Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies.

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed "starch-mannose-fermentation" biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System.

Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained.IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

Development of food-grade expression system for d-allulose 3-epimerase preparation with tandem isoenzyme genes in Corynebacterium glutamicum and its application in conversion of cane molasses to D-allulose.

D-Allulose 3-epimerase (DAE) has been applied to produce D-allulose, a low-calorie and functional sweetener. In this study, a new DAE from Paenibacillus senegalensis was characterized in Escherichia coli. Furthermore, we presented a tandem isoenzyme gene expression strategy to express multiple DAEs in one cell and construct food-grade expression systems based on Corynebacterium glutamicum. Seventeen expression cassettes based on three DAE genes from different organisms were constructed. Among all recombinant strains, DAE16 harboring three DAE genes in an expression vector exhibited the highest enzyme activity with 22.7 U/mg. Whole-cell transformation of DAE16 produced 225 g/L D-allulose with a volumetric productivity of 353 g·g -1 ·hr -1 . The catalytic efficiency of strain C-DAE9 integrating total 11 DAE genes in chromosome was 16.4-fold higher than strains carrying one DAE. Fed-batch culture of C-DAE9 gave enzyme activity of 44,700 U/L. We also expressed a thermostable invertase in C. glutamicum and obtained enzyme activity of 29 U/mg. Immobilized cells expressing DAE or invertase exhibited 80% of retained activity after 30 cycles of catalytic reactions. Those immobilized cells were coupled to produce 61.2 g/L D-allulose from cane molasses in a two-step reaction process. This study provided an efficient approach for enzyme preparation and allowed access to produce D-allulose from other abundant and low-cost feedstock enriched with sucrose.

Engineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars.

Naturally, haloacid dehalogenase superfamily phosphatases have been evolved with broad substrate promiscuity; however, strong specificity to a particular substrate is required for developing thermodynamically driven routes for manufacturing sugars. How to alter the intrinsic substrate promiscuity of phosphatases and fit the "one enzyme-one substrate" model remains a challenge. Herein, we report the structure-guided engineering of a phosphatase, and successfully provide variants with tailor-made preference for three widespread phosphorylated sugars, namely, glucose 6-phosphate, fructose 6-phosphate, and mannose 6-phosphate, while simultaneously enhancement in catalytic efficiency. A 12000-fold switch from unfavorite substrate to dedicated one is generated. Molecular dynamics simulations reveal the origin of improved activity and substrate specificity. Furthermore, we develop four coordinated multienzyme systems and accomplish the conversion of inexpensive sucrose and starch to fructose and mannose in excellent yield of 94-96%. This innovative sugar-biosynthesis strategy overcomes the reaction equilibrium of isomerization and provides the promise of high-yield manufacturing of other monosaccharides and polyols.

Novel catalytic property of fructose-6-phosphate aldolase in directly conversion of two 1-hydroxyalkanones to diketones.

Asymmetric CC bond formation catalyzed by aldolases requires the supplementation of nucleophiles and receptors in the reaction medium. However, aldol condensation using a single ketone as substrate has never been reported yet. In this work, we discovered that d-fructose-6-phosphate aldolase (FSA) could convert two 1-hydroxyalkanones, such as hydroxyacetone (HA) and 1-hydroxy-2-butanone, into two type of diketones. The initial product synthesis rate increased 3-fold and the yield reached to 56 %, when pure oxygen was directly inputted into the reaction medium. The results confirmed that oxygen participated in this reaction and hydrogen peroxide was generated. Metal ions Co2+ and Cu2+ remarkably increased the conversion yield compared with the control. For this reaction mechanism, we conjectured that HA may be oxidized to methylglyoxal by enzyme FSA in the presence of oxygen in the medium, and then FSA catalyzes the aldol addition between HA and its oxidative product MG to form diketone products. The obtained diketones could serve as important precursors for preparing furans and pyrroles.

High-Yield Biosynthesis of Glucosylglycerol through Coupling Phosphorolysis and Transglycosylation Reactions.

Glucosylglycerol is a powerful osmolyte that has attracted attention as a useful moisturizing ingredient in the cosmetic industry. This study demonstrates two artificially designed synthetic routes for manufacturing glucosylglycerol by combining phosphorolysis and transglycosylation reactions. The overall Gibbs energy change of the synthetic routes was negative, indicating that they are thermodynamically favorable. In vitro biosystems were constructed through combining the phosphorolysis ability of sucrose/maltose phosphorylase and the transglycosylation capacity of glucosylglycerol phosphorylases from different organisms. A near-stoichiometric conversion of sucrose and glycerol with a high product yield of 98% was achieved under optimal reaction conditions. The large-scale glucosylglycerol production of this biosystem was investigated under a high concentration of substrates (2 mol/L sucrose and 2.4 mol/L glycerol), and the titer reached 1.78 mol/L (452 g/L) with a productivity of 24.3 g/L/h. To the best of our knowledge, this value presented the highest glucosylglycerol production level until now, which indicated a great industrial application potential for glucosylglycerol manufacturing.

Multi-enzyme systems and recombinant cells for synthesis of valuable saccharides: Advances and perspectives.

Saccharides have recently attracted considerable attention because of their biological functions and potential applications in the pharmaceutical, cosmetic and food industries. Over the decades, a large amount of enzymes involved in saccharide synthesis have been discovered and characterised with the aid of available genome sequences. The advancement of metabolic engineering and synthetic biology strategies facilitated the artificial pathway design and construction for production of multiple sugars in vitro and in vivo based on those characterized enzymes. This review presented a panoramic view of enzymes related to saccharide synthesis and gave the detailed information. Furthermore, we provide an extensive overview of the recent advances in the construction of cell-free reaction systems and engineering of microbial cells for the production of natural or unnatural saccharides. In addition, the future trends in the synthesis of sugars with high structural diversity through the combination of multiple pathways are presented and evaluated.

Biosynthesis of dendroketose from different carbon sources using in vitro and in vivo metabolic engineering strategies.

BACKGROUND: Asymmetric aldol-type C-C bond formation with ketones used as electrophilic receptor remains a challenging reaction for aldolases as biocatalysts. To date, only one kind of dihydroxyacetone phosphate (DHAP)-dependent aldolases has been discovered and applied to synthesize branched-chain sugars directly using DHAP and dihydroxyacetone (DHA) as substrate. However, the unstable and high-cost properties of DHAP limit large-scale application. Therefore, biosynthesis of branched-chain sugar from low-cost and abundant carbon sources is essential. RESULTS: The detailed catalytic property of l-rhamnulose-1-phosphate aldolase (RhaD) and l-fuculose-1-phosphate aldolase (FucA) from Escherichia coli in catalyzing the aldol reactions with DHA as electrophilic receptors was characterized. Furthermore, we calculated the Bürgi-Dunitz trajectory using molecular dynamics simulations, thereby revealing the original sources of the catalytic efficiency of RhaD and FucA. A multi-enzyme reaction system composed of formolase, DHA kinase, RhaD, fructose-1-phosphatase, and polyphosphate kinase was constructed to in vitro produce dendroketose, a branched-chain sugar, from one-carbon formaldehyde. The conversion rate reached 86% through employing a one-pot, two-stage reaction process. Moreover, we constructed two artificial pathways in Corynebacterium glutamicum to obtain this product in vivo starting from glucose or glycerol. Fermentation with glycerol as feedstock produced 6.4 g/L dendroketose with a yield of 0.45 mol/mol glycerol, representing 90% of the maximum theoretical value. Additionally, the dendroketose production reached 36.3 g/L with a yield of 0.46 mol/mol glucose when glucose served as the sole carbon resource. CONCLUSIONS: The detailed enzyme kinetics data of the two DHAP-dependent aldolases with DHA as electrophilic receptors were presented in this study. In addition, insights into this catalytic property were given via in silico simulations. Moreover, the cost-effective synthesis of dendroketose starting from one-, three-, and six-carbon resources was achieved through in vivo and in vitro metabolic engineering strategies. This rare branched-chain ketohexose may serve as precursor to prepare 4-hydroxymethylfurfural and branched-chain alkanes using chemical method.