Integrated pathway mining and selection of an artificial CYP79-mediated bypass to improve benzylisoquinoline alkaloid biosynthesis.

BACKGROUND: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct. RESULTS: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway. CONCLUSIONS: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.

Structural study of a polysaccharide component of nfnB mutant of Shewanella vesiculosa HM13.

Shewanella vesiculosa HM13 is a Gram-negative bacterium able to produce a large amount of extracellular membrane vesicles. These nanoparticles carry a major protein P49, the loading of which seems to be influenced by the glycans decorating the membrane. Here we report the structural characterization, using chemical analyses and NMR spectroscopy, of the capsular polysaccharides isolated from the nfnB-mutant strain of S. vesiculosa HM13, which is unable to load P49 on the membrane vesicles. In addition to the polysaccharide corona isolated and characterized from the parental strain, the nfnB-mutant strain released another polysaccharide composed of disaccharide repeating units having the following structure. →4)-ß-D-Glc-(1 â†’ 3)-ß-D-GlcNAc-(1→.

Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains.

Extracellular vesicles (EVs) have attracted an attention as important targets in the fields of biology and medical science because they contain physiologically active molecules. Curvature-sensing peptides are currently used as novel tools for marker-independent EV detection techniques. A structure-activity correlation study demonstrated that the α-helicity of the peptides is prominently involved in peptide binding to vesicles. However, whether a flexible structure changing from a random coil to an α-helix upon binding to vesicles or a restricted α-helical structure is an important factor in the detection of biogenic vesicles is still unclear. To address this issue, we compared the binding affinities of stapled and unstapled peptides for bacterial EVs with different surface polysaccharide chains. We found that unstapled peptides showed similar binding affinities for bacterial EVs regardless of surface polysaccharide chains, whereas stapled peptides showed substantially decreased binding affinities for bacterial EVs covered with capsular polysaccharides. This is probably because curvature-sensing peptides must pass through the layer of hydrophilic polysaccharide chains prior to binding to the hydrophobic membrane surface. While stapled peptides with restricted structures cannot easily pass through the layer of polysaccharide chains, unstapled peptides with flexible structures can easily approach the membrane surface. Therefore, we concluded that the structural flexibility of curvature-sensing peptides is a key factor for governing the highly sensitive detection of bacterial EVs.

Polysaccharide corona: The acetyl-rich envelope wraps the extracellular membrane vesicles and the cells of Shewanella vesiculosa providing adhesiveness.

Bacterial extracellular membrane vesicles (EMVs) play an active role in many physiological and pathogenic processes. Here, we report the identification and the detailed structural characterization of the capsular polysaccharide from both cells and EMVs from Shewanella vesiculosa by NMR and chemical analysis. The polysaccharide consists of a pentasaccharide repeating unit containing neutral monosaccharides together with amino sugars, of which one has never been isolated from a natural source. The adhesion ability of the polymer both on synthetic surfaces, such as polystyrene nanoparticles and on vesicles with a bilayer mimicking the bacterial membrane in the presence and absence of lipopolysaccharide was investigated. In both cases, a "CPS-corona" that could be the first stage of biofilm formation was observed. The polymer also activates Caspases on colon cancer cells, making S. vesiculosa EMVs as natural nanocarriers for drug delivery.

Design of the N-Terminus Substituted Curvature-Sensing Peptides That Exhibit Highly Sensitive Detection Ability of Bacterial Extracellular Vesicles.

Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.

Genetic characterization and functional implications of the gene cluster for selective protein transport to extracellular membrane vesicles of Shewanella vesiculosa HM13.

A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, lptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs.

Structural Elucidation of a Novel Lipooligosaccharide from the Antarctic Bacterium OMVs Producer Shewanella sp. HM13.

Shewanella sp. HM13 is a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel. It produces a large amount of outer membrane vesicles (OMVs), which are particles released in the medium where the bacterium is cultured. This strain biosynthesizes a single major cargo protein in the OMVs, a fact that makes Shewanella sp. HM13 a good candidate for the production of extracellular recombinant proteins. Therefore, the structural characterization of the components of the vesicles, such as lipopolysaccharides, takes on a fundamental role for understanding the mechanism of biogenesis of the OMVs and their applications. The aim of this study was to investigate the structure of the oligosaccharide (OS) isolated from Shewanella sp. HM13 cells as the first step for a comparison with that from the vesicles. The lipooligosaccharide (LOS) was isolated from dry cells, purified, and hydrolyzed by alkaline treatment. The obtained OS was analyzed completely, and the composition of fatty acids was obtained by chemical methods. In particular, the OS was investigated in detail by ¹H and 13C NMR spectroscopy and MALDI-TOF mass spectrometry. The oligosaccharide was characterized by the presence of a residue of 8-amino-3,8-dideoxy-manno-oct-2-ulosonic acid (Kdo8N) and of a d,d-heptose, with both residues being identified in other oligosaccharides from Shewanella species.