Maltoheptaose

Enzymatic Assembly of Chitosan-Based Network Polysaccharides and Their Encapsulation and Release of Fluorescent Dye.[Pubmed:38675624]

Molecules. 2024 Apr 16;29(8):1804.

We prepared network polysaccharide nanoscopic hydrogels by crosslinking water-soluble chitosan (WSCS) with a carboxylate-terminated maltooligosaccharide crosslinker via condensation. In this study, the enzymatic elongation of amylose chains on chitosan-based network polysaccharides by glucan phosphorylase (GP) catalysis was performed to obtain assembly materials. Maltoheptaose (Glc(7)) primers for GP-catalyzed enzymatic polymerization were first introduced into WSCS by reductive amination. Crosslinking of the product with the above-mentioned crosslinker by condensation was then performed to produce Glc(7)-modified network polysaccharides. The GP-catalyzed enzymatic polymerization of the alpha-d-glucose 1-phosphate monomer from the Glc(7) primers on the network polysaccharides was conducted, where the elongated amylose chains formed double helices. Enzymatic disintegration of the resulting network polysaccharide assembly successfully occurred by alpha-amylase-catalyzed hydrolysis of the double helical amyloses. The encapsulation and release of a fluorescent dye, Rhodamine B, using the CS-based network polysaccharides were also achieved by means of the above two enzymatic approaches.

Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths.[Pubmed:38211531]

Biochem Biophys Res Commun. 2024 Feb 5;695:149467.

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to Maltoheptaose with affinities up to Ka = 9.02 +/- 0.49 x 10(5) M(-1). SAmalE binding to maltotriose-Maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 +/- 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 +/- 0.49 x 10(5) M(-1). In the plot of DeltaH-T*DeltaS, the of Enthalpy-Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

In-Source Decay MALDI and High-Energy Collision-Induced Dissociation Mass Spectrometry of Alkali Metal-Adducted Underivatized Oligosaccharides.[Pubmed:37812625]

J Am Soc Mass Spectrom. 2023 Nov 1;34(11):2594-2606.

In-source decay (ISD) and high-energy collision-induced dissociation (HE-CID) were explored to provide structural information on alkali metal-adducted linear and stacked oligosaccharides (oligosaccharides with increased flexibility due to linkage type). These oligosaccharides include isomeric tetrasaccharides, Maltoheptaose, and several human milk oligosaccharides (HMOs). Matrix-assisted laser desorption ionization (MALDI) ion production efficiency, as well as the product ion intensities, and the number of product ions formed in ISD and HE-CID of these oligosaccharides were influenced by the matrix, the ionic radius of the metal ion used for adduction, and the affinity of metal ions for specific functional groups in the oligosaccharides. 2,4,6-Trihydroxyacetophenone (THAP) was the best matrix for HE-CID of oligosaccharides, 4-dimethylaminobenzaldehyde (DMABA) worked best for ISD of tetrasaccharides and pentasaccharides, while 2,5-dihydroxybenzoic acid (DHB) was the best matrix for ISD and HE-CID of long chain oligosaccharides. In general, the number of product ions formed followed the trend Li(+) > Na(+) > K(+) > Rb(+) > Cs(+), except for HMOs where Na(+) >/= Li(+) > K(+) > Rb(+) > Cs(+) occurred. The type of product ions formed and their intensities varied based on the position of the glycosidic bond linkage and the content of the monosaccharide. ISD and HE-CID produced diagnostic ions that could structurally differentiate isomers. Overall, HE-CID of alkali-metal adducted oligosaccharides produces intense glycosidic bond cleavages and low intensity cross-ring and internal cleavages. In contrast, ISD generates mainly cross-ring cleavages and internal cleavages at intensities higher than in HE-CID. In addition, ISD produced unique product ions that complement results from HE-CID.

Permeabilized whole cells containing co-expressed cyclomaltodextrinase and maltooligosyltrehalose synthase facilitate the synthesis of nonreducing maltoheptaose (N-G7) from beta-cyclodextrin.[Pubmed:37337412]

J Sci Food Agric. 2023 Nov;103(14):7061-7069.

BACKGROUND: Maltodextrin is an important bulk ingredient in food and other industries; however, drawbacks such as uneven polymerization and high reducibility limit its utilization. Nonreducing Maltoheptaose (N-G7) is a good substitute for maltodextrin owing to its single degree of polymerization and its nonreducing properties. In this study, in vitro cell factory biotransformation of beta-cyclodextrin (beta-CD) to N-G7 is demonstrated using coexpressed cyclomaltodextrinase (CDase, EC 3.2.1.54) and maltooligosyltrehalose synthase (MTSase, EC 5.4.99.15). However, the cell membrane prevents beta-CD from entering the cell owing to its large diameter. RESULTS: The amylase-deficient permeabilized host DeltaycjM-DeltamalS-DeltalpxM is utilized for the coexpression of recombinant CDase and MTSase. Deletion of lpxM effectively allows the entry of beta-cyclodextrin into the cell, despite its large diameter, without requiring any relevant cell membrane permeability-promoting reagent. This results in a 28.44% increase in the efficiency of beta-CD entry into the cell, thus enabling intracellular N-G7 synthesis without the extracellular secretion of recombinant CDase and MTSase. After reacting for 5.5 h, the highest purity of N-G7 (65.50%) is obtained. However, hydrolysis decreases the purity of N-G7 to 49.30%, thus resulting in a conversion rate of 40.16% for N-G7 when the reaction lasts 6 h. Precise control of reaction time is crucial for obtaining high-purity N-G7. CONCLUSION: Whole-cell catalysis avoids cell fragmentation and facilitates the creation of an eco-friendly, energy-efficient biotransformation system; thus, it is a promising approach for N-G7 synthesis. (c) 2023 Society of Chemical Industry.

Synthesis of Glycopolymer Micelles for Antibiotic Delivery.[Pubmed:37241780]

Molecules. 2023 May 11;28(10):4031.

In this work, we designed biodegradable glycopolymers consisting of a carbohydrate conjugated to a biodegradable polymer, poly(lactic acid) (PLA), through a poly(ethylene glycol) (PEG) linker. The glycopolymers were synthesized by coupling alkyne end-functionalized PEG-PLA with azide-derivatized mannose, trehalose, or Maltoheptaose via the click reaction. The coupling yield was in the range of 40-50% and was independent of the size of the carbohydrate. The resulting glycopolymers were able to form micelles with the hydrophobic PLA in the core and the carbohydrates on the surface, as confirmed by binding with the lectin Concanavalin A. The glycomicelles were ~30 nm in diameter with low size dispersity. The glycomicelles were able to encapsulate both non-polar (rifampicin) and polar (ciprofloxacin) antibiotics. Rifampicin-encapsulated micelles were much smaller (27-32 nm) compared to the ciprofloxacin-encapsulated micelles (~417 nm). Moreover, more rifampicin was loaded into the glycomicelles (66-80 mug/mg, 7-8%) than ciprofloxacin (1.2-2.5 mug/mg, 0.1-0.2%). Despite the low loading, the antibiotic-encapsulated glycomicelles were at least as active or 2-4 times more active than the free antibiotics. For glycopolymers without the PEG linker, the antibiotics encapsulated in micelles were 2-6 times worse than the free antibiotics.

Interface Manipulations Using Cross-Linked Underlayers and Surface-Active Diblock Copolymers to Extend Morphological Diversity in High-chi Diblock Copolymer Thin Films.[Pubmed:37134266]

ACS Appl Mater Interfaces. 2023 May 17;15(19):23736-23748.

Top and bottom interfaces of high-chi cylinder-forming polystyrene-block-Maltoheptaose (PS-b-MH) diblock copolymer (BCP) thin films are manipulated using cross-linked copolymer underlayers and a fluorinated phase-preferential surface-active polymer (SAP) additive to direct the self-assembly (both morphology and orientation) of BCP microdomains into sub-10 nm patterns. A series of four photo-cross-linkable statistical copolymers with various contents of styrene, a 4-vinylbenzyl azide cross-linker, and a carbohydrate-based acrylamide are processed into 15 nm-thick cross-linked passivation layers on silicon substrates. A partially fluorinated analogue of the PS-b-MH phase-preferential SAP additive is designed to tune the surface energy of the top interface. The self-assembly of PS-b-MH thin films on top of different cross-linked underlayers and including 0-20 wt % of SAP additive is investigated by atomic force microscopy and synchrotron grazing incidence small-angle X-ray scattering analysis. The precise manipulation of the interfaces of ca. 30 nm thick PS-b-MH films not only allows the control of the in-plane/out-of-plane orientation of hexagonally packed (HEX) cylinders but also promotes epitaxial order-order transitions from HEX cylinders to either face-centered orthorhombic or body-centered cubic spheres without modifying the volume fraction of both blocks. This general approach paves the way for the controlled self-assembly of other high-chi BCP systems.

Mass Spectrometry Approach for Differentiation of Positional Isomers of Saccharides: Toward Direct Analysis of Rare Sugars.[Pubmed:36947664]

Anal Chem. 2023 Apr 4;95(13):5635-5642.

Rare sugars have gained popularity in recent years due to their use in antiaging treatments, their ability to sweeten with few calories, and their ability to heal infections. Rare sugars are found in small quantities in nature, and they exist typically as isomeric forms of traditional sugars, rendering some challenges in their isolation, synthesis, and characterization. In this work, we present the first direct mass spectrometric approach for differentiating structural isomers of sucrose that differ only by their glycosidic linkages. The method employed a noncontact nanoelectrospray (nESI) platform capable of analyzing minuscule volumes (5 muL) of saccharides via the formation of halide adducts ([M+X](-); X = Cl and Br). Tandem mass spectrometry analysis of the five structural isomers of sucrose afforded diagnostic fragment ions that can be used to distinguish each isomer. Detailed mechanisms showcasing the distinct fragmentation pattern for each isomer are discussed. The method was applied to characterize and confirm the presence of all five selected rare sugars in raw honey complex samples. Aside from the five natural alpha isomers of sucrose, the method was also suitable for differentiating some beta isomers of the same glycosidic linkages, provided the monomeric sugar units are different. The halide adduct formation via the noncontact nESI source was also proven to be effective for oligosaccharides such as raffinose, beta-cyclodextrin, and Maltoheptaose. The results from this study encourage the future development of methods that function with simple operation to enable straightforward characterization of small quantities of rare sugars.

Cost-effective and controllable synthesis of isomalto/malto-polysaccharides from beta-cyclodextrin by combined action of cyclodextrinase and 4,6-alpha-glucanotransferase GtfB.[Pubmed:36925243]

Carbohydr Polym. 2023 Jun 15;310:120716.

Isomalto/malto-polysaccharides (IMMPs) derived from malto-oligosaccharides such as Maltoheptaose (G7) are elongated non-branched gluco-oligosaccharides produced by 4,6-alpha-glucanotransferase (GtfB). However, G7 is expensive and cumbersome to produce commercially. In this study, a cost-effective enzymatic process for IMMPs synthesis is developed that utilizes the combined action of cyclodextrinase from Palaeococcus pacificus (PpCD) and GtfB-DeltaN from Limosilactobacillus reuteri 121 to convert beta-cyclodextrin into IMMPs with a maximum yield (16.19 %, w/w). The purified IMMPs synthesized by simultaneous or sequential treatments, designated as IMMP-Sim and IMMP-Seq, possess relatively high contents of alpha-(1 --> 6) glucosidic linkages. By controlling the release of G7 and smaller malto-oligosaccharides by PpCD, IMMP-Seq was obtained of DP varying from 12.9 to 29.5. Enzymatic fingerprinting revealed different linkage-type distribution of alpha-(1 --> 6) linked segments with alpha-(1 --> 4) segments embedded at the reducing end and middle part. The proportion of alpha-(1 --> 6) segments containing the non-reducing end was 56.76 % for IMMP-Sim but 28.98 % for IMMP-Seq. Addition of G3 or G4 as specific acceptors resulted in IMMPs exhibiting low polydispersity. This procedure can be applied as a novel bioprocess that does not require costy high-purity malto-oligosaccharides and with control of the average DP of IMMPs by adjusting the substrate composition.

Vine-Twining Inclusion Behavior of Amylose towards Hydrophobic Polyester, Poly(beta-propiolactone), in Glucan Phosphorylase-Catalyzed Enzymatic Polymerization.[Pubmed:36836651]

Life (Basel). 2023 Jan 20;13(2):294.

This study investigates inclusion behavior of amylose towards, poly(beta-propiolactone) (PPL), that is a hydrophobic polyester, via the vine-twining process in glucan phosphorylase (GP, isolated from thermophilic bacteria, Aquifex aeolicus VF5)-catalyzed enzymatic polymerization. As a result of poor dispersibility of PPL in sodium acetate buffer, the enzymatically produced amylose by GP catalysis incompletely included PPL in the buffer media under the general vine-twining polymerization conditions. Alternatively, we employed an ethyl acetate-sodium acetate buffer emulsion system with dispersing PPL as the media for vine-twining polymerization. Accordingly, the GP (from thermophilic bacteria)-catalyzed enzymatic polymerization of an alpha-d-glucose 1-phosphate monomer from a Maltoheptaose primer was performed at 50 degrees C for 48 h in the prepared emulsion to efficiently form the inclusion complex. The powder X-ray diffraction profile of the precipitated product suggested that the amylose-PPL inclusion complex was mostly produced in the above system. The (1)H NMR spectrum of the product also supported the inclusion complex structure, where a calculation based on an integrated ratio of signals indicated an almost perfect inclusion of PPL in the amylosic cavity. The prevention of crystallization of PPL in the product was suggested by IR analysis, because it was surrounded by the amylosic chains due to the inclusion complex structure.

Extracellular recombinant production of 4,6 and 4,3 alpha-glucanotransferases in Lactococcus lactis.[Pubmed:36516732]

Enzyme Microb Technol. 2023 Mar;164:110175.

4,6 alpha-Glucanotransferase (4,6-alpha-GTase) and 4,3 alpha-glucanotransferases (4,3-alpha-GTase) produced by Lactic Acid Bacteria (LAB) in the GH70 enzyme family have become important due to their catalytic effect on starch and maltodextrins. Their high level of production is necessary for their application at industrial scale. In this respect, both enzymes were expressed extracellularly using Lactococcus lactis as GRAS host. 4,6-alpha-GTase and 4,3-alpha-GTase genes from Limosilactobacillus reuteri E81 and Limosilactobacillus fermentum PFC282 respectively were transformed into the plasmid pLEB124 vector having the signal peptide usp45 under the P45 continuous promoter and successfully expressed in Lactococcus lactis MG1363. Western blot screening showed that the relevant enzymes were able to be successfully secreted extracellularly. The Vmax and Km of 4,6-alpha-GTase were 2.58 micromol min(-1) and 0054 mg min(-1) whereas 3369 micromol min(-1) and 0032 mg min(-1) for 4,3-alpha-GTase respectively. NMR analysis demonstrated the formation of new bonds within the corresponding enzymes. Also, both enzymes were active on maltose, Maltoheptaose, maltohexaose and starch and produced malto-oligosaccarides observed by TLC analysis. In conclusion, this study demonstrated first time the extracellular production of 4,6-alpha-GTase and 4,3-alpha-GTase with GRAS status that can be useful for starch retrogradation delay and glycaemic index reduction.

Improving the Product Specificity of Maltotetraose-Forming Amylase from Pseudomonas saccharophila STB07 by Removing the Carbohydrate-Binding Module.[Pubmed:36238980]

J Agric Food Chem. 2022 Oct 26;70(42):13709-13718.

Maltotetraose (G4) is composed of four glucose units linked by the alpha-1,4-glycosidic bond, which has excellent adaptability in food processing and specific physiological functions. Maltotetraose-forming amylases (MFAses) are used in the industry as a promising tool for G4 production. The MFAse from Pseudomonas saccharophila STB07 (MFA(PS)), which belongs to the GH13, can preferentially hydrolyze substrates to G4. MFA(PS) contains a carbohydrate-binding module (CBM). In this study, we removed the CBM to obtain the mutant MFA(PS)-DeltaCBM. We explored the aspects affecting the catalytic performance of enzymes through structural simulations and molecular docking. Results showed that when the CBM was removed, the thermal stability of MFA(PS) was slightly reduced, and its catalytic ability for long-chain substrates, such as corn starch, was significantly reduced. However, the catalytic ability and product specificity of the substrates with shorter chain length, such as maltodextrin (DE 7-9), were improved. The G1-G7 (glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), and Maltoheptaose (G7)) contents and G4 proportion of the mutant MFA(PS)-DeltaCBM reaction at 24 h were 11.1 and 11.6% higher than those of MFA(PS), respectively. The results also showed that the forces of MFA(PS) on the substrate near the -4, -1, +1, and +3 subsites were critical for its product specificity.

Measuring key human carbohydrate digestive enzyme activities using high-performance anion-exchange chromatography with pulsed amperometric detection.[Pubmed:36180531]

Nat Protoc. 2022 Dec;17(12):2882-2919.

Carbohydrate digestion in the mammalian gastrointestinal tract is catalyzed by alpha-amylases and alpha-glucosidases to produce monosaccharides for absorption. Inhibition of these enzymes is the major activity of the drugs acarbose and miglitol, which are used to manage diabetes. Furthermore, delaying carbohydrate digestion via inhibition of alpha-amylases and alpha-glucosidases is an effective strategy to blunt blood glucose spikes, a major risk factor for developing metabolic diseases. Here, we present an in vitro protocol developed to accurately and specifically assess the activity of alpha-amylases and alpha-glucosidases, including sucrase, maltase and isomaltase. The assay is especially suitable for measuring inhibition by compounds, drugs and extracts, with minimal interference from impurities or endogenous components, because the substrates and digestive products in the enzyme activity assays are quantified directly by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Multiple enzyme sources can be used, but here we present the protocol using commercially available human alpha-amylase to assess starch hydrolysis with Maltoheptaose as the substrate, and with brush border sucrase-isomaltase (with maltase, sucrase and isomaltase activities) derived from differentiated human intestinal Caco-2(/TC7) cells to assess hydrolysis of disaccharides. The wet-lab assay takes ~2-5 h depending on the number of samples, and the HPAE-PAD analysis takes 35 min per sample. A full dataset therefore takes 1-3 d and allows detection of subtle changes in enzyme activity with high sensitivity and reliability.

Glass Transition Temperatures of Individual Submicrometer Atmospheric Particles: Direct Measurement via Heated Atomic Force Microscopy Probe.[Pubmed:35993793]

Anal Chem. 2022 Sep 6;94(35):11973-11977.

The phase (solid, semisolid, or liquid) of atmospheric aerosols is central to their ability to take up water or undergo heterogeneous reactions. In recent years, the unexpected prevalence of viscous organic particles has been shown through field measurements and global atmospheric modeling. The aerosol phase has been predicted using glass transition temperatures (T(g)), which were estimated based on molecular weight, oxygen:carbon ratio, and chemical formulae of organic species present in atmospheric particles via studies of bulk materials. However, at the most important sizes for cloud nucleation ( approximately 50-500 nm), particles are complex mixtures of numerous organic species, inorganic salts, and water with substantial particle-to-particle variability. To date, direct measurements of T(g) have not been feasible for individual atmospheric particles. Herein, nanothermal analysis (NanoTA), which uses a resistively heated atomic force microscopy (AFM) probe, is combined with AFM photothermal infrared (AFM-PTIR) spectroscopy to determine the T(g) and composition of individual particles down to 76 nm in diameter at ambient temperature and pressure. Laboratory-generated proxies for organic aerosol (sucrose, ouabain, raffinose, and Maltoheptaose) had similar T(g) values to bulk T(g) values measured with differential scanning calorimetry (DSC) and the T(g) predictions used in atmospheric models. Laboratory-generated phase-separated particles and ambient particles were analyzed with NanoTA + AFM-PTIR showing intraparticle variation in composition and T(g). These results demonstrate the potential for NanoTA + AFM-PTIR to increase our understanding of viscosity within submicrometer atmospheric particles with complex phases, morphologies, and compositions, which will enable improved modeling of aerosol impacts on clouds and climate.

Harnessing of Spatially Confined Perovskite Nanocrystals Using Polysaccharide-based Block Copolymer Systems.[Pubmed:35737998]

ACS Appl Mater Interfaces. 2022 Jul 6;14(26):30279-30289.

Metal halide perovskite nanocrystals (PVSK NCs) are generally unstable upon their transfer from colloidal dispersions to thin film devices. This has been a major obstacle limiting their widespread application. In this study, we proposed a new approach to maintain their exceptional optoelectronic properties during this transfer by dispersing brightly emitting cesium lead halide PVSK NCs in polysaccharide-based Maltoheptaose-block-polyisoprene-block-Maltoheptaose (MH-b-PI-b-MH) triblock copolymer (BCP) matrices. Instantaneous crystallization of ion precursors with favorable coordination to the sugar (Maltoheptaose) domains produced ordered NCs with varied nanostructures of controlled domain size ( approximately 10-20 nm). Confining highly ordered and low dimension PVSK NCs in polysaccharide-based BCPs constituted a powerful tool to control the self-assembly of BCPs and PVSK NCs into predictable structures. Consequently, the hybrid thin films exhibited excellent durability to humidity and stretchability with a relatively high PL intensity and photoluminescence quantum yield (>70%). Furthermore, stretchable phototransistor memory devices were produced and maintained with a good memory ratio of 10(5) and exhibited a long-term memory retention over 10(4) s at a high strain of 100%.

Permeabilized whole-cell biocatalyst containing co-expressed two enzymes facilitates the synthesis of maltoheptaose (G7) from starch.[Pubmed:35605493]

Enzyme Microb Technol. 2022 Sep;159:110057.

Maltoheptaose (G7) is one of the mixtures of maltodextrin widely used in the food, pharmaceutical, and cosmetics industries. A genetically engineered strain, which simultaneously expressed cyclodextrin glucanotransferase (CGTase) from Gracilibacillus alcaliphilus SK51.001 and cyclomaltodextrinase (CDase) from Bacillus sphaericus E-244, two enzymes, was constructed by cloning the above two genes into a plasmid and transformed into the host Escherichia coli BL21(DE3) (E. coli) strain, resulted in recombinant cells harboring the vector pETDuet-GaCGT/BsCD (pGaBs). These cells were used as whole-cell catalysts for the biotransformation of G7 from the inexpensive substrate (starch). Due to the high molecular weight of starch, the cell membrane prevents the entry of starch into the cellular system. Therefore, the pGaBs cell wall was permeabilized by lysozyme, EDTA, and heat treatment. After reaching the optimized conditions of permeabilized pGaBs cell amount, lysozyme amount, reaction temperature, and metal ion concentration, approximately 4.1 g/L of G7 was produced from 30 g/L starch in 1 h with the addition of Ca(2+). This co-expression system offers a one-pot synthesis approach to the production of G7 using an inexpensive substrate, avoiding enzyme purification steps.