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

Abstract

BackgroundAsymmetric 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1293-7) contains supplementary material, which is available to authorized users.

One-Pot Multienzyme Synthesis of Rare Ketoses from Glycerol

“…The activities of RhaD from E. coli, FucA from T. thermophiles HB8, and FruA from S. carnosus were determined by measuring the decrease of D-GA concentration in the aldol reactions. 22 Briefly, the reaction was carried out in a 50 mM Tris-HCl buffer (pH 7.0) containing 50 mM of DHAP, 100 mM of D-GA, and 0.5 mg/mL of each aldolase in a final volume of 1 mL. After 5 min, the reaction was terminated by heating at 100 °C for 5 min, and then the concentration of D-GA was determined by highperformance liquid chromatography (HPLC).…”

“…thermophiles HB8, and FruA from S. carnosus were determined by measuring the decrease of d -GA concentration in the aldol reactions . Briefly, the reaction was carried out in a 50 mM Tris-HCl buffer (pH 7.0) containing 50 mM of DHAP, 100 mM of d -GA, and 0.5 mg/mL of each aldolase in a final volume of 1 mL.…”

“…Apart from the “Izumoring strategy”, aldol additions catalyzed by aldolases afford a promising alternative for rare sugar synthesis through joining two smaller fragments. − Among all aldolases, the dihydroxyacetone phosphate (DHAP)-dependent aldolases are very popularly used for building carbon–carbon bonds in synthesizing carbohydrates and their derivatives. − In our previous study, we adopted a one-pot four-enzyme system based on different DHAP-dependent aldolases and successfully used it to produce various rare ketoses. − In this system (Scheme ), we had to supply both fragments for the aldol reaction: glycerol 3-phosphate was used to produce DHAP through oxidation by glycerol phosphate oxidase (GPO), whereas the acceptor aldehyde had to be added externally. The by-product hydrogen peroxide from the oxidation step must be degraded into oxygen and water by catalase.…”

Production of l-Ribulose Using an Encapsulated l-Arabinose Isomerase in Yeast Spores

“…Currently, rare sugars have become very attractive in the food and medical industries due to their unique functions and potential application prospects. − Izumori established the “Izumoring strategy”, which is a very useful approach for producing all rare sugars through enzymatic conversion . As an improvement and extension of the Izumoring strategy”, Wen and co-workers developed an enzymatic phosphorylation–dephosphorylation cascade reaction to prepare various rare ketoses efficiently and cost-effectively. − In addition to the “Izumoring strategy”, aldol reactions catalyzed by aldolases provide an alternative and valuable method for the synthesis of rare sugars and their derivatives. − l -Ribulose is a rare sugar that has been employed as the precursor for the synthesis of l -ribose, which is widely used to prepare nucleoside analogues with good antiviral activity. − l -Ribulose is generated via the dehydrogenation of ribitol catalyzed by the whole cells of Gluconobacter oxidans . However, this method is not practical for mass production due to the expense associated with the large scale preparation of ribitol .…”

Artificial ATP-Free in Vitro Synthetic Enzymatic Biosystems Facilitate Aldolase-Mediated C–C Bond Formation for Biomanufacturing

“…GA3P is an important substrate (receptor) for some aldolases (e.g., fructose 6-phosphate aldolase and 2-deoxy- d -ribose 5-phosphate aldolase) to produce fructose 6-phosphate, 2-deoxy- d -ribose 5-phosphate, and 2′-deoxyribonucleosides . DHAP is another important substrate (donor) for DHAP-dependent aldolase (e.g., d -fructose 1,6-bisphosphate aldolase, l -fuculose 1-phosphate aldolase, l -rhamnulose 1-phosphate aldolase, and d -tagatose 1,6-bisphosphate aldolase) to synthesize saccharides, such as tagatose, allulose, erythrulose, dendroketose, and 3R,4R­(S),5R,6R-heptulose. − However, current methods to produce GA3P and DHAP are costly . Because these two compounds are labile, chemical synthesis methods usually focus on making more stable precursors, such as 3-phosphoryl d -glyceraldehyde dimethyl acetal (for GA3P) and 2,5-diethoxy- p -dioxane-2,5-dimethanol-O2 1 -O5 1 -bisphosphate (for DHAP), that can be stored at room temperature and can be simply transformed to GA3P and DHAP when necessary .…”