are highly valuable building blocks for the synthesis of many different natural products and pharmaceuticals, and biocatalytic methods for their synthesis have received considerable interest. This review emphasizes the application of biocatalytic approaches as an efficient alternative or complement to conventional chemical reactions, with a special focus on the asymmetric reductions catalyzed by ketoreductases, kinetic resolution catalyzed using lipases or esterases, stereoselective biotransformation catalyzed by halohydrin dehalogenases, asymmetric hydroxylation catalyzed by cytochrome P450 monooxygenases, asymmetric dehalogenation catalyzed by haloalkane dehalogenases, and aldehyde condensation catalyzed by aldolases. Although many chiral vic-halohydrins have been successfully synthesized using wild-type biocatalysts, their enantioselectivity is often too low for enantiopure synthesis. To overcome these limitations, catalytic properties of wild-type enzymes have been improved by rational and semi-rational protein design or directed evolution. This review briefly introduces the research status in this field, highlighting aspects of basic academic research in the biocatalytic synthesis of optically active vic-halohydrins by employing such unconventional approaches. KEY POINTS: • Outlines the enzymatic strategies for the production of enantiopure vic-halohydrins • Highlights recent advances in biocatalytic production of enantiopure vic-halohydrins • Provide guidance for efficient preparation of enantiopure vic-halohydrins.

Improving the enantioselectivity of halohydrin dehalogenase for the synthesis of (R)-benzyl glycidyl ether via biocatalytic azidolysis.

Halohydrin dehalogenases (HHDHs) are valuable biocatalysts for the synthesis of enantiopure benzyl glycidyl ether (BGE) and its derivatives, which are important synthetic intermediates for anti-cancer and anti-obesity drugs. However, all the reported HHDHs exhibit low enantioselectivity. In this study, we screened site-saturation mutagenesis libraries of AbHHDH at positions R89, A136, V137, P178, N179, F180, I181, Y186 and F187 for mutants with enhanced enantioselectivity toward BGE. The four improved variant R89V, R89Y, R89K and V137I were identified, and the double mutant R89Y/V137I showed 2.9-fold higher enantioselectivity than the wild type. The regions of HHDH containing the identified mutations were analyzed by homology modeling to explain the changes of enantioselectivity. Kinetic resolution of 20 to 100 mM BGE using whole cells of Escherichia coli expressing the mutant R89Y/V137I resulted in (R)-BGE yields of 42 to 32.5%, with ee >99%. This study improves our understanding of the enantioselectivity of HHDHs and contributes improved biocatalysts for the kinetic resolution of BGE.

Targeting annexin A2 reduces tumorigenesis and therapeutic resistance of nasopharyngeal carcinoma.

The expression of annexin A2 (ANXA2) in nasopharyngeal carcinoma (NPC) cells induces the immunosuppressive response in dendritic cells; however, the oncogenic effect and clinical significance of ANXA2 have not been fully investigated in NPC cells. Immunohistochemical staining for ANXA2 was performed in 61 patients and the association with clinicopathological status was determined. Short hairpin (sh)RNA knockdown of ANXA2 was used to examine cellular effects of ANXA2, by investigating alterations in cell proliferation, migration, invasion, adhesion, tube-formation assay, and chemo- and radiosensitivity assays were performed. RT-qPCR, Western blotting, and immunofluorescence were applied to determine molecular expression levels. Clinical association studies showed that the expression of ANXA2 was significantly correlated with metastasis (p = 0.0326) and poor survival (p = 0.0256). Silencing of ANXA2 suppressed the abilities of cell proliferation, adhesion, migration, invasion, and vascular formation in NPC cell. ANXA2 up-regulated epithelial-mesenchymal transition associated signal proteins. Moreover, ANXA2 reduced sensitivities to irradiation and chemotherapeutic drugs. These results define ANXA2 as a novel prognostic factor for malignant processes, and it can serve as a molecular target of therapeutic interventions for NPC.