Enzymatic Synthesis Of Rare Sugars With Dihydroxyacetone Phosphate-Dependent Aldolases In Vitro And In Vivo

Asymmetric carbon-carbon bond construction has long been regarded as the most challenging issue in organic chemistry. Aldol additions catalyzed by aldolases have become an indispensable tool for the formation of asymmetric carbon-carbon bonds. Among the aldolase family, dihydroxyacetone phosphate (DHAP)-dependent aldolases are the most synthetically useful. This group of aldolases catalyze the aldol reaction between the donor DHAP and the acceptor aldehyde, generating the product with two stereocenters, which are usually determined by enzymes. Fortunately, four reported DHAP-dependent aldolases are stereocomplementary, therefore a complete set of diastereomers of vicinal diols can be achieved. Over the past two decades, aldolases of this group have been applied successfully in producing many rare monosaccharides and their derivatives. However, for this group of aldolases, the main drawback is the strict requirement for the donor substrate DHAP, a rather expensive and unstable compound, which limits their use in large-scale preparation.Rare sugars are monosaccharides and their derivatives, that are particularly uncommon in nature. Compared with sucrose, rare sugars usually have similar sweetness but provide less or no energy. Importantly, rare sugars possess many potential applications in the food, pharmaceutical and nutrition industries. In addition, rare sugars can be used as starting materials for the synthesis of intriguing natural products with important biological activities. Unfortunately, most rare sugars are quite expensive, and their synthetic routes are both limited and costly due to the expense of costly starting materials.This dissertation can be divided into two parts:the first part (chapter2) is in vitro synthesis of rare sugars using DHAP-dependent aldolases; the second part (chapter3) is the synthesis of rare sugars using DHAP-dependent aldolases in Escherichia coli, which is based on in vitro study. However, our ultimate purpose is in vivo synthesis of rare sugars with DHAP-dependent aldolases.In the first part, in order to investigate the synthetic applications of DHAP-dependent aldolases in vitro, four DHAP-dependent aldolases (L-rhamnulose-1-phosphate aldolase (RhaD), L-fuculose-1-phosphate aldolase (FucA), D-fructose-1,6-bisphophate aldolase (FruA) and D-tagatose-1,6-bisphosphate aldolase (TagA) were overexpressed and purified from E.coli BL21(DE3). In Chapter2(Section1), aldolase RhaD was used for in vitro rare sugar synthesis. It was previously reported that RhaD selectively choses L-glyceraldehyde from racemic glyceraldehyde to produce L-fructose exclusively. Contrastingly, we discovered that D-glyceraldehyde was also tolerated as an acceptor and the stereoselectivity of the enzyme was lost in the corresponding aldol addition. Furthermore, we applied this property to efficiently synthesize two rare sugars D-sorbose and D-psicose. To improve the practicality of the reaction by avoiding the use of DHAP, a rather unstable and expensive substrate, we adopted a one-pot four-enzyme strategy for the synthesis of D-sorbose and D-psicose. L-glycerol3-phosphate, which is cheap and stable, was used as the starting material to generate DHAP. DHAP was produced from the oxidation of L-glycerol3-phosphate by glycerol phosphate oxidase (GPO). The by-product of this oxidation, H2O2, being harmful for GPO activity, was thus selectively degraded by adding catalase. The DHAP generated in situ was coupled with D-glyceraldehyde by RhaD and following acid phosphatase catalyzed dephosphorylation furnished D-sorbose and D-psicose in a one-pot fashion. The product ratio (D-sorbose/D-psicose=～1/1) was determined by HPLC. The reaction mixture was purified by silica gel column chromatography and Bio-gel P-2column to get the mixture of D-sorbose and D-psicose. The total yield for these two sugars was48%. D-sorbose and D-psicose could be further purified by Ca2+exchange resin to get each pure sugar. Under the same conditions, when L-glyceraldehyde was used, instead of generating two sugars with D-glyceraldehyde as the acceptor, L-fructose was produced exclusively with a66%overall yield. This demonstrated that the configuration of the aldehyde could affect the stereoselectivity of RhaD. As GPO is selective toward L-glycerol-3-phosphate, a much cheaper starting material DL-glycerol-3-phosphate was used instead of L-glycerol-3-phosphate to further reduce the cost.In Chapter2(Section2), aldolase FucA was used for our in vitro synthesis of rare sugars. Firstly, FucA from E.coli (FucAE.coli) was employed in the synthesis of D-psicose via one-pot four-enzyme reaction with L-glycerol-3-phosphate as the starting material and D-glyceraldehyde as the acceptor. Unfortunately, after silica gel and gel filtration purification, only a12%yield of D-psicose could be obtained. It was reported that enzymes from thermophilic bacteria showed great potentials for biotechnological applications. To improve the overall yield for this synthesis, we turned our attention towards aldolase FucA from thermophilic source. Upon investigation, we noticed that the crystal structure of FucA from Thermus thermophilus HB8(FucAT.HN8) was recently reported, but the enzyme had yet to be used for synthetic purposes. Consequently, we expressed and purified FucAT.HN8in E.coli BL21(DE3) and then employed it in the one-pot reaction under the same conditions. To our delight, the yield was greatly improved compared with the E.coli counterpart. Our results demonstrated that FucAT.HN8showed a relaxed stereoselectivity toward D-glyceraldehdye with D-psicose as the dominated product and D-sorbose as the minor product. The product ratio of D-psicose/D-sorbose was～5:1as determined by HPLC. To further reduce the cost, L-glycerol-3-phosphate could be replaced by DL-glycerol-3-phosphate. We also discovered that when L-glyceraldehyde was used instead of D-glyceraldehyde in the one-pot reaction, FucAT.HN8lost its stereoselectivity and two rare sugars L-fructose and L-tagatose were generated simultaneously. The ratio of L-fructose/L-tagatose was1.2:1as determined by HPLC. The reaction mixture was purified by silica gel column chromatography and Bio-gel P-2column to get the mixture of L-fructose and L-tagatose. The total yield for these two sugars is47%. L-fructose and L-tagatose can be further separated by Ca2+exchange resin to get each pure sugar.In Chapter2(Section3), various ketoses were synthesized with glyceraldehydes and aliphatic aldehydes (acetaldehyde, propionaldehyde, butyraldehyde, and pentanaldehyde) as acceptors using D-fructose-1,6-bisphosphate aldolases (FruA) from rabbit muscle and Staphylococcus carnosus. The one-pot four-enzyme system was employed to prepare these carbohydrate molecules. FruAS.car demonstrated similar activity as RAMA under our reaction conditions. Unlike RhaD and FucA, which generate a mixture of diastereomers in our previous study and the stereoselectivities of which were influenced by the configuration of acceptors, FruAS.car and RAMA demonstrated excellent stereoselectivity:only single product was isolated for either reaction and the products were verified by1H NMR. The configuration of vicinal diols produced from the aldol addition with aliphatic aldehydes follows that of the glyceraldehydes. Considering the broad applications of FruA in synthetic chemistry, this approach could ultimately contribute to the synthesis of other carbohydrates and their derivatives with biological functions.In Chapter2(Section4), D-tagatose-1,6-bisphosphate aldolases (TagA) from three different bacterial sources were overexpressed and purified from E.coli BL21(DE3). Unfortunately, the activities of these three enzymes were low detected by TLC. Therefore, TagA was not investigated in detail for its application in rare sugar synthesis. In Chapter3, we established a synthetic platform with the potential for the large-scale production of uncommon sugars and their derivatives in E.coli. Recently, production of useful chemicals from cheap and renewable resources via microbial fermentation has become more and more popular. Compared with traditional chemical synthesis, microbial processes are mild, cost-efficient, more environmentally friendly and promising. Our strategy was as follows:firstly a recombinant plasmid was constructed to co-express DHAP-dependent aldolase and phosphatase in E.coli. The donor substrate DHAP was generated in this recombinant E.coli strain via glycolytic pathway with glucose as the green carbon source (or via dissimilation pathway of glycerol with glycerol as the carbon source). The acceptor substrate aldehyde, which could be transported into the E.coli, was provided by adding into the medium continuously when necessary. Then, the intracellular DHAP was condensed with aldehyde catalyzed by overexpressed aldolase to give the aldol product, which was further dephosphorylated by appropriate phosphatase to furnish the final products. The sugar molecule without a highly polar phosphate group can escape from the cell membrane into the medium, which makes the procedure for product harvest and purification very easy. Significantly, the phosphate could be regenerated in the cell. In this chapter, we successfully introduced aldol reaction into the E.coli cells and established a synthetic platform with the potential for the large-scale production of rare sugars and their derivatives (D-psicose or D-sorbose for instance). Our products contained two parts:C1,2,3of the product were from the donor DHAP and the other carbons were from the acceptor aldehyde. Theoretically, if E.coli was fed with [U-13C6] labeled glucose as the sole carbon source,13C3labeled DHAP would be generated within the cell. As a result, C1,2,3of the product would be labeled derived from DHAP. To prove that these three carbons of the product are ultimately from glucose,[U-13C6] labeled glucose was used as the sole carbon source for the isotope experiment. In this experiment, the E.coli strain with co-expression of aldolase FruA and phosphatase YqaB was used as the target strain and propionaldehyde was chosen as the acceptor. After fermentation and purification, the product was detected with13C NMR. The result demonstrated that C1,2,3of the product were labeled indeed with13C and these three carbons of the product were ultimately from glucoseOur future plan mainly includes two aspects:the use of system biology and synthetic biology to improve the yields of rare monosaccharides and their derivatives; the synthesis of other bioactive carbohydrate derivatives with our "E.coli machinery", glycosidase inhibitors iminocyclitol for instance. We believe that this research will provide a good technology platform for food and pharmaceutical industry.