Identification and Validation of Magnolol Biosynthesis Genes in Magnolia officinalis.

Bacterial infections pose a significant risk to human health. Magnolol, derived from Magnolia officinalis, exhibits potent antibacterial properties. Synthetic biology offers a promising approach to manufacture such natural compounds. However, the plant-based biosynthesis of magnolol remains obscure, and the lack of identification of critical genes hampers its synthetic production. In this study, we have proposed a one-step conversion of magnolol from chavicol using laccase. After leveraging 20 transcriptomes from diverse parts of M. officinalis, transcripts were assembled, enriching genome annotation. Upon integrating this dataset with current genomic information, we could identify 30 laccase enzymes. From two potential gene clusters associated with magnolol production, highly expressed genes were subjected to functional analysis. In vitro experiments confirmed MoLAC14 as a pivotal enzyme in magnolol synthesis. Improvements in the thermal stability of MoLAC14 were achieved through selective mutations, where E345P, G377P, H347F, E346C, and E346F notably enhanced stability. By conducting alanine scanning, the essential residues in MoLAC14 were identified, and the L532A mutation further boosted magnolol production to an unprecedented level of 148.83 mg/L. Our findings not only elucidated the key enzymes for chavicol to magnolol conversion, but also laid the groundwork for synthetic biology-driven magnolol production, thereby providing valuable insights into M. officinalis biology and comparative plant science.

Optimizing Trilobatin Production via Screening and Modification of Glycosyltransferases.

Trilobatin (TBL) is a key sweet compound from the traditional Chinese sweet tea plant (Rubus suavissimus S. Lee). Because of its intense sweetness, superior taste profile, and minimal caloric value, it serves as an exemplary natural dihydrochalcone sweetener. It also has various health benefits, including anti-inflammatory and glucose-lowering effects. It is primarily produced through botanical extraction, which impedes its scalability and cost-effectiveness. In a novel biotechnological approach, phloretin is used as a precursor that is transformed into TBL by the glycosyltransferase enzyme ph-4'-OGT. However, this enzyme's low catalytic efficiency and by-product formation limit the large-scale synthesis of TBL. In our study, the enzyme Mdph-4'-OGT was used to screen 17 sequences across species for TBL synthesis, of which seven exhibited catalytic activity. Notably, PT577 exhibited an unparalleled 97.3% conversion yield within 3 h. We then optimized the reaction conditions of PT577, attaining a peak TBL bioproduction of 163.3 mg/L. By employing virtual screening, we identified 25 mutation sites for PT577, thereby creating mutant strains that reduced by-products by up to 50%. This research enhances the enzymatic precision for TBL biosynthesis and offers a robust foundation for its industrial-scale production, with broader implications for the engineering and in silico analysis of glycosyltransferases.

Identification and Optimization of more Efficient Olivetolic Acid Synthases.

Introduction: Olivetolic acid (OLA) is a key intermediate in cannabidiol (CBD) synthesis, and cannabinoids are important neuroactive drugs. However, the catalytic activity of olivetolic acid synthase (OLS), the key enzyme involved in OLA biosynthesis, remains low and its catalytic mechanism is unclear. Materials and Methods: In this study, we conducted a scrupulous screening of the pivotal rate-limiting enzyme and analyzed its amino acid sites that are critical to enzyme activity as validated by experiments. Results: Through stringent enzyme screening, we pinpointed a highly active OLS sequence, OLS4. Then, we narrowed down three critical amino acid sites (I258, D198, E196) that significantly influence the OLS activity. Conclusions: Our findings laid the groundwork for the efficient biosynthesis of OLA, and thereby facilitate the biosynthesis of CBD.

[Discussion on the location of Dazhui (GV 14) and Yaoyangguan (GV 3)].

Since the anatomical location of acupoints was recorded in The latest Practice of Western Acupuncture in 1915, and Lecture Notes on Advanced Acupuncture in 1931, the Japanese acupuncture works of Chinese translation version, the location of Dazhui (GV 14) (under the spinous process of the 7th cervical vertebra) and Yaoyangguan (GV 3) (under the spinous process of the 4th lumbar vertebra) had rarely been questioned for nearly a century. In order to confirm the above statement, the writers have reviewed ancient literature, combined with the modern anatomical knowledge and searched the evidences from the core arguments of the acupuncture Mingtang chart and the bronze acupuncture statue. It is believed that Dazhui （GV 14） should be positioned under the spinous process of the 1st thoracic vertebra, and Yaoyangguan（GV 3） be under the spinous process of the 5th lumbar vertebra. Accordingly, all of the other acupoints of these meridians should be moved down by 1 vertebra, i.e. those on the governor vessel from Dazhui (GV 14) to Yaoyangguan (GV 3), those on the 1st lateral line of the bladder meridian of foot-taiyang from Dazhu (BL 11) to Baihuanshu (BL 30) and those on the 2nd lateral line of the bladder meridian from Fufen (BL 41) to Zhibian (BL 54).