Jon D. Stewart

Dehydrogenases and transaminases in asymmetric synthesis

Improved stereoselectivity in dehydrogenase-mediated reductions has been achieved by rationally designed gene overexpression and knockouts in Saccharomyces cerevisiae cells and by isolating and characterizing novel dehydrogenases from other organisms. Transaminases have been used to prepare unnatural amines and amino acids in good yields, particularly when the equilibria are shifted by selective product removal.

Understanding and Improving NADPH-Dependent Reactions by Nongrowing Escherichia coli Cells

We have shown that whole Escherichia coli cells overexpressing NADPH‐dependent cyclohexanone monooxygenase carry out a model Baeyer‐Villiger oxidation with high volumetric productivity (0.79 g ϵ‐caprolactone/L·h ) under nongrowing conditions (Walton, A. Z.; Stewart, J. D. Biotechnol. Prog. 2002, 18 , 262–268). This is approximately 20‐fold higher than the space‐time yield for reactions that used growing cells of the same strain. Here, we show that the intracellular stability of cyclohexanone monooxygenase and the rate of substrate transport across the cell membrane were the key limitations on the overall reaction duration and rate, respectively. Directly measuring the levels of intracellular nicotinamide cofactors under bioprocess conditions suggested that E. coli cells could support even more efficient NADPH‐dependent bioconversions if a more suitable enzyme‐substrate pair were identified. This was demonstrated by reducing ethyl acetoacetate with whole cells of an E. coli strain that overexpressed an NADPH‐dependent, short‐chain dehydrogenase from bakerapos;s yeast ( Saccharomyces cerevisiae). Under glucose‐fed, nongrowing conditions, this reduction proceeded with a space‐time yield of 2.0 g/L·h and a final product titer of 15.8 g/L using a biocatalyst:substrate ratio (g/g) of only 0.37. These values are significantly higher than those obtained previously. Moreover, the stoichiometry linking ketone reduction and glucose consumption (2.3 ± 0.1) suggested that the citric acid cycle supplied the bulk of the intracellular NADPH under our process conditions. This information can be used to improve the efficiency of glucose utilization even further by metabolic engineering strategies that increase carbon flux through the pentose phosphate pathway.

Site-Saturation Mutagenesis of Tryptophan 116 of Saccharomyces pastorianus Old Yellow Enzyme Uncovers Stereocomplementary Variants

Site-saturation mutagenesis was used to generate all possible replacements for Trp 116 of Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis ) old yellow enzyme (OYE). Our original hypothesisthat smaller amino acids at position 116 would allow better acceptance of bulky 3-alkyl-substituted 2-cyclohexenonesproved incorrect. Instead, Phe and Ile replacements favored the binding of some substrates in an opposite orientation, which yielded reversed stereochemical outcomes compared to that of the wild-type OYE. For example, W116I OYE reduced (R)- and (S)-carvone to enantiomeric products, rather than the diastereomers produced by the wild-type OYE. Deuterium labeling revealed that (S)-carvone reduction by the W116I OYE occurred by the same pathway as that by the wild type (net trans-addition of H(2)), proving that different substrate binding orientations were responsible for the divergent products. Trp 116 mutants also afforded different stereochemical outcomes for reductions of (R)-perillaldehyde and neral. Preliminary studies of an OYE family member whose native sequence contains Ile at position 116 ( Pichia stipitis OYE 2.6) revealed that this enzymes stereoselectivity matched that of the wild-type S. pastorianus OYE, showing that the identity of the residue at position 116 does not solely determine the substrate binding orientation. Computational docking studies using an induced fit methodology successfully reproduced the majority of the experimental outcomes. These computational tools will allow preliminary in silico screening of additional residues to identify those most likely to control the substrate binding orientation and provide some guidance to future experimental studies.

Organic transformations catalyzed by engineered yeast cells and related systems.

Asymmetric ketone reductions remain the most popular application of bakers yeast (Saccharomyces cerevisiae) in organic synthesis and data from the genome sequencing project is beginning to have an impact on improving the stereoselectivities of these reactions, augmenting traditional approaches based on selective inhibition. In addition, the catalytic repertoire of yeast has been expanded to include chiral ketone oxidations by overexpression of a bacterial Baeyer-Villiger monooxygenase.

New insight into the catalytic mechanism of chorismate mutases from structural studies

Chorismate mutase catalyzes the rearrangement of chorismic acid to prephenic acid, which is the first committed step in the biosynthesis of aromatic amino acids. Its catalytic mechanism has been much studied, but is poorly understood. Recent structural information on enzymes from two species, and on an antibody that catalyzes the same reaction, has shed new light on this topic.

An efficient enzymatic Baeyer-Villiger oxidation by engineered Escherichia coli cells under non-growing conditions

Economical methods of supplying NADPH must be developed before biotransformations involving this cofactor can be considered for large‐scale applications. We have studied the enzymatic Baeyer–Villiger oxidation of cyclohexanone as a model for this class of reactions and developed a simple approach that uses whole, non‐growing Escherichia coli cells to provide high productivity (0.79 g ϵ‐caprolactone/L/h ≡ 18 μmol ϵ‐caprolactone/min/g dcw) and an 88% yield. Glucose supplied the reducing equivalents for this process, and no exogenous cofactor was required. The volumetric productivity of non‐growing cells was an order of magnitude greater than that achieved with growing cells of the same strain. Cells of an engineered E. coli strain that overexpresses Acinetobacter sp. cyclohexanone monooxygenase were grown under inducing conditions in rich medium until the entry to stationary phase; the subsequent cyclohexanone oxidation was carried out in minimal salts medium lacking a nitrogen source. After the biotransformation was complete, the lactone product was adsorbed to a solid support and recovered by washing with an organic solvent.

ASYMMETRIC OXIDATIONS AT SULFUR CATALYZED BY ENGINEERED STRAINS THAT OVEREXPRESS CYCLOHEXANONE MONOOXYGENASE

Recombinant strains of bakers yeast (Saccharomycescerevisiae) and Escherichiacoli expressing cyclohexanone monooxygenase from Acinetobacter sp. NCIB 9871 have been used as whole-cell biocatalysts for oxidations of several sulfides, dithianes and dithiolanes to the corresponding sulfoxides. The enantio- and diastereoselectivities of these reactions compare favorably with oxidations catalyzed by the purified monooxygenase or the parent microorganism (a class II pathogen). The facility of handling yeast reactions makes these biotransformations an attractive alternative route to optically pure sulfoxides.

Enantioselective reductions of ethyl 2-oxo-4-phenylbutyrate by Saccharomyces cerevisiae dehydrogenases

Abstract A set of fusion proteins consisting of glutathione S -transferase linked to the N-terminus of putative dehydrogenases produced by baker’s yeast ( Saccharomyces cerevisiae ) was screened for the reduction of ethyl 2-oxo-4-phenylbutyrate in the presence of NADH and NADPH. Two dehydrogenases—Ypr1p and Gre2p—rapidly reduced this α-ketoester, providing the ( R )- and ( S )-alcohol, respectively, with high stereoselectivities. The same enzymes were over-expressed in their native forms in Escherichia coli and growing cells of the engineered strains could also be used to carry out the reductions without the need for exogenous cofactor. These results demonstrate the power of genomic fusion protein libraries to identify appropriate biocatalysts rapidly and expedite process development.

‘Designer yeast’: a new reagent for enantioselective Baeyer–Villiger oxidations

The catalytic repertoire of bakers yeast has been expanded to include enantioselective Baeyer–Villiger oxidations. To create this catalyst, the Acinetobacter sp. cyclohexanone monooxygenase gene was inserted into a yeast expression vector and this was used to create a ‘designer yeast’ that performed oxidation reactions. Whole cell-mediated Baeyer–Villiger reactions were carried out on a 1.0 mmol scale and several cyclic ketones were converted in 20–30 h into the corresponding lactones in isolated yields of 60–83%. Under the reaction conditions, ketone reduction constituted only a minor side-reaction. Oxidation of prochiral 4-substituted cyclohexanones produced lactones with very high enantioselectivities.

A shrunken-2 Transgene Increases Maize Yield by Acting in Maternal Tissues to Increase the Frequency of Seed Development

This work examines the function of a maize heat-stable, less inhibitor–sensitive form of ADP-glucose pyrophosphorylase, which increases maize yield by increasing seed number. This work shows that this increase requires high temperature during early seed development and results from transgene function in maternal tissues to increase the probability that an ovary will produce a seed. The maize (Zea mays) shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme, ADP-glucose pyrophosphorylase. Expression of a transgenic form of the enzyme with enhanced heat stability and reduced phosphate inhibition increased maize yield up to 64%. The extent of the yield increase is dependent on temperatures during the first 4 d post pollination, and yield is increased if average daily high temperatures exceed 33°C. As found in wheat (Triticum aestivum) and rice (Oryza sativa), this transgene increases maize yield by increasing seed number. This result was surprising, since an entire series of historic observations at the whole-plant, enzyme, gene, and physiological levels pointed to Sh2 playing an important role only in the endosperm. Here, we present several lines of evidence that lead to the conclusion that the Sh2 transgene functions in maternal tissue to increase seed number and, in turn, yield. Furthermore, the transgene does not increase ovary number; rather, it increases the probability that a seed will develop. Surprisingly, the number of fully developed seeds is only ∼50% of the number of ovaries in wild-type maize. This suggests that increasing the frequency of seed development is a feasible agricultural target, especially under conditions of elevated temperatures.