Production of ginsenoside compound K by microbial cell factory using synthetic biology-based strategy: a review.

Ginsenoside compound K (CK) is a major intestinal bacterial metabolite of the protopanaxadiol-type ginsenoside family that can be absorbed in the systemic circulation. CK possesses diverse and important pharmacological properties. The low production and high cost of traditional manufacturing methods based on the extraction and biotransformation of total ginsenosides from ginseng have limited their medical application. However, considerable progress has been made in the area of de novo CK production via microbial cell factories using synthetic biology-based strategies. By introducing key enzymes responsible for CK biosynthesis into microbial cells, CK was produced via a series of in vivo enzymatic reactions that utilize the inherent precursors in microbial cells. After systematic optimization using various metabolic engineering strategies, the yield of CK increased significantly and exceeded the traditional plant extraction-biotransformation method, implying the commercial feasibility of this approach. This review summarizes recent novel advancements in the production of CK using microbial cell factories.

Regeneration of Phytochemicals by Structure-Driven Organization of Microbial Biosynthetic Steps.

Medicinal phytochemicals, such as artemisinin and taxol, have impacted the world, and hypericin might do so if its availability issue could be addressed. Hypericin is the hallmark component of Saint John's wort (Hypericum perforatum L.), an approved depression alleviator documented in the US, European, and British pharmacopoeias with its additional effectiveness against diverse cancers and viruses. However, the academia-to-industry transition of hypericin remain hampered by its low in planta abundance, unfeasible bulk chemical synthesis, and unclear biosynthetic mechanism. Here, we present a strategy consisting of the hypericin-structure-centered modification and reorganization of microbial biosynthetic steps in the repurposed cells that have been tamed to enable the designed consecutive reactions to afford hypericin (43.1 mg L-1 ), without acquiring its biosynthetic knowledge in native plants. The study provides a synthetic biology route to hypericin and establishes a platform for biosustainable access to medicinal phytochemicals.

Intertwined Biosynthesis of Skyrin and Rugulosin A Underlies the Formation of Cage-Structured Bisanthraquinones.

Skyrin and rugulosin A are bioactive bisanthraquinones found in many fungi, with the former suggested as a precursor of hypericin (a diversely bioactive phytochemical) and the latter characterized by its distinct cage-like structure. However, their biosynthetic pathways remain mysterious, although they have been characterized for over six decades. Here, we present the rug gene cluster that governs simultaneously the biosynthesis of skyrin and rugulosin A in Talaromyces sp. YE3016, a fungal endophyte residing in Aconitum carmichaeli. A combination of genome sequencing, gene inactivation, heterologous expression, and biotransformation tests allowed the identification of the gene function, biosynthetic precursor, and enzymatic sets involved in their molecular architecture constructions. In particular, skyrin was demonstrated to form from the 5,5'-dimerization of emodin radicals catalyzed by RugG, a cytochrome P450 monooxygenase evidenced to be potentially applicable for the (chemo)enzymatic synthesis of dimeric polyphenols. The fungal aldo-keto reductase RugH was shown to be capable of hijacking the closest skyrin precursor (CSP) immediately after the emodin radical coupling, catalyzing the ketone reduction of CSP to inactivate its tautomerization into skyrin and thus allowing for the spontaneous intramolecular Michael addition to cyclize the ketone-reduced form of CSP into rugulosin A, a representative of diverse cage-structured bisanthraquinones. Collectively, the work updates our understanding of bisanthraquinone biosynthesis and paves the way for synthetic biology accesses to skyrin, rugulosin A, and their siblings.

Structural Insights into the Broad Substrate Specificity of a Novel Endoglycoceramidase I Belonging to a New Subfamily of GH5 Glycosidases.

Endoglycoceramidases (EGCases) specifically hydrolyze the glycosidic linkage between the oligosaccharide and the ceramide moieties of various glycosphingolipids, and they have received substantial attention in the emerging field of glycosphingolipidology. However, the mechanism regulating the strict substrate specificity of these GH5 glycosidases has not been identified. In this study, we report a novel EGCase I from Rhodococcus equi 103S (103S_EGCase I) with remarkably broad substrate specificity. Based on phylogenetic analyses, the enzyme may represent a new subfamily of GH5 glycosidases. The X-ray crystal structures of 103S_EGCase I alone and in complex with its substrates monosialodihexosylganglioside (GM3) and monosialotetrahexosylganglioside (GM1) enabled us to identify several structural features that may account for its broad specificity. Compared with EGCase II from Rhodococcus sp. M-777 (M777_EGCase II), which possesses strict substrate specificity, 103S_EGCase I possesses a longer α7-helix and a shorter loop 4, which forms a larger substrate-binding pocket that could accommodate more extended oligosaccharides. In addition, loop 2 and loop 8 of the enzyme adopt a more open conformation, which also enlarges the oligosaccharide-binding cavity. Based on this knowledge, a rationally designed experiment was performed to examine the substrate specificity of EGCase II. The truncation of loop 4 in M777_EGCase II increased its activity toward GM1 (163%). Remarkably, the S63G mutant of M777_EGCase II showed a broader substrate spectra and significantly increased activity toward bulky substrates (up to >1370-fold for fucosyl-GM1). Collectively, the results presented here reveal the exquisite substrate recognition mechanism of EGCases and provide an opportunity for further engineering of these enzymes.

A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production.

Lyso-glycosphingolipids (lyso-GSLs), the N-deacylated forms of glycosphingolipids (GSLs), are important synthetic intermediates for the preparation of GSL analogs. Although lyso-GSLs can be produced by hydrolyzing natural GSLs using sphingolipid ceramide N-deacylase (SCDase), the yield for this reaction is usually low because SCDase also catalyzes the reverse reaction, ultimately establishing an equilibrium between hydrolysis and synthesis. In the present study, we developed an efficient method for controlling the reaction equilibrium by introducing divalent metal cation and detergent in the enzymatic reaction system. In the presence of both Ca(2+) and taurodeoxycholate hydrate, the generated fatty acids were precipitated by the formation of insoluble stearate salts and pushing the reaction equilibrium toward hydrolysis. The yield of GM1 hydrolysis can be achieved as high as 96%, with an improvement up to 45% compared with the nonoptimized condition. In preparative scale, 75 mg of lyso-GM1 was obtained from 100 mg of GM1 with a 90% yield, which is the highest reported yield to date. The method can also be used for the efficient hydrolysis of a variety of GSLs and sphingomyelin. Thus, this method should serve as a facile, easily scalable, and general tool for lyso-GSL production to facilitate further GSL research.

Comprehensive characterization of sphingolipid ceramide N-deacylase for the synthesis and fatty acid remodeling of glycosphingolipids.

Sphingolipid ceramide N-deacylase (SCDase) catalyzes reversible reactions in which the amide linkage in glycosphingolipids is hydrolyzed or synthesized. While SCDases show great value for the enzymatic synthesis of glycosphingolipids, they are relatively poorly characterized enzymes. In this work, the enzymatic properties of SCDase from Shewanella alga G8 (SA_SCD) were systematically characterized and compared with the commercially available SCDase from Pseudomonas sp. TK4 (PS_SCD). The optimal pH values for the hydrolytic and synthetic activity of SA_SCD were pH 6.0 and pH 7.5, respectively. Both activities were strongly inhibited by Zn(2+) and Cu(2+), while Fe(2+), Co(2+), Ni(2+), Mn(2+), Ca(2+), and Mg(2+) promoted the hydrolytic activity but inhibited the synthetic activity. SA_SCD showed very broad substrate specificity both in hydrolysis and synthesis. Importantly, SA_SCD has a broader specificity for acyl donor acceptance than does PS_SCD, especially for unsaturated fatty acids and fatty acids with very short or long acyl chains. Further kinetic analysis revealed that the k cat/K M value for the hydrolytic activity of SA_SCD was 8.9-fold higher than that of PS_SCD for GM1a, while the values for the synthetic activity were 38-fold higher for stearic acid and 23-fold higher for lyso-GM1a (d18:1) than those of PS_SCD, respectively. The broad fatty acid specificity and high catalytic efficiency, together with the ease of expression of SA_SCD in Escherichia coli, make it a better biocatalyst than is PS_SCD for the synthesis and structural remodeling of glycosphingolipids.