Combining Aldolases and Transaminases for the Synthesis of 2-Amino-4-hydroxybutanoic Acid

Abstract

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)-and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a systems biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an S-or R-selective, pyridoxal phosphate dependent transaminase was used as a catalyst to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the class II pyruvate aldolase was found to tolerate formaldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)-or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L −1 d −1 , and ee values of >99%.

Nucleophile Promiscuity of Natural and Engineered Aldolases

“…4‐Hydroxy‐2‐keto acid derivatives are important building blocks for the synthesis of amino acids, hydroxycarboxylic acids, and aldehydes . Chemical or enzymatic decarboxylation leads to aldehydes and carboxylic acids, which are also valuable synthetic intermediates.…”

“…A class II pyruvate aldolase from E. coli , YfaU (EC 4.1.2.53) has recently been exploited for synthetic purposes . In the aldol addition of 6 a to formaldehyde, YfaU fused with maltose‐binding protein from E. coli (MBP‐YfaU), retains its activity at unexpectedly high formaldehyde concentrations (>1 m ), which is remarkable for such a strongly inactivating compound that often causes enzyme denaturation, even at very low concentrations. MBP‐YfaU showed broad electrophile selectivity for aldol additions of 6 a and 2‐oxobutyrate .…”

Whole‐Cell Cascade Biotransformations for One‐Pot Multistep Organic Synthesis

“…Biotransformation of l ‐Phe (40–140 m m ) with the corresponding resting E. coli cells (20 g L −1 ) provided these five chiral compounds in 62–78 % isolated yield and 96–99 % ee . Recently, Clapés et al combined an aldolase and a transaminase in vitro to establish a 3‐step recycling cascade to convert l ‐alanine and formaldehyde to enantiopure ( S )‐2‐amino‐4‐hydroxybutanoic acid in high concentration (400 m m ) . The same cascade was also reported to produce l ‐4‐hydroxy glutamic acid from l ‐aspartate and glyoxylic acid, as well as l ‐4,5‐dihydroxynorvaline from l ‐alanine and glycolaldehyde …”

Donor‐Promiskuität einer thermostabilen Transketolase durch gelenkte Evolution – effektive Komplementierung der 1‐Desoxy‐d‐ xylulose‐5‐phosphat‐Synthase‐Aktivität

“…Erstaunlicherweise zeigte die geplante H102F-Variante nur eine leicht verbesserte Aktivitätm it 2a als Donor (Tabelle 1). Abschließend wurden die Top-Kandidaten aus beiden vorangegangenen Te strunden auch gegenüber 2-Oxobutanoat (2b)u nd 4-Hydroxy-2-oxobutanoat (2e) [20] (jeweils 10 mm)a ls Donorsubstrate mittlerer Grçße kinetisch analysiert (Abbildung 3, Tabelle S5 der SI). [18] Deshalb wählten wir in einem semi-rationalen Ansatz alle diejenigen Reste,d ie im direkten Kontakt zur CH 2 OH-Einheit von 1 stehen (His68, His102, Gly116 und H474), als Ziele Abbildung 1.…”

“…Die Graphik wurde mit PyMOL erstellt. [ [20] (jeweils 10 mm)a ls Donorsubstrate mittlerer Grçße kinetisch analysiert (Abbildung 3, Tabelle S5 der SI). Der beste für 2b gefundene Kandidat (hçchster k cat -Wert) war die Doppelvariante H102L/H474S, die 2b und 2a mit vergleichbarer Katalyserate bei signifikant erhçhter Substrataffinitätu msetzen konnte.B emerkenswerterweise wurde das polare Substratanalogon 2e nicht akzeptiert.…”