Highly regio- and enantioselective multiple oxy- and amino-functionalizations of alkenes by modular cascade biocatalysis

Abstract

New types of asymmetric functionalizations of alkenes are highly desirable for chemical synthesis. Here, we develop three novel types of regio- and enantioselective multiple oxy- and amino-functionalizations of terminal alkenes via cascade biocatalysis to produce chiral α-hydroxy acids, 1,2-amino alcohols and α-amino acids, respectively. Basic enzyme modules 1-4 are developed to convert alkenes to (S)-1,2-diols, (S)-1,2-diols to (S)-α-hydroxyacids, (S)-1,2-diols to (S)-aminoalcohols and (S)-α-hydroxyacids to (S)-α-aminoacids, respectively. Engineering of enzyme modules 1 &2, 1 &3 and 1, 2 &4 in Escherichia coli affords three biocatalysts over-expressing 4-8 enzymes for one-pot conversion of styrenes to the corresponding (S)-α-hydroxyacids, (S)-aminoalcohols and (S)-α-aminoacids in high e.e. and high yields, respectively. The new types of asymmetric alkene functionalizations provide green, safe and useful alternatives to the chemical syntheses of these compounds. The modular approach for engineering multi-step cascade biocatalysis is useful for developing other new types of one-pot biotransformations for chemical synthesis.

Figure 1. Regio- and enantioselective multiple oxy- and amino-functionalizations of terminal alkenes by modular cascade biocatalysis.

(a) One-pot conversion of terminal alkene to chiral α-hydroxy acid, 1,2-amino alcohol and α-amino acid with E. coli cells containing multiple basic enzyme modules, respectively. (b) Four general basic enzyme modules and their cascade biotransformations. Module 1: epoxidase (EP) and epoxide hydrolase (EH) for epoxidation–hydrolysis of terminal alkene to 1,2-diol; Module 2: alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) for terminal double oxidation of 1,2-diol to α-hydroxy acid; Module 3: ADH, ω-transaminase (ω-TA) and alanine dehydrogenase (AlaDH) for oxidation–transamination of 1,2-diol to 1,2-amino alcohol; Module 4: hydroxy acid oxidase (HO), α-transaminase (α-TA), catalase (CAT) and glutamate dehydrogenase (GluDH) for oxidation–transamination of α-hydroxy acid to α-amino acid.

Figure 2. Regio- and enantioselective multiple oxy- and amino-functionalizations of styrenes by modular cascade biocatalysis.

(a) Conversion of styrenes to (S)-α-hydroxy acids with E. coli strains containing enzyme module 1 and 2. (b) Conversion of styrenes to (S)-1,2-amino alcohols with E. coli strains containing enzyme module 1 and 3. (c) Conversion of styrenes to (S)-α-amino acids with E. coli strains containing enzyme module 1, 2 and 4. SMO: styrene monooxygenase from Pseudomonas sp. VLB120; SpEH: epoxide hydrolase from Sphingomonas sp. HXN-200; AlkJ: alcohol dehydrogenase from P. putida GPo1; EcALDH: phenylacetaldehyde dehydrogenase from E. coli; CvωTA: ω-transaminase from C. violaceum; AlaDH: alanine dehydrogenase from B. subtilis; HMO: hydroxymandelate oxidase from S. coelicolor A3(2); EcαTA: branch chain amino acid transaminase from E. coli; GluDH: glutamate dehydrogenase from E. coli; and CAT: catalase from E. coli.

Figure 3. SDS–PAGE and biotransformation time course of E. coli strains containing individual enzyme modules.

(a) E. coli (R-M1) cells containing enzyme module 1 (SMO and SpEH); and biotransformation of styrene 1a to (S)-1-phenyl-1,2-ethanediol 3a. (b) E. coli (R-M2) cells containing enzyme module 2 (AlkJ and EcALDH); and biotransformation of (S)-1-phenyl-1,2-ethanediol 3a to (S)-mandelic acid 5a. (c) E. coli (R-M3) cells containing enzyme module 3 (AlkJ, CvωTA and AlaDH); and biotransformation of (S)-1-phenyl-1,2-ethanediol 3a to (S)-phenylethanolamine 6a. (d) E. coli (R-M4) cells containing enzyme module 4 (HMO, EcαTA, GluDH and CAT); and biotransformation of (S)-mandelic acid 5a to (S)-phenylglycine 8a (blue arrow: adding additional 0.5% glucose at 22 h). All biotransformations were performed in triplicate, and error bars show±s.d.

Figure 4. Regio- and enantioselective multiple oxy- and amino-functionalizations of styrene 1a with E. coli strains containing multiple enzyme modules.

(a) Product concentration of biotransformation of 100 mM 1a to (S)-5a with twelve E. coli strains (10 g cdw l⁻¹), each containing both enzyme module 1 and 2, respectively. (b) Time course of biotransformation of 120 mM 1a to (S)-5a with E. coli (A-M1_R-M2) cells (15 g cdw l⁻¹) in a two-liquid-phase system (KP buffer containing 0.25% glucose and n-hexadecane; 1:1) at 30 °C. (c) Product concentration of biotransformation of 50 mM 1a to (S)-6a with twelve E. coli strains (10 g cdw l⁻¹), each containing both enzyme module 1 and 3, respectively. (d) Time course of biotransformation of 60 mM 1a to (S)-6a with E. coli (A-M1_E-M3) cells (15 g cdw l⁻¹) in a two-liquid-phase system (NaP buffer containing 1% glucose and 200 mM NH₃/NH₄Cl and n-hexadecane; 1:1) at 25 °C. (e) Product concentration of biotransformation of 50 mM 1a to (S)-8a with eight E. coli strains (10 g cdw l⁻¹), each containing enzyme module 1, 2 and 4, respectively. (f) Time course of biotransformation of 60 mM 1a to (S)-8a with E. coli (A-M1_R-M2_C-M4) cells (15 g cdw l⁻¹) in a two-liquid-phase system (KP buffer containing 0.5% glucose and 100 mM NH₃/NH₄Cl and n-hexadecane; 1:1) at 30 °C (arrow: adding additional 0.5% glucose and 100 mM NH₃/NH₄Cl at 20 h). All biotransformations were performed in triplicate, and error bars show±s.d.