Bacterial whole-cell biotransformation
Utilization of microorganisms for reductive whole-cell biotransformation under resting cell conditions has become an important method in chemoenzymatic synthesis, e. g. for the production of chiral intermediates required in the synthesis of pharmaceuticals. Prominent products whose synthesis by biotransformation requires NAD(P)H are chiral alcohols that serve as building blocks in the synthesis of statins, compounds that dominate the world market for cholesterol-lowering drugs (1, 2).
A basic requirement for the efficiency of reductive whole-cell biotransformations is the reducing capacity of the host. We apply the pentose phosphate pathway (PPP) for NADPH regeneration with glucose as the electron-donating co-substrate using Escherichia coli as host. Reduction of the prochiral α-keto ester methyl acetoacetate to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate serves as a model reaction, catalyzed by an R-specific alcohol dehydrogenase (Fig. 1).
Our main focus is maximization of the reduced product per glucose yield of this pathway-coupled cofactor regeneration with resting cells. With a strain lacking the phosphoglucose isomerase, the yield of the reference strain was increased from 2.44 to 3.78 mol MHB/mol glucose (Fig. 2). Even higher yields were obtained with strains lacking either phosphofructokinase I (4.79 mol MHB/mol glucose) or phosphofructokinase I and II (5.46 mol MHB/mol glucose). These results persuasively demonstrate the potential of NADPH generation by the PPP in whole-cell biotransformations (3).
1 Liljeblad A, Kallinen A, Kanerva LT (2009) Biocatalysis in the preparation of the statin side chain. Curr Org Synth 6:362-379
2 Panke S, Wubbolts M (2005) Advances in biocatalytic synthesis of pharmaceutical intermediates. Curr Opin Chem Biol 9:188-194
3 Siedler S, Bringer S, Blank LM, Bott M (2011) Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport. Appl Microbiol Biotechnol DOI:10.1007/s00253-011-3626-3
4 Siedler S, Bringer S, Bott M (2011) Increased NADPH availability in Escherichia coli: improvement of the product per glucose ratio in reductive whole-cell biotransformation. Appl Microbiol Biotechnol 92 (2011) 5, 929–937, DOI:10.1007/s00253-011-3374-4
Gluconobacter oxydans strain development
Cytoplasmic utilization of sugars of the obligatory aerobic acetic acid bacterium Gluconobacter oxydans is restricted to only two functional pathways, the Entner-Doudoroff pathway and the pentose-phosphate pathway (Fig. 1). Lack of a gene for a phospofructokinase disables the Embden-Meyerhof-Parnas pathway and absence of succinyl-CoA synthetase and succinate dehydrogenase impede a cyclic operation of the tricarboxylic acid cycle. Sugars are primarily oxidized in the periplasm by respiratory chain associated, membrane-bound dehydrogenases to the end products 2-ketogluconate and 2,5-diketogluconate with intermediate formation of gluconate. Growth with glucose and mannitol proceeds biphasic with a first phase of rapid growth and a second with slow, linear growth. As a consequence of the restrictions of cytoplasmic sugar degradation, G. oxydans has a low growth yield. In order to improve it, we constructed mutants in which the genes encoding the membrane-bound glucose dehydrogenase and the cytoplasmic glucose dehydrogenase were deleted. The growth yield of the mutant was increased by 271%. In addition, the growth rate improved by 78% compared to the parental strain (1).
Besides the above-mentioned membrane-bound substrate dehydrogenases, the respiratory chain of G. oxydans contains a non-proton pumping NADH dehydrogenase II, a cytochrome bo3-type ubiquinol oxidase and a cytochrome bd-type quinol oxidase. In addition, G. oxydans also possesses the genes qcrABC for a cytochrome bc1 complex and cycA for a soluble cytochrome c552 (2). However, genes for a terminal cytochrome c oxidase are absent in the genome and therefore, the function of the cytochrome bc1 complex is unclear (Fig.2).
Regulation of carbon flux through the two catabolic pathways, of the respiratory chain constituents and of substrate transport was not yet studied in G. oxydans. A better knowledge of the regulatory mechanisms in G. oxydans would allow a rational strain design, e.g. the development of a strain with optimized electron flux and energy yield from the incomplete oxidations. Genome-wide transcriptional analyses with the help of DNA-microarrays and proteomics with the help of 2-D-gels are in progress.
1 Krajewski V, Simić P, Mouncey N, Bringer S, Sahm H, Bott M (2010) Metabolic engineering of Gluconobacter oxydans for improved growth rate and growth yield on glucose by elimination of gluconate formation. Appl Environ Microbiol: 76, 4369–4376, Doi:10.1128/AEM.03022-09
2 Prust C,. Hoffmeister M, Liesegang H, Wiezer A, Fricke WF, Ehrenreich A, Gottschalk G, Deppenmeier U (2005) Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nat Biotechnol 23,195-200