Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy.

Rapid evolution of enzyme activities is often hindered by the lack of efficient and affordable methods to identify beneficial mutants. We report the development of a new growth-coupled selection method for evolving NADPH-consuming enzymes based on the recycling of this redox cofactor. The method relies on a genetically modified Escherichia coli strain, which overaccumulates NADPH. This method was applied to the engineering of a carboxylic acid reductase (CAR) for improved catalytic activities on 2-methoxybenzoate and adipate. Mutant enzymes with up to 17-fold improvement in catalytic efficiency were identified from single-site saturated mutagenesis libraries. Obtained mutants were successfully applied to whole-cell conversions of adipate into 1,6-hexanediol, a C6 monomer commonly used in polymer industry.

Utilization of naproxen by Amycolatopsis sp. Poz 14 and detection of the enzymes involved in the degradation metabolic pathway.

The pollution of aquatic environments by drugs is a problem for which scarce research has been conducted in regards of their removal. Amycolatopsis sp. Poz 14 presents the ability to biotransformation naphthalene at high efficiency, therefore, in this work this bacterium was proposed as an assimilator of naproxen and carbamazepine. Growth curves at different concentrations of naproxen and carbamazepine showed that Amycolatopsis sp. Poz 14 is able to utilize these drugs at a concentration of 50 mg L-1 as a source of carbon and energy. At higher concentrations, the bacterial growth was inhibited. The transformation kinetics of naproxen showed the total elimination of the compound in 18 days, but carbamazepine was only eliminated in 19.9%. The supplementation with cometabolites such as yeast extract and naphthalene (structure similar to naproxen) at 50 mg L-1, showed that the yeast extract shortened the naproxen elimination to 6 days and reached a higher global consumption rate compared to the naphthalene cometabolite. The biotransformation of carbamazepine was not improved by the addition of cometabolites. The partial sequencing of the genome of Amycolatopsis sp. Poz 14 detected genes encoding putative enzymes for the degradation of cyclic aromatic compounds and the activities of aromatic monooxygenase, catechol 1,2-dioxygenase and gentisate 1,2-dioxygenase exhibited their involving in the naproxen biodegradation. The HPLC-MS analysis detected the 5-methoxysalicylic acid at the end of the biotransformation kinetics. This work demonstrates that Amycolatopsis sp. Poz 14 utilizes naproxen and transforms it to 5-methoxysalicylic acid which is the initial compound for the catechol and gentisic acid metabolic pathway.

Methane production from coal by a single methanogen.

Coal-bed methane is one of the largest unconventional natural gas resources. Although microbial activity may greatly contribute to coal-bed methane formation, it is unclear whether the complex aromatic organic compounds present in coal can be used for methanogenesis. We show that deep subsurface-derived Methermicoccus methanogens can produce methane from more than 30 types of methoxylated aromatic compounds (MACs) as well as from coals containing MACs. In contrast to known methanogenesis pathways involving one- and two-carbon compounds, this "methoxydotrophic" mode of methanogenesis couples O-demethylation, CO2 reduction, and possibly acetyl-coenzyme A metabolism. Because MACs derived from lignin may occur widely in subsurface sediments, methoxydotrophic methanogenesis would play an important role in the formation of natural gas not limited to coal-bed methane and in the global carbon cycle.

Benzenoid biosynthesis in the flowers of Eriobotrya japonica: molecular cloning and functional characterization of p-methoxybenzoic acid carboxyl methyltransferase.

MAIN CONCLUSION: p -Methoxybenzoic acid carboxyl methyltransferase (MBMT) was isolated from loquat flowers. MBMT displayed high similarity to jasmonic acid carboxyl methyltransferases, but exhibited high catalytic activity to form methyl p -methoxybenzoate from p -methoxybenzoic acid. Volatile benzenoids impart the characteristic fragrance of loquat (Eriobotrya japonica) flowers. Here, we report that loquat produces methyl p-methoxybenzoate, along with other benzenoids, as the flowers bloom. Although the adaxial side of flower petals is covered with hairy trichomes, the trichomes are not the site of volatile benzenoid formation. Here we identified four carboxyl methyltransferase (EjMT1 to EjMT4) genes from loquat and functionally characterized EjMT1 which we found to encode a p-methoxybenzoic acid carboxyl methyltransferase (MBMT); an enzyme capable of converting p-methoxybenzoic acid to methyl p-methoxybenzoate via methylation of the carboxyl group. We found that transcript levels of MBMT continually increased throughout the flower development with peak expression occurring in fully opened flowers. Recombinant MBMT protein expressed in Escherichia coli showed the highest substrate preference toward p-methoxybenzoic acid with an apparent K m value of 137.3 µM. In contrast to benzoic acid carboxyl methyltransferase (BAMT) and benzoic acid/salicylic acid carboxyl methyltransferase, MBMT also displayed activity towards both benzoic acid and jasmonic acid. Phylogenetic analysis revealed that loquat MBMT forms a monophyletic group with jasmonic acid carboxyl methyltransferases (JMTs) from other plant species. Our results suggest that plant enzymes with same BAMT activity have evolved independently.

Screening of Burkholderia sp. WGB31 producing anisic acid from anethole and optimization of fermentation conditions.

Anisic acid, the precursor of a variety of food flavors and industrial raw materials, can be bioconversed from anethole which extracted from star anise fruits. WGB31 strain with anisic acid molar production rate of 10.25% was isolated and identified as Burkholderia sp. Three significant influential factors, namely, glucose concentration, initial pH value, and medium volume were selected and their effects were evaluated by Box-Behnken Design (BBD). Regression analysis was performed to determine response surface methodology and the significance was tested to obtain the process model of optimal conditions for producing anisic acid. The fermentation conditions at the stable point of the model were obtained: glucose 6 g L(-1) , pH 6.2, culture medium volume 61 mL in a triangular flask with 250 ml volume. Verification test indicated that the production rate of anisic acid was 30.7%, which was three times of that before optimizing. The results provide a basis and reference for producing anisic acid by microbial transformation.

Production of natural fragrance aromatic acids by coexpression of trans-anethole oxygenase and p-anisaldehyde dehydrogenase genes of Pseudomonas putida JYR-1 in Escherichia coli.

A gene encoding p-anisaldehyde dehydrogenase (PAADH), which catalyzes the oxidation of p-anisaldehyde to p-anisic acid, was identified to be clustered with the trans-anethole oxygenase (tao) gene in Pseudomonas putida JYR-1. Heterologously expressed PAADH in Escherichia coli catalyzed the oxidation of vanillin, veratraldehyde, and piperonal to the corresponding aromatic acids vanillic acid, veratric acid, and piperonylic acid, respectively. Coexpression of trans-anethole oxygenase (TAO) and PAADH in E. coli also resulted in the successful transformation of trans-anethole, isoeugenol, O-methyl isoeugenol, and isosafrole to p-anisic acid, vanillic acid, veratric acid, and piperonylic acid, respectively, which are compounds found in plants as secondary metabolites. Because of the relaxed substrate specificity and high transformation rates by coexpressed TAO and PAADH in E. coli , the engineered strain has potential to be applied in the fragrance industry.