Dirk Franke

Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions

Expanding the scope of substrate specificity and stereoselectivity is of current interest in enzyme catalysis. Using error-prone PCR for in vitro directed evolution, the Neu5Ac aldolase from Escherichia coli has been altered to improve its catalytic activity toward enantiomeric substrates including N-acetyl-L-mannosamine and L-arabinose to produce L-sialic acid and L-KDO, the mirror-image sugars of the corresponding naturally occurring D-sugars. The first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the (alpha,beta)(8)-barrel active site exhibits an inversion of enantioselectivity toward KDO and the second generation variant contains an additional amino acid change Val251Ile outside the alpha,beta-barrel active site that improves the enantiomeric formation of L-sialic acid and L-KDO. The X-ray structure of the triple mutant epNanA.2.5 at 2.3A resolution showed no significant difference between the wild-type and the mutant enzymes. We probed the potential structural hot spot of enantioselectivity with saturation mutagenesis at Val251, the mutated residue most proximal to the Schiff base forming Lys165. The selected variant had an increase in k(cat) via replacement with another hydrophobic residue, leucine. Further sampling of a larger sequence space with error-prone PCR selected a third generation variant with significant improvement in L-KDO catalysis and a complete reversal of enantioselectivity.

Diversity‐Oriented Production of Metabolites Derived from Chorismate and Their Use in Organic Synthesis

According to Corey s retrosynthetic approach, the chemical synthesis of individual target compounds requires the use of specific starting synthons in most cases. In contrast, the matrix approach inspired by nature s biosynthetic machinery is based on a diversity-oriented strategy. Indeed, the in vivo synthesis of many natural metabolites is subject to a complex matrix of dependencies and regulations. Thus, natural metabolites may generally be biosynthesized by alternative pathways, starting either from different or from the same compounds. The biosynthesis of a metabolite may require one specific enzymatic transformation or a biotransformation step that is catalyzed by several enzymes acting together in a cascade of reactions. Moreover, the activity of some enzymes may differ from substrate to substrate, some enzymes may simultaneously be involved in several different pathways, and other enzymes may be diversified in posttranslational modification steps. Furthermore, the status of the matrix is generally controlled on the DNA level by regulating the enzyme expression, and on the metabolite level by activating or inhibiting enzyme activities. The shikimate pathway is one prominent example of a matrix-based biosynthesis, which is essential in plants, bacteria, and fungi, and has been described as a branched metabolic tree for the synthesis of a wide range of (mostly aromatic) compounds. Several enzymes that modify chorismate exhibit structural and mechanistical similarities. The biosynthesis of chorismate and its precursors is subject to complex regulations. 6] Some important enzymatic transformations that start from chorismate (1) are depicted in Scheme 1. The proteinogenic aromatic amino acids, folates, ubiquinones, menaquinones, enterobactin, and many secondary metabolites are biosynthesized in a few steps starting from chorismate. 8]

Directed evolution of aldolases

Publisher Summary This chapter describes the directed evolution of aldolases. The detailed protocols for generating aldolases with new catalytic properties are presented. The gene encoding aldolase can be amplified using the standard polymerase chain reaction (PCR) from a genomic DNA preparation using the Qiagen kit and primers flanking the gene with engineered restriction sites. Purify the resulting fragment on an agarose gel, digest with appropriate restriction enzymes, and ligate into the expression vector. The ligation product can then be transformed into the appropriate E. coli cloning strain by electroporation. Gene disruption of a chromosomally encoded wild-type enzyme should be performed without prior cloning via recombination with linear DNA in recBCD -deficient E. coli strains. Substrates for the retroaldol reaction can be synthesized enzymatically from aldehyde and keto compounds using wild-type aldolase or advantageously evolved mutants. It is found that in these caged substrates, fluorogenic umbelliferone is released through β elimination from the primary or secondary carbonyl retro-aldol reaction product.

Synthesis of Functionalized Cyclohexadiene‐trans‐Diols with Recombinant Cells of Escherichia coli

As valuable chiral building blocks for the syntheses of natural products and pharmacologically active substances, especially of carbohydrate mimetics, functionalized cyclohexadiene-trans-diols such as (2S,3S)-dihydroxy-2,3-dihydrobenzoic acid (1) can be prepared easily in 17u2009% yield starting from glucose by using metabolically deregulated, recombinant microorganisms such as the Escherichia coli strain AN193.

Regioselective oxidation of terfenadine with Cunninghamella blakesleeana

Abstract The regioselective oxidation of terfenadine with the fungi Cunninghamella blakesleeana was studied as a biochemical alternative for the chemical synthesis of the antihistaminic drug fexofenadine. It was demonstrated that C. blakesleeana oxidises the tert-butyl group of terfenadine to the corresponding alcohol 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenyl]-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-butanol. A continuous process for regioselective oxidation of terfenadine was developed. Terfenadine was supplied micro-crystalline due to the low solubility in water. Optimum reaction conditions with respect to medium composition, temperature, pH, pO2, co-substrate and feeding rates were found by means of reaction engineering studies. A cross-flow microfiltration unit was operated in a by-pass of a lab-scale stirred tank reactor for retention of the biocatalysts and the micro-crystalline substrate. The alcohol was continuously removed with the filtrate to minimise product inhibition. Continuous biotransformation of micro-crystalline terfenadine with C. blakesleeana in the membrane reactor system with a dilution rate of 33 h at co-substrate concentrations of about 1 up to 3 g/l glycerol in the reactor resulted in a space–time yield of 145 mg of alcohol/l/day and an alcohol yield of 71%. The produced alcohol was easily isolated from the filtrate by adsorption on XAD-4 resin followed by eluation with methanol (concentration factor 7).

Easy Access to (R,R)-3,4-Dihydroxy-3,4-dihydrobenzoic Acid with Engineered Strains of Escherichia coli

We recently described a preparative route to (S,S)-2,3-dihydroxy2,3-dihydrobenzoic acid (2,3-trans-CHD [CHD: cyclohexadienediol] , 1), that makes use of genetically engineered cells of Escherichia coli, based on work previously described by Leistner et al. The potential of 2,3-trans-CHD (1) as a starting material in natural product chemistry has been demonstrated by the syntheses of iso-crotepoxide and ent-senepoxide, and the diversity-oriented synthesis of new (amino)carbasugar building blocks. (R,R)-trans-3,4-Dihydroxy-3,4-dihydrobenzoic acid (3,4trans-CHD, 2), the regioisomer of 1, promises a variety of synthetic applications similar to those of 1 or to the corresponding cis-CHD. In contrast to 1, which is the metabolic precursor in the biosynthesis of the iron chelator enterobactin, compound 2 has not been found to have a biological function in E. coli. Nevertheless, in the presence of isochorismatase (encoded by entB), whose physiological function is the hydrolysis of isochorismate (3) to form 1, 3,4-trans-CHD (2) can be produced from chorismate (4) in vitro as well as in vivo with strains of Klebsiella pneumoniae (Scheme 1). We herein report an efficient approach for the production of 3,4-trans-CHD (2) by redirecting the post-chorismate pathways of aromatic amino acid biosynthesis in E. coli. Different approaches for the generation of microbial producers of 2 were examined. Our first approach was analogous to the strategy we used for the production of 1. Only the pathway for the Scheme 1. Substrates and products of E. coli isochorismatase (EntB)-catalyzed transformations.

Cyclohexadiene-trans-diols as versatile starting material in natural product synthesis: short and efficient synthesis of iso-crotepoxide and ent-senepoxide

A new synthesis of ent-senepoxide and iso-crotepoxide starting from microbially produced(+)-trans-2,3-dihydroxy-2,3-dihydrobenzoic acid via regio- and stereoselective epoxidation is described.