Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate

Abstract

The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a non-functionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the KM value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of kcat/KM up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced “uniform binding” of the substrates, which is the first step in the evolution of novel catalytic activity.

A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols

“…Protein directed evolution, on the other hand, offers the potential to improve the function of a specific enzyme (Cadwell and Joyce, 1992;Leung et al, 1989;Stemmer, 1994). Various techniques for directed evolution have been shown to be highly successful to improve or modify the function of the targeted enzyme (Cheriyan et al, 2011;Meyer et al, 2002;Zhao and Arnold, 1999) and by extension to improve the production of a desired product in a pathway (Atsumi and Liao, 2008;Christ et al, 2010;Wang and Liao, 2001). However, a major challenge in either bioprospecting or directed evolution is the effectiveness of the screening strategy, which determines the throughput of the effort.…”

Improving upon Nature: Active Site Remodeling Produces Highly Efficient Aldolase Activity toward Hydrophobic Electrophilic Substrates

“…Previous data suggested that this selection method mainly identifies enhancements in substrate binding due, presumably, to the low in vivo concentration of the substrates. ( 15 ) To circumvent this issue, KDPG aldolase was fused to the signal sequence from the filamentous phage fd pIII gene which localizes the protein to the periplasm( 31 ) where the substrate concentration reflects the concentration in the medium. Using this method E. coli KDPG aldolase variants were selected for improved catalysis of two hydrophobic analogs of KDPG: (1) 2-keto-4-hydroxy-octonoate (KHO), a hydrophobic analog that lacks the C5 hydroxyl and C6 phosphate of KDPG; and (2) (4 S )-2-keto-4-hydroxy-4-(2′-pyridyl)butyrate ( S -KHPB), an analog containing a pyridyl group that is an important precursor for nikkomycin synthesis (Figure 3).…”

“…( 15 ) Therefore random mutagenesis coupled to in vivo selection identified mutations that lowered the value of K M , but did not improve k cat . Albery and Knowles( 38 ) suggest that the first mutations in the evolution of a new activity often result in “uniform binding” where a mutation has a similar stabilizing effect on both the ground and transition states of the rate-limiting step, leaving the value of k cat unchanged.…”

“…While whole gene directed evolution experiments are extremely useful for improving thermal and chemical stability of enzymes,( 39 ) employing a similar strategy to alter the specificity of aldolase/transaldolase genes has generally led to only modest improvements (6 to 80-fold) in substrate selectivity and reactivity. ( 13, 15, 16, 40-43 ) Additionally, the optimized mutant enzymes often possess improved k cat / K M and K M values but lower catalytic turnover numbers. In contrast, efforts combining structural information and site-specific saturation mutagenesis have led to large alterations in the enzymatic properties of various aldolase/transaldolases with minimal mutagenesis.…”

Engineering aldolases as biocatalysts

“…Recent engineering has used both directed evolution [21] and structure-based mutagenesis [20 •• ] to expand its substrate range to non-functionalized electrophilic substrates and pyridine carboxaldehyde substrates, respectively. Furthermore, the activity of the variant KDPGA with the pyridine carboxaldehyde substrate (4 S )-2-keto-4-hydroxy-4-(2′-pyridyl) butyrate ( S -KHPB) maintains high stereoselectivity at a similar rate to that of the wild-type enzyme with KDPG.…”