Outer membrane mutation effects on UDP-glucose permeability and whole-cell catalysis rate

In whole-cell biocatalysis, cell envelopes represent a formidable barrier for substrates to permeate. The present research addresses this critical issue by investigating the effects of outer membrane mutation on uridine diphosphate (UDP)-glucose-utilizing enzymes in whole-cell systems. Owing to the severe limitation in substrate permeability, the wild-type Escherichia coli cells only exhibited as low as 4% of available enzyme activities. The reduction of the barriers of the outer membrane permeability (by mutations in its structure) led to a striking acceleration (up to 14-fold) of the reaction rate in cells expressing UDP-glucose dehydrogenase. Mutations in the lipopolysaccharide synthesis pathway or Braun’s lipoprotein are both effective. The acceleration was dependent upon the substrate concentrations as well as the enzyme expression level. In addition, the mutation has been demonstrated to be much more effective than the freeze–thaw permeabilizing method. An application of outer membrane mutants was illustrated with the synthesis of a disaccharide (N-acetyllactosamine) from UDP-glucose. Both reaction rate and product yield were enhanced significantly (more than twofold) in the lipoprotein mutant, demonstrating the importance of the outer membrane permeability barrier and the advantages of using outer membrane mutants in synthesis. This research and the results outlined in this paper point to a valid strategy in addressing permeability issues in whole-cell biocatalysis. It also highlights a need for an assessment of substrate permeability in biocatalysis research and development.     

Lipoprotein mutation accelerates substrate permeability-limited toluene dioxygenase-catalyzed reaction

One of the major problems in whole-cell biocatalysis is its low reaction rate. The underlying cause is the substrate permeation barrier presented by cell envelopes. The present research investigates mutation effects of the Braun's lipoprotein, the most abundant outer membrane structural protein in Escherichia coli, on toluene dioxyengase (TDO)-catalyzed reaction. Dramatic enhancement of the reaction rate, an increase of up to 6-fold, was observed with the mutant for all three small, hydrophobic substrates tested (toluene, ethylbenzene, and 2-indanone). The increase was observed over a wide range of substrate concentrations (0.1-5 mM). The mutant exhibited a normal growth rate and expressed the recombinant multicomponent enzyme as well as the isogenic parent strain. Taken together, the lipoprotein mutant expressing TDO is a much better whole-cell catalyst for the oxidation reaction. The beneficial effect of the lipoprotein mutation may be general for a broad range of substrates and enzyme systems as the mutation affects the global integrity of the cell membrane. A comparison of the mutation effect with a common permeabilizing procedure, the EDTA treatment, further illustrates the clear advantages of using genetic modification in cellular membrane engineering for improved whole-cell catalysts.     

Microbial steroid transformations: current state and prospects

Studies of steroid modifications catalyzed by microbial whole cells represent a well-established research area in white biotechnology. Still, advances over the last decade in genetic and metabolic engineering, whole-cell biocatalysis in non-conventional media, and process monitoring raised research in this field to a new level. This review summarizes the data on microbial steroid conversion obtained since 2003. The key reactions of structural steroid functionalization by microorganisms are highlighted including sterol side-chain degradation, hydroxylation at various positions of the steroid core, and redox reactions. We also describe methods for enhancement of bioprocess productivity, selectivity of target reactions, and application of microbial transformations for production of valuable pharmaceutical ingredients and precursors. Challenges and prospects of whole-cell biocatalysis applications in steroid industry are discussed.     

lpp deletion as a permeabilization method

Our earlier studies with outer membrane permeability in E. coli showed that an insertion mutation in lpp gene (encoding Braun's lipoprotein) drastically changed the outer membrane permeability, resulting in significant acceleration of whole-cell catalyzed reactions. In order to gain a mechanistic understanding of the nature of permeability change, the lpp region was sequenced. The results revealed that Lpp was not expressed in the insertion mutant, suggesting that the absence, rather than the alteration, of Lpp is responsible for the observed permeability change. This surprising result prompts us to investigate the possibility of establishing lpp deletion as a general permeabilization method. Two lpp deletion mutants were generated from strains with different genetic background and the effect of lpp deletion on cell physiology was investigated. While lpp deletion had no significant effect on cell growth, carbon metabolism, and fatty acid compositions, it enhanced permeability of various small molecules, consistent with the results with the insertion mutant. This phenotype is useful in a wide range of biotechnological applications. We illustrate here the use of the mutant with organophosphate hydrolysis and L-carnitine synthesis, where permeability is known to be a limiting factor. Both processes were significantly improved with the mutant because of enhanced permeability through the outer membrane. Therefore, this study has established an easy yet generally applicable method for permeabilizing E. coli cells without significant adverse effects. Further, as lpp homolog is known to exist in gram-negative bacteria, we expect that this method will be applicable to other gram-negative bacteria.     

A window into biocatalysis and biotransformations

Eight papers were presented in this year's symposium "Advances in Biocatalysis" at the 232nd ACS National Meeting, accentuating the most recent development in biocatalysis. Researchers from both industry and academia are addressing several fundamental problems in biocatalysis, including the limited number of commercially available enzymes that can be provided in bulk quantities, the limited enzyme stability and activity in nonaqueous environments, and the permeability issue and cell localization problems in whole-cell systems. A trend that can be discerned from these eight talks is the infusion of new tools and technologies in addressing various challenges facing biocatalysis. Nanotechnology, bioinformatics, cellular membrane engineering and metabolic engineering (for engineering whole-cell catalysts), and protein engineering (to improve enzymes and create novel enzymes) are becoming more routinely used in research laboratories and are providing satisfactory solutions to the problems in biocatalysis. Significant progress in various aspects of biocatalysis from discovery to industrial applications was highlighted in this symposium.     

Advancing biocatalysis through enzyme, cellular, and platform engineering

Biocatalysis offers opportunities for highly selective chemical reactions with high turnover rates under relatively mild conditions. Use of whole-cell or multi-enzyme systems enables transformations of complexity unmatched by nonbiological routes. However, advantages of biocatalysis are frequently compromised by poor enzymatic performance under non-native reaction conditions, the absence of enzymes with desired substrate or reaction specificities, and low metabolic fluxes or competing pathways. During the 234th National Meeting of the American Chemical Society, these issues were addressed in the "Advances in Biocatalysis" sessions. Protein engineering and metabolic pathway engineering were used to develop efficient enzymes and whole-cell catalysts. Novel strategies for the use of enzymes at solid interfaces and in nonaqueous environments were discussed, and efficient biotransformation platforms were demonstrated. These advances broaden the applications of biocatalysis in biofuels, pharmaceuticals, fine chemicals, and human health.     

Permeability issues in whole-cell bioprocesses and cellular membrane engineering

Nutrient uptake and waste excretion are among the many important functions of the cellular membrane. While permitting nutrients into the cell, the cellular membrane system evolves to guide against noxious agents present in the environment from entering the intracellular milieu. The semipermeable nature of the membrane is at odds with biomolecular engineers in their endeavor of using microbes as cell factory. The cellular membrane often retards the entry of substrate into the cellular systems and prevents the product from being released from the cellular system for an easy recovery. Consequently, productivities of whole-cell bioprocesses such as biocatalysis, fermentation, and bioremediations are severely compromised. For example, the rate of whole-cell biocatalysis is usually 1–2 orders of magnitude slower than that of the isolated enzymes. When product export cannot keep pace with the production rate, intracellular product accumulation quickly leads to a halt of production due to product inhibition. While permeabilization via chemical or physical treatment of cell membrane is effective in small-scale process, large-scale implementation is problematic. Molecular engineering approach recently emerged as a much better alternative. Armed with increasingly sophisticated tools, biomolecular engineers are following nature’s ingenuity to derive satisfactory solutions to the permeability problem. This review highlights these exciting molecular engineering achievements.     

Identification and Characterization of a New Gene of Escherichia Coli K-12 Involved in Outer Membrane Permeability

Using a genetic selection for mutations which allow large maltodextrins to cross the outer membrane of Escherichia coli in the absence of the LamB maltoporin, we have obtained and characterized two mutations that define a new locus of E. coli. We have designated this locus imp for increased membrane permeability. Mapping studies show that the imp gene resides at approximately 1.2 min on the E. coli chromosome. The mutations alter the permeability of the outer membrane resulting in increased sensitivity to detergents, antibiotics and dyes. The mutations are nonreverting and codominant. Genetic analysis of the mutations suggest that the imp gene is an essential gene. We describe a general cloning strategy that can be used to clone both dominant and recessive alleles. Using this technique, we have cloned the wild-type and mutant imp alleles onto a low copy number plasmid.     

Improving simvastatin bioconversion in Escherichia coli by deletion of bioH

Simvastatin is an important cholesterol lowering compound and is currently synthesized from the natural product lovastatin via multistep chemical synthesis. We have previously reported the use of an Escherichia coli strain BL21(DE3)/pAW31 as the host for whole-cell biocatalytic conversion of monacolin J acid to simvastatin acid. During fermentation and bioconversion, unknown E. coli enzyme(s) hydrolyzed the membrane permeable thioester substrate dimethylbutyryl-S-methyl mercaptopropionate (DMB-S-MMP) to the free acid, significantly decreased the efficiencies of the whole-cell bioconversion and the downstream purification steps. Using the Keio K-12 Singe-Gene Knockout collection, we identified BioH as the sole enzyme responsible for the observed substrate hydrolysis. Purification and reconstitution of E. coli BioH activity in vitro confirmed its function. BioH catalyzed the rapid hydrolysis of DMB-S-MMP with kcat and Km values of 260+/-45 s(-1) and 229+/-26 microM, respectively. This is in agreement with previous reports that BioH can function as a carboxylesterase towards fatty acid esters. YT2, which is a delta bioH mutant of BL21(DE3), did not hydrolyze DMB-S-MMP during prolonged fermentation and was used as an alternative host for whole-cell biocatalysis. The rate of simvastatin acid synthesis in YT2 was significantly faster than in BL21(DE3) and 99% conversion of 15 mM simvastatin acid in less than 12 h was achieved. Furthermore, the engineered host required significantly less DMB-S-MMP to be added to accomplish complete conversion. Finally, simvastatin acid synthesized using YT2 can be readily purified from fermentation broth and no additional steps to remove the hydrolyzed dimethylbutyryl-S-mercaptopropionic acid is required. Together, the proteomic and metabolic engineering approaches render the whole-cell biocatalytic process more robust and economically attractive.     

Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates

The substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S and S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (S-KHPB). These mutations improve the value of k(cat)/K(M)(S-KHPB) by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of k(cat)(S-KHPB) for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximal value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate, a nonfunctionalized hydrophobic analogue. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate aldolases. Furthermore, targeting mutations to the active site provides a marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to those of naturally occurring enzymes.     

Structural biology of membrane-intrinsic beta-barrel enzymes: sentinels of the bacterial outer membrane

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel beta-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three beta-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipidColon, two colonslipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane beta-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two beta-barrel enzymes of unknown structure; namely, the Salmonella enterica 3'-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O(2) to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how beta-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger beta-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.     

Engineering of Phenylacetaldehyde Reductase for Efficient Substrate Conversion in Concentrated 2-Propanol

Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.     

Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol

Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.     

Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.     

Isolation and characterization of outer membrane permeability mutants in Escherichia coli K-12.

Escherichia coli normally requires the lamB gene for the uptake of maltodextrins. We have identified and characterized three independent mutations that allow E. coli to grow on maltodextrin in the absence of a functional lamB gene by allowing maltodextrins with a molecular weight greater than 1,000 to cross the outer membrane barrier. Two of the mutations map to the structural gene for the outer membrane porin OmpF, and the remaining mutation maps to the structural gene for the second major outer membrane porin, OmpC. These mutations increase the permeability of the outer membrane to small hydrophilic substances, antibiotics, and detergents. These mutations alter the electrophoretic mobility of the respective porin proteins.     

The hydrophobic uptake pathway across the outer membrane of the antibiotic supersusceptible Pseudomonas aeruginosa mutant Z61

Abstract The Pseudomonas aeruginosa antibiotic supersusceptible mutant Z61 was 50–400-fold more susceptible than its wild-type parent K799 to 5 hydrophobic antibiotics. The strain Z61 outer membrane also demonstrated enhanced permeability towards a hydrophobic fluorescent probe. Strain Z61 cells had an altered cell surface, as revealed by phase-partitioning experiments, a lower amount of Lipid A phosphate, and a reduction in the number of Mg²⁺ binding sites in Lipid A, as demonstrated by dansyl polymyxin competition experiments. An antibiotic permation pathway directly through the outer membrane bilayer, rather than through porin proteins, is proposed for strain Z61.     

Structural biology of membrane-intrinsic β-barrel enzymes: Sentinels of the bacterial outer membrane

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel β-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three β-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipid∷lipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane β-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two β-barrel enzymes of unknown structure; namely, the Salmonella enterica 3′-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O₂ to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how β-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger β-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.     

Engineering the CYP101 system for in vivo oxidation of unnatural substrates.

The protein engineering of CYP enzymes for structure-activity studies and the oxidation of unnatural substrates for biotechnological applications will be greatly facilitated by the availability of functional, whole-cell systems for substrate oxidation. We report the construction of a tricistronic plasmid that expresses the CYP101 monooxygenase from Pseudomonas putida, and its physiological electron transfer co-factor proteins putidaredoxin reductase and putidaredoxin in Escherichia coli, giving a functional in vivo catalytic system. Wild-type CYP101 expressed in this system efficiently transforms camphor to 5-exo-hydroxycamphor without further oxidation to 5-oxo-camphor until >95% of camphor has been consumed. CYP101 mutants with increased activity for the oxidation of diphenylmethane (the Y96F-I395G mutant), styrene and ethylbenzene (the Y96F-V247L mutant) have been engineered. In particular, the Y96F-V247L mutant shows coupling efficiency of approximately 60% for styrene and ethylbenzene oxidation, with substrate oxidation rates of approximately 100/min. Escherichia coli cells transformed with tricistronic plasmids expressing these mutants readily gave 100-mg quantities of 4-hydroxydiphenylmethane and 1-phenylethanol in 24-72 h. This new in vivo system can be used for preparative scale reactions for product characterization, and will greatly facilitate directed evolution of the CYP101 enzyme for enhanced activity and selectivity of substrate oxidation.     

Purification of a class C A-type beta-lactamase from a derepressed strain of Enterobacter cloacae. Comparison of the wild-type and mutant enzyme with those from strains P99, 208 and GN7471.

Enterobacter cloacae strain 5822 expresses low levels of a class C beta-lactamase which can be induced 100-fold by imipenem. Mutants that constitutively express high levels of beta-lactamase can be selected on aztreonam or cefotaxime. The beta-lactamase from one such mutant (5822M2) has been purified to homogeneity and compared on the basis of subunit Mr, pI, substrate specificity, inhibitor sensitivity and immunological cross-reactivity with the enzyme from strains P99, GN7471 and 208, which have been studied previously. The enzyme from strain 5822M2 is clearly related to these other forms and is of the A-type according to the criteria of Seeberg, Tolxdorff-Neutzling & Wiedemann [Antimicrob. Agents Chemother. (1983) 23, 918-925]. The enzyme from the wild-type strain (5822) is shown to be identical to that found in the depressed strain (5822M2), indicating that the mutation is in a regulatory gene. A detailed analysis of the kinetics of the enzyme from strain 5822M2 shows that all of the beta-lactams studied are substrates and that a mechanism involving the formation of an acyl-enzyme is probably applicable in every case. The substrates however can clearly be grouped into two classes, i.e. 'good' substrates with kcat. values of 80-1200 s-1 and 'poor' substrates/good inhibitors with kcat. values of 0.009-0.00007 s-1. The permeability barrier to aztreonam is 4-fold less in the derepressed strain when compared with the wild-type strain. This is associated with significant changes in the expression of outer membrane porins. The observed resistance in the derepressed mutant appears to be linked to the elevated levels of beta-lactamase (3000-fold) rather than to the modest changes in the permeability barrier.     

The role of lipopolysaccharides in the action of the bactericidal/permeability-increasing neutrophil protein on the bacterial envelope

The killing of gram-negative bacteria by the bactericidal/permeability-increasing protein ( BPI ) of neutrophils requires surface binding, and is accompanied by a discrete increase in outer membrane permeability to small hydrophobic substances. This outer membrane alteration appears to be related to perturbation of outer membrane lipopolysaccharides (LPS). BPI causes extracellular release of LPS, but only at supra-saturating doses. Nevertheless, because the organization of LPS in the outer membrane is altered by pretreatment of bacteria with saturating doses of BPI (producing maximal bactericidal and permeability-increasing effects), the amount of LPS released during Tris-EDTA treatment is reduced by 80%. BPI markedly (approximately 50%) and selectively stimulates biosynthesis of LPS, suggesting an attempt by BPI -killed bacteria to repair outer membrane damage. The removal of surface-bound BPI by 40 mM Mg2+ initiates time- and temperature-dependent repair of the outer membrane permeability barrier and a further increase (approximately 170% of control) in LPS synthesis, even though the bacteria are no longer viable. Mg2+-induced repair is blocked when: 1) a temperature-sensitive mutant (Salmonella typhimurium HD50 ) with a conditional defect in LPS synthesis is incubated at the nonpermissive temperature (42 degrees C); and 2) LPS synthesis is selectively inhibited by a diazaborine derivative (Sandoz drug No. 84474). In contrast, repair is normal by the mutant at permissive temperatures (30 degrees C) and by the parent strain (S. typhimurium AG701 ) at both 30 degrees C and 42 degrees C. Inhibition (greater than 85%) of protein synthesis by chloramphenicol has little or no effect on repair. These findings indicate that the repair of the permeability barrier after the removal of BPI from the surface requires newly made LPS, but apparently no biosynthesis of other outer membrane constituents, which strongly suggests that the effects of BPI on LPS are mainly responsible for the break-down of the outer membrane permeability barrier.