Regiospecific Oxidation of Chlorobenzene to 4-Chlororesorcinol, Chlorohydroquinone, 3-Chlorocatechol and 4-Chlorocatechol by Engineered Toluene o-Xylene Monooxygenases.

Toluene o-xylene monooxygenase (ToMO) was found to oxidize chlorobenzene to form 2-chlorophenol (2-CP, 4%), 3-CP (12%), and 4-CP (84%) with a total product formation rate of 1.2 ± 0.17 nmol/min/mg protein. It was also discovered that ToMO forms 4-chlorocatechol (4-CC) from 3-CP and 4-CP with initial rates of 0.54 ± 0.10 and 0.40 ± 0.04 nmol/min/mg protein, respectively, and chlorohydroquinone (CHQ, 13%), 4-chlororesorcinol (4-CR, 3%), and 3-CC (84%) from 2-CP with an initial product formation rate of 1.1 ± 0.32 nmol/min/mg protein. To increase the oxidation rate and alter the oxidation regiospecificity of chloroaromatics, as well as to study the roles of active site residues L192 and A107 of the alpha hydroxylase fragment of ToMO (TouA), we used the saturation mutagenesis approach of protein engineering. Thirteen TouA variants were isolated, among which some of the best substitutions uncovered here have never been studied before. Specifically, TouA variant L192V was identified which had 1.8-, 1.4-, 2.4-, and 4.8-fold faster hydroxylation activity toward chlorobenzene, 2-CP, 3-CP, and 4-CP, respectively, compared to the native ToMO. The L192V variant also had the regiospecificity of chlorobenzene changed from 4% to 13% 2-CP and produced the novel product 3-CC (4%) from 3-CP. Most of the isolated variants were identified to change the regiospecificity of oxidation. For example, compared to the native ToMO, variants A107T, A107N, and A107M produced 6.3-, 7.0-, and 7.3-fold more 4-CR from 2-CP, respectively, and variants A107G and A107G/L192V produced 3-CC (33 and 39%, respectively) from 3-CP whereas native ToMO did not. IMPORTANCE Chlorobenzene is a commonly used toxic solvent and listed as a priority environmental pollutant by the US Environmental Protection Agency. Here, we report that Escherichia coli TG1 cells expressing toluene o-xylene monooxygenase (ToMO) can successfully oxidize chlorobenzene to form dihydroxy chloroaromatics, which are valuable industrial compounds. ToMO performs this at room temperature in water using only molecular oxygen and a cofactor supplied by the cells. Using protein engineering techniques, we also isolated ToMO variants with enhanced oxidation activity as well as fine-tuned regiospecificities which make direct microbial oxygenations even more attractive. The significance of this work lies in the ability to degrade environmental pollutants while at the same time producing valuable chemicals using environmentally benign biological methods rather than expensive, complex chemical processes.

Cavity residue leucine 95 and channel residues glutamine 204, aspartic acid 211, and phenylalanine 269 of toluene o-xylene monooxygenase influence catalysis.

Structural analysis of toluene-o-xylene monooxygenase (ToMO) hydroxylase revealed the presence of three hydrophobic cavities, a channel, and a pore leading from the protein surface to the active site. Here, saturation mutagenesis was used to investigate the catalytic roles of alpha-subunit (TouA) second cavity residue L95 and TouA channel residues Q204, D211, and F269. By testing the substrates toluene, phenol, nitrobenzene, and/or naphthalene, these positions were found to influence the catalytic activity of ToMO. Several regiospecific variants were identified from TouA positions Q204, F269, and L95. For example, TouA variant Q204H had the regiospecificity of nitrobenzene changed significantly from 30 to 61 % p-nitrophenol. Interestingly, a combination of mutations at Q204H and A106V altered the regiospecificity of nitrobenzene back to 27 % p-nitrophenol. TouA variants F269Y, F269P, Q204E, and L95D improved the meta-hydroxylating capability of nitrobenzene by producing 87, 85, 82, and 77 % m-nitrophenol, respectively. For naphthalene oxidation, TouA variants F269V, Q204A, Q204S/S222N, and F269T had the regiospecificity changed from 16 to 9, 10, 23, and 25 % 2-naphthol, respectively. Here, two additional TouA residues, S222 and A106, were also identified that may have important roles in catalysis. Most of the isolated variants from D211 remained active, whereas having a hydrophobic residue at this position appeared to diminish the catalytic activity toward naphthalene. The mutational effects on the ToMO regiospecificity described here suggest that it is possible to further fine tune and engineer the reactivity of multicomponent diiron monooxygenases toward different substrates at positions that are relatively distant from the active site.

Saturation mutagenesis of Bradyrhizobium sp. BTAi1 toluene 4-monooxygenase at alpha-subunit residues proline 101, proline 103, and histidine 214 for regiospecific oxidation of aromatics.

A novel toluene monooxygenase (TMO) six-gene cluster from Bradyrhizobium sp. BTAi1 having an overall 35, 36, and 38 % protein similarity with toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, and toluene-para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, respectively, was cloned and expressed in Escherichia coli TG1, and its potential activity was investigated for aromatic hydroxylation and trichloroethylene (TCE) degradation. The natural substrate toluene was hydroxylated to p-cresol, indicating that the new toluene monooxygenase (T4MO·BTAi1) acts as a para hydroxylating enzyme, similar to T4MO and TpMO. Some shifts in regiospecific hydroxylations were observed compared to the other wild-type TMOs. For example, wild-type T4MO·BTAi1 formed catechol (88 %) and hydroquinone (12 %) from phenol, whereas all the other wild-type TMOs were reported to form only catechol. Furthermore, it was discovered that TG1 cells expressing wild-type T4MO·BTAi1 mineralized TCE at a rate of 0.67 ± 0.10 nmol Cl(-)/h/mg protein. Saturation and site directed mutagenesis were used to generate eight variants of T4MO·BTAi1 at alpha-subunit positions P101, P103, and H214: P101T/P103A, P101S, P101N/P103T, P101V, P103T, P101V/P103T, H214G, and H214G/D278N; by testing the substrates phenol, nitrobenzene, and naphthalene, positions P101 and P103 were found to influence the regiospecific oxidation of aromatics. For example, compared to wild type, variant P103T produced four fold more m-nitrophenol from nitrobenzene as well as produced mainly resorcinol (60 %) from phenol whereas wild-type T4MO·BTAi1 did not. Similarly, variants P101T/P103A and P101S synthesized more 2-naphthol and 2.3-fold and 1.6-fold less 1-naphthol from naphthalene, respectively.

The role of substrate binding pocket residues phenylalanine 176 and phenylalanine 196 on Pseudomonas sp. OX1 toluene o-xylene monooxygenase activity and regiospecificity.

Saturation mutagenesis was used to generate eleven substitutions of toluene-o-xylene monooxygenase (ToMO) at alpha subunit (TouA) positions F176 and F196 among which nine were novel: F176H, F176N, F176S, F176T, F196A, F196L, F196T, F196Y, F196H, F196I, and F196V. By testing the substrates phenol, toluene, and naphthalene, these positions were found to influence ToMO oxidation activity and regiospecificity. Specifically, TouA variant F176H was identified that had 4.7-, 4.3-, and 1.8-fold faster hydroxylation activity towards phenol, toluene, and naphthalene, respectively, compared to native ToMO. The F176H variant also produced the novel product hydroquinone (61%) from phenol, made twofold more 2-naphthol from naphthalene (34% vs. 16% by the wild-type ToMO), and had the regiospecificity of toluene changed from 51% to 73% p-cresol. The TouA F176N variant had the most para-hydroxylation capability, forming p-cresol (92%) from toluene and hydroquinone (82%) from phenol as the major product, whereas native ToMO formed 30% o-cresol, 19% m-cresol, and 51% of p-cresol from toluene and 100% catechol from phenol. For naphthalene oxidation, TouA variant F176S exhibited the largest shift in the product distribution by producing threefold more 2-naphthol. Among the other F196 variants, F196L produced catechol from phenol two times faster than the wild-type enzyme. The TouA F196I variant produced twofold less o-cresol and 19% more p-cresol from toluene, and the TouA F196A variant produced 62% more 2-naphthol from naphthalene compared to wild-type ToMO. Both of these positions have never been studied through the saturation mutagenesis and some of the best substitutions uncovered here have never been predicted and characterized for aromatics hydroxylation.

Metabolically engineered bacteria for producing hydrogen via fermentation.

Hydrogen, the most abundant and lightest element in the universe, has much potential as a future energy source. Hydrogenases catalyse one of the simplest chemical reactions, 2H(+) + 2e(-) ↔ H(2), yet their structure is very complex. Biologically, hydrogen can be produced via photosynthetic or fermentative routes. This review provides an overview of microbial production of hydrogen by fermentation (currently the more favourable route) and focuses on biochemical pathways, theoretical hydrogen yields and hydrogenase structure. In addition, several examples of metabolic engineering to enhance fermentative hydrogen production are presented along with some examples of expression of heterologous hydrogenases for enhanced hydrogen production.

A homogeneous fluorometric assay platform based on novel synthetic proteins.

Novel synthetic recombinant sensor proteins have been created to detect analytes in solution, in a rapid single-step "mix and read" noncompetitive homogeneous assay process, based on modulating the Förster resonance energy transfer (FRET) property of the sensor proteins upon binding to their targets. The sensor proteins comprise a protein scaffold that incorporates a specific target-capturing element, sandwiched by genetic fusion between two molecules that form a FRET pair. The utility of the sensor proteins was demonstrated via three examples, for detecting an anti-biotin Fab antibody, a His-tagged recombinant protein, and an anti-FLAG peptide antibody, respectively, all done directly in solution. The diversity of sensor-target interactions that we have demonstrated in this study points to a potentially universal applicability of the biosensing concept. The possibilities for integrating a variety of target-capturing elements with a common sensor scaffold predict a broad range of practical applications.