Abstract
Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.
Highlights
Carbon– carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry
We identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs)
We demonstrated that E. coli cells expressing the DERA-AKR pathway with engineered DERA variants produced 5– 6 times more 1,3BDO from glucose compared with cells with the WT BH1352
Summary
To provide insight into the phylogenetic diversity of DERAs, 2,553 sequences of putative DERAs were extracted from the Kyoto Encyclopedia of Genes and Genomes Orthology database using the identifier K01619 for the E. coli DERA (DeoC), which is the best-characterized DERA enzyme [23]. Because B. halodurans is an alkaliphilic bacterium (grows well at pH Ͼ9.0), we determined the optimal pH range for BH1352 using the retro-aldol DRP cleavage reaction coupled with glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase [16, 24, 43, 44] These assays revealed a broad pH range with the maximal activity of BH1352 at pH 7.2–9.5 (Fig. S3), whereas the previously reported DERA enzymes from other bacteria showed the highest activity at pH 6.0 –7.5 [24, 28]. Steady-state kinetic parameters of BH1352 and its variants were determined using the DRP cleavage reaction (Table 1) These experiments revealed that BH1352 exhibits typical Michaelis–Menten kinetics with the apparent Km ϭ 0.22 mM, which is close to that for E. coli DeoC and more than 10 times lower than that for the L. brevis DERA (3.3 mM) [24]. Crystallographic data collection and model refinement statistics for the crystal structures of BH1352 (wildtype and K184L) and E. coli DeoC (EcDERA)
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