Cytochrome P450 ΒΜ-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio-and enantioselectively using atmospheric dioxygen as an oxidant. BM-3 variant 9-10A-A328V hydroxylates octane at the 2-position to form S-2-octanol (40% ee). Another variant, 1-12G, also hydroxylates alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active (rates up to 400 min -1 ) and support thousands of product turnovers. The regio-and enantioselectivities are retained in whole-cell biotransformations with Escherichia coli, where the engineered P450s can be expressed at high levels and the cofactor is supplied endogenously.

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Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k cat , the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.Directed evolution by random mutagenesis and recombination followed by screening or selection is a valuable tool for the engineering of enzymes (2,3,16,61). Functional gene expression in a suitable host is a prerequisite for directed evolution. Considering transformation efficiency, stability of plasmid DNA, and growth rate, Escherichia coli and Saccharomyces cerevisiae are best suited for these experiments. Heterologous expression in these hosts, however, is often limited by differences in the expression systems from the native organism (50). Different codon usage, missing chaperones, and posttranslational modifications such as disulfide bridges or glycosylation can all cause low expression levels and misfolded proteins that are degraded or driven into inclusion bodies (20). Finding the bottlenecks of a specific expression system requires consideration of many possibilities whose impact is hard to predict. Some incompatibilities between the expressed gene and heterologous host, such as codon usage or the recognition of signal sequences, can be overcome by changing the gene sequence. Thus, achieving functional expression is a good target for directed evolution (13,42,43).Laccases, like other ligninolytic enzymes, are notoriously difficult to express in nonfungal systems. The laccase from Myceliophthora thermophila (MtL) used in this work was previously expressed only in Aspergillus oryzae (6). Expression in S. cerevisiae has been reported for other laccase genes (11,33,34,60). Kojima et al. demonstrated expression of...

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a caseby-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.branched-chain amino acid pathway | cofactor imbalance K etol-acid reductoisomerases (KARI; EC 1.1.1.86) are a family of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidoreductases that catalyze an alkyl-migration followed by a ketol-acid reduction of (S)-2-acetolactate (S2AL) and 2-aceto-2-hydroxybutyrate to yield (R)-2,3-dihydroxy-isovalerate and (R)-2,3-dihydroxy-3-methylvalerate, respectively (1), which are essential intermediates in the biosynthesis of branched-chain amino acids (BCAAs) (2, 3). The demand for these essential amino acids, used in the preparation of animal feed, human dietary supplements, and pharmaceuticals, is currently estimated to exceed 1,500 tons per year (4). In addition, the BCAA pathway has been engineered to produce fine chemicals and biofuels, including 1-butanol and isobutanol (5, 6). Under the anaerobic conditions preferred for large-scale fermentations, biosynthesis of BCAAs and other products that use this pathway is limited by the pathway's cofactor imbalance and reduced cellular production of NADPH (7,8). One approach to overcoming the cofactor imbalance is to engineer KARI to use NADH generated in glycolysis, thereby enabling anaerobic production of BCAA pathway products (7,8).Efforts to switch the cofactor specificity of oxidoreductases from NADPH to NADH have been made with varying degrees of success (8-17). The three reports of cofactor-switched KARIs (7,8,15) from two different organisms show few commonalities in terms of residues targeted for engineering. A general recipe for switching KARI cofactor specificity would allow metabolic engineers to take advantage of the natural sequence diversity of the KARI family, with concomitant diversity in properties such as expression level, pH tolerance, or thermal stability. By combining a systematic analysis of all reviewed and manually annotated [SwissProt (18)] KARIs, information from our previous work on switching the cofactor specificity of the Escherichia coli KARI (7), and available KARI structures, we have identified a subset of residues in ...

Picking on someone smaller. Cytochromes P450 catalyze the hydroxylation of thousands of substrates, including alkanes. No naturally occurring P450, however, is known to oxidize the smallest alkanes, ethane and methane. Here we report the direct and selective oxidation of ethane to ethanol using dioxygen, catalyzed by a cytochrome P450 BM‐3 variant engineered for high activity towards small alkanes (see scheme). Achieving P450‐catalyzed oxidation of ethane is a key step in the pathway to P450‐catalyzed methane oxidation and opens new opportunities for the bioconversion of natural gas to fuels and chemicals.