Synthesis Of Angiotensin-converting Enzyme Inhibitors Research Articles

Integrative Biosynthetic Gene Cluster Mining to Optimize a Metabolic Pathway to Efficiently Produce l-Homophenylalanine in Escherichia coli.

Mining biosynthetic genes for the exploration of hybrid metabolic pathways is a promising approach in heterologous production of natural and unnatural products. Here, we developed an integrative biosynthetic gene cluster (BGC) mining strategy to engineer the biosynthesis of l-homophenylalanine (l-Hph), an important intermediate for the synthesis of angiotensin-converting enzyme inhibitors. We assembled the putative l-Hph BGCs and integrated phylogenetic analysis with target metabolite abundance mapping to prioritize candidate BGCs. To obtain an effective l-Hph pathway, various combinations of candidate genes from different species were screened in an iterative design-build-test stepwise manner. After the pathway was strength balanced and the metabolic flux was enhanced, engineered Escherichia coli produced 1.41 g/L of l-Hph from glucose in feeding shake-flask fermentation. Our cluster mining strategy enabled optimization of the target metabolic pathway, and it would be promising for production of other valuable products in the postgenomic era.

Redesign of a short-chain dehydrogenase/reductase for asymmetric synthesis of ethyl (R)-2-hydroxy-4-phenylbutanoate based on per-residue free energy decomposition and sequence conservatism analysis

As an important building block for the synthesis of angiotensin-converting enzyme inhibitors, ethyl (R)-2-hydroxyl-4-phenylbutanoate [(R)-HPBE] has attracted increasing attention. The key to industrial biosynthesis of (R)-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate (OPBE) with high R-enantioselectivity. This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis. Using this strategy, 4 nonconservative sites with high energy contribution to binding of OPBE were chosen as engineering targets, generating variant Mu27 with 99% conversion and 98% (R) ee value at substrate loading of up to 500 ​mmol/L. MD simulations suggested the higher stability and formation probability of Mu27-OPBEproR prereaction state as key reasons for the excellent R-enantioselectivity of Mu27 towards OPBE. The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.

Efficient production of (R)-(-)-2-hydroxy-4-phenylbutyric acid by recombinant Pichia pastoris expressing engineered D-lactate dehydrogenase from Lactobacillus plantarum with a single-site mutation.

(R)-2-hydroxy-4-phenylbutyric acid (R-HPBA) is a valuable intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The asymmetric reduction of 2-oxo-4-phenylbutyric acid (OPBA) by oxidoreductases is an efficient approach for its synthesis. Here, we report a novel biocatalytic approach for asymmetric synthesis of R-HPBA using recombinant Pichia pastoris expressing the Tyr52Leu variant of D-lactate dehydrogenase (D-LDH) from Lactobacillus plantarum. The recombinant yeast cells showed impressive catalytic activity at a high concentration of NaOPBA (380mM, 76g/L) and achieved full conversion starting with 40g/L NaOPBA or even at higher concentration. Under optimized reaction conditions (pH 7.5, 37°C, and 2% glucose), a full conversion with > 95% reaction yield and ~ 100% product enantiomeric excess (ee) was achieved for the preparation of R-HPBA on a 2-g scale. The findings of this study promote both the biotransformation of R-HPBA and an extension of the application of recombinant yeast as biocatalysts.

Enhancement of biocatalytic efficiency by increasing substrate loading: enzymatic preparation of l-homophenylalanine

Enantiomerically pure L-homophenylalanine (L-HPA) is a key building block for the synthesis of angiotensin-converting enzyme inhibitors and other chiral pharmaceuticals. Among the processes developed for the L-HPA production, biocatalytic synthesis employing phenylalanine dehydrogenase has been proven as the most promising route. However, similar to other dehydrogenase-catalyzed reactions, the viability of this process is markedly affected by insufficient substrate loading and high costs of the indispensable cofactors. In the present work, a highly efficient and economic biocatalytic process for L-HPA was established by coupling genetically modified phenylalanine dehydrogenase and formate dehydrogenase. Combination of fed-batch substrate addition and a continuous product removal greatly increased substrate loading and cofactor utilization. After systemic optimization, 40g (0.22mol) of keto acid substrate was transformed to L-HPA within 24h and a total of 0.2mM NAD(+) was reused effectively in eight cycles of fed-batch operation, consequently giving an average substrate concentration of 510mM and a productivity of 84.1gl(-1)day(-1) for L-HPA. The present study provides an efficient and feasible enzymatic process for the production of L-HPA and a general solution for the increase of substrate loading.

Stereoselective esterase from Pseudomonas putida IFO12996 reveals alpha/beta hydrolase folds for D-beta-acetylthioisobutyric acid synthesis.

Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-beta-acetylthioisobutyrate (dl-MATI) to produce d-beta-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67 degrees C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 A, confirming that EST is a member of the alpha/beta hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket approximately 12 A deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.

Enantioselective Synthesis ofN-[(S)-Ethoxycarbonyl-3-phenylpropyl]-L-alanyl-L-proline from Chiral Synthon Prepared Enzymatically; A Practical Method for Large-Scale Synthesis

Efficient procedures have been developed for the stereospecific synthesis of Angiotensin-converting enzyme inhibitor, Enalapril. The starting material, (R)-2-hydroxy-4-phenylbutyronitrile, is prepared from lipase-catalyzed acetylation. This process is useful for multigram-scale synthesis.

Synthesis of Angiotensin-Converting Enzyme Inhibitors and its Application to Experimental Hypertensioe Rat for Understanding the Pathogenesis of Renovascular Hypertension

Synthesis of Angiotensin-Converting Enzyme Inhibitors and its Application to Experimental Hypertensioe Rat for Understanding the Pathogenesis of Renovascular Hypertension