Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases.

Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 from Rhodopseudomonas palustris can also accept bulkier substrates. In this study, we explored the substrate promiscuity of microbial FADs and their ability to defluorinate polyfluorinated organic acids. Enzymatic screening of eight purified dehalogenases with reported fluoroacetate defluorination activity revealed significant hydrolytic activity against difluoroacetate in three proteins. Product analysis using liquid chromatography-mass spectrometry identified glyoxylic acid as the final product of enzymatic DFA defluorination. The crystal structures of DAR3835 from Dechloromonas aromatica and NOS0089 from Nostoc sp. were determined in the apo-state along with the DAR3835 H274N glycolyl intermediate. Structure-based site-directed mutagenesis of DAR3835 demonstrated a key role for the catalytic triad and other active site residues in the defluorination of both fluoroacetate and difluoroacetate. Computational analysis of the dimer structures of DAR3835, NOS0089, and RPA1163 indicated the presence of one substrate access tunnel in each protomer. Moreover, protein-ligand docking simulations suggested similar catalytic mechanisms for the defluorination of both fluoroacetate and difluoroacetate, with difluoroacetate being defluorinated via two consecutive defluorination reactions producing glyoxylate as the final product. Thus, our findings provide molecular insights into substrate promiscuity and catalytic mechanism of FADs, which are promising biocatalysts for applications in synthetic chemistry and bioremediation of fluorochemicals.

Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling.

The steady growth in industrial production of synthetic plastics and their limited recycling have resulted in severe environmental pollution and contribute to global warming and oil depletion. Currently, there is an urgent need to develop efficient plastic recycling technologies to prevent further environmental pollution and recover chemical feedstocks for polymer re-synthesis and upcycling in a circular economy. Enzymatic depolymerization of synthetic polyesters by microbial carboxylesterases provides an attractive addition to existing mechanical and chemical recycling technologies due to enzyme specificity, low energy consumption, and mild reaction conditions. Carboxylesterases constitute a diverse group of serine-dependent hydrolases catalysing the cleavage and formation of ester bonds. However, the stability and hydrolytic activity of identified natural esterases towards synthetic polyesters are usually insufficient for applications in industrial polyester recycling. This necessitates further efforts on the discovery of robust enzymes, as well as protein engineering of natural enzymes for enhanced activity and stability. In this essay, we discuss the current knowledge of microbial carboxylesterases that degrade polyesters (polyesterases) with focus on polyethylene terephthalate (PET), which is one of the five major synthetic polymers. Then, we briefly review the recent progress in the discovery and protein engineering of microbial polyesterases, as well as developing enzyme cocktails and secreted protein expression for applications in the depolymerisation of polyester blends and mixed plastics. Future research aimed at the discovery of novel polyesterases from extreme environments and protein engineering for improved performance will aid developing efficient polyester recycling technologies for the circular plastics economy.

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins.

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical α/ß-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal ß-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications, including recycling of synthetic and biobased polyesters increasingly used in textiles, fibers, coatings and adhesives. Here, we identified three novel thermotolerant carboxylesterases (IS10, IS11, and IS12) from high-temperature enrichment cultures from Ischia hydrothermal vents and incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest effective volumes among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous than IS10 and IS12, IS11 had a higher thermostability with a high temperature optimum (80 to 90°C) for activity and hydrolyzed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity of these enzymes makes them attractive candidates for further optimization and potential application in plastics recycling.

The HydS C-terminal domain of the Thiocapsa bogorovii HydSL hydrogenase is involved in membrane anchoring and electron transfer.

Thiocapsa bogorovii BBS (former name Thiocapsa roseopersicina) contains HydSL hydrogenase belonging to 1e subgroup of NiFe hydrogenases (isp-type). The operon of these hydrogenases contains gene for small subunit (hydS), gene for large subunit (hupL), and genes isp1 and isp2 between them. It is predicted that last two genes code electron transport careers for electron transfer from/to HydSL hydrogenase. However, the interaction between them is unclear. The aim of this study was to determine structural and functional role of T. bogorovii HydS C-terminal end. For this purpose, we modelled all subunits of the complex HydS-HydL-Isp1-Isp2. Hydrophobicity surface analysis of the Isp1 model revealed highly hydrophobic helices suggesting potential membrane localization, as well as the hydrophilic C-terminus, which is likely localized outside of membrane. Isp1 model was docked with models of full length and C-terminal truncated HydSL hydrogenases and results illustrate the possibility of HydSL membrane anchoring via transmembrane Isp1 with essential participation of C-terminal end of HydS in the interaction. C-terminal end of HydS subunit was deleted and our studies revealed that the truncated HydSL hydrogenase detached from cellular membranes in contrast to native hydrogenase. It is known that HydSL hydrogenase in T. bogorovii performs the reaction of elemental sulfur reduction (S0 + H2 = ≥H2S). Cells with truncated HydS produced much less H2S in the presence of H2 and S0. Thus, our data support the conclusion that C-terminal end of HydS subunit participates in interaction of HydSL hydrogenase with Isp1 protein for membrane anchoring and electron transfer.

Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

Opportunities and Challenges for Microbial Synthesis of Fatty Acid-Derived Chemicals (FACs).

Global warming and uneven distribution of fossil fuels worldwide concerns have spurred the development of alternative, renewable, sustainable, and environmentally friendly resources. From an engineering perspective, biosynthesis of fatty acid-derived chemicals (FACs) is an attractive and promising solution to produce chemicals from abundant renewable feedstocks and carbon dioxide in microbial chassis. However, several factors limit the viability of this process. This review first summarizes the types of FACs and their widely applications. Next, we take a deep look into the microbial platform to produce FACs, give an outlook for the platform development. Then we discuss the bottlenecks in metabolic pathways and supply possible solutions correspondingly. Finally, we highlight the most recent advances in the fast-growing model-based strain design for FACs biosynthesis.

Lignin-oxidizing activity of bacterial laccases characterized using soluble substrates and polymeric lignin.

Efficient biotransformation of lignin requires the activity of different oxidative enzymes. In this work, 19 bacterial multi-copper oxidases were screened for oxidase activity against 19 soluble substrates and revealed the highest activity in the laccase CotABsu (BSU0630) from Bacillus subtilis. Structure-based site-directed mutagenesis of CotABsu identified four conserved residues (His419, Cys492, His497, and Met502) as critical for activity against 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS). Greatly reduced oxidase activity was found in the CotABsu mutant proteins E213A, N214A, C229A, N264A, E298A, T415A, R416A, Q468A, and T480A. We also designed a lignin-agarose plate screen for detecting oxidase activity of purified proteins against polymeric lignin, which confirmed the results obtained with ABTS and identified three mutant variants with increased activity toward kraft lignin (E213A, T415A, and T260A). X-ray photoelectron spectroscopy analysis of low sulfonate kraft lignin after incubation with CotABsu revealed a reduction in the content of CC/CC bonds and increase in CO/CO bonds. Product analyses using mass spectrometry, liquid chromatography, and bright-field microscopy revealed an increased polymerization state of reaction products suggesting that formation of radical intermediates was followed by radical coupling. Our results provide further insights into the mechanisms of lignin oxidation by laccases.

A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution.

BACKGROUND: Aldehyde decarbonylases (ADs), which convert acyl aldehydes into alkanes, supply promising solution for producing alkanes from renewable feedstock. However the instability of ADs impedes their further application. Therefore, the current study aimed to investigate the degradation mechanism of ADs and engineer it towards high stability. RESULTS: Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus using error-prone PCR-based directed evolution system. Bioinformatic analysis revealed that this C-terminal degron is common in bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli, thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo. CONCLUSION: This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.

Biocatalytic in Vitro and in Vivo FMN Prenylation and (De)carboxylase Activation.

Reversible UbiD-like (de)carboxylases represent a large family of mostly uncharacterized enzymes, which require the recently discovered prenylated FMN (prFMN) cofactor for activity. Functional characterization of novel UbiDs is hampered by a lack of robust protocols for prFMN generation and UbiD activation. Here, we report two systems for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions. The in vitro one-pot prFMN cascade includes five enzymes: FMN prenyltransferase (UbiX), prenol kinase, polyphosphate kinase, formate dehydrogenase, and FMN reductase, which use prenol, polyphosphate, formate, ATP, NAD+, and FMN as substrates and cofactors. Under aerobic conditions, this cascade produced prFMN from FMN with over 98% conversion and activated purified ferulic acid decarboxylase Fdc1 from Aspergillus niger and protocatechuic acid decarboxylase ENC0058 from Enterobacter cloaceae. The in vivo system for FMN prenylation and UbiD activation is based on the coexpression of Fdc1 and UbiX in Escherichia coli cells under aerobic conditions in the presence of prenol. The in vitro and in vivo FMN prenylation cascades will facilitate functional characterization of novel UbiDs and their applications.

Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol.

Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.

Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 µg/mL) beyond that of the three-kinase system (∼400 µg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

One-Pot Biocatalytic Transformation of Adipic Acid to 6-Aminocaproic Acid and 1,6-Hexamethylenediamine Using Carboxylic Acid Reductases and Transaminases.

Production of platform chemicals from renewable feedstocks is becoming increasingly important due to concerns on environmental contamination, climate change, and depletion of fossil fuels. Adipic acid (AA), 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD) are key precursors for nylon synthesis, which are currently produced primarily from petroleum-based feedstocks. In recent years, the biosynthesis of adipic acid from renewable feedstocks has been demonstrated using both bacterial and yeast cells. Here we report the biocatalytic conversion/transformation of AA to 6-ACA and HMD by carboxylic acid reductases (CARs) and transaminases (TAs), which involves two rounds (cascades) of reduction/amination reactions (AA → 6-ACA → HMD). Using purified wild type CARs and TAs supplemented with cofactor regenerating systems for ATP, NADPH, and amine donor, we established a one-pot enzyme cascade catalyzing up to 95% conversion of AA to 6-ACA. To increase the cascade activity for the transformation of 6-ACA to HMD, we determined the crystal structure of the CAR substrate-binding domain in complex with AMP and succinate and engineered three mutant CARs with enhanced activity against 6-ACA. In combination with TAs, the CAR L342E protein showed 50-75% conversion of 6-ACA to HMD. For the transformation of AA to HMD (via 6-ACA), the wild type CAR was combined with the L342E variant and two different TAs resulting in up to 30% conversion to HMD and 70% to 6-ACA. Our results highlight the suitability of CARs and TAs for several rounds of reduction/amination reactions in one-pot cascade systems and their potential for the biobased synthesis of terminal amines.

Site-directed mutagenesis and stability of the carboxylic acid reductase MAB4714 from Mycobacterium abscessus.

Carboxylic acid reductases (CARs) catalyze ATP- and NADPH-dependent reduction of carboxylic acids to corresponding aldehydes. Although successful applications of these enzymes for the bioconversion of monocarboxylic acids have already been reported, their applicability for the reduction of dicarboxylic acids is not well understood. Here, we explored the possibility of engineering CARs for enhanced activity toward succinic acid for potential applications in 1,4-butanediol production. Structural models of the carboxylate-binding pocket of the CAR enzyme MAB4714 from Mycobacterium abscessus suggested that its reactivity toward succinic acid could be enhanced by reducing the pocket volume. Using site-directed mutagenesis, we introduced larger side chains into the MAB4714 carboxylate binding pocket and compared the activity of 16 mutant proteins against cinnamic and succinic acids. These experiments revealed that, although the reaction rates remain low, the replacement of Leu284 or Thr285 with Trp increased activity toward succinic acid more than two times. The T285E mutant protein also showed increased activity toward succinic acid, but it was lower than that of T285W. The mutated residues of MAB4714 are located on the flexible loop covering the carboxylate-binding pocket, which appears to contribute to substrate preference of CARs. Thus, reductase activity of CARs against succinic acid can be improved by introducing large side chains into the carboxylate-binding pocket. We also discovered that alanine replacement of the conserved Ser713 in the CAR phosphopantetheine attachment site resulted in complete degradation of the full-length protein into separate A and R domains, suggesting that CAR phosphopantetheinylation is important for its stability in solution.

Prenylated FMN: Biosynthesis, purification, and Fdc1 activation.

Prenylated flavin mononucleotide (prFMN) is a recently discovered flavin cofactor produced by the UbiX family of FMN prenyltransferases, and is required for the activity of UbiD-like reversible decarboxylases. The latter enzymes are known to be involved in ubiquinone biosynthesis and biotransformation of lignin, aromatic compounds, and unsaturated aliphatic acids. However, exploration of uncharacterized UbiD proteins for biotechnological applications is hindered by our limited knowledge about the biochemistry of prFMN and prFMN-dependent enzymes. Here, we describe experimental protocols and considerations for the biosynthesis of prFMN in vivo and in vitro, in addition to cofactor extraction and application for activation of UbiD proteins.

Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters.

The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 Å resolution and revealed a modified α/ß hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.

Biocatalytic production of adipic acid from glucose using engineered Saccharomyces cerevisiae.

Adipic acid is an important industrial chemical used in the synthesis of nylon-6,6. The commercial synthesis of adipic acid uses petroleum-derived benzene and releases significant quantities of greenhouse gases. Biocatalytic production of adipic acid from renewable feedstocks could potentially reduce the environmental damage and eliminate the need for fossil fuel precursors. Recently, we have demonstrated the first enzymatic hydrogenation of muconic acid to adipic acid using microbial enoate reductases (ERs) - complex iron-sulfur and flavin containing enzymes. In this work, we successfully expressed the Bacillus coagulans ER in a Saccharomyces cerevisiae strain producing muconic acid and developed a three-stage fermentation process enabling the synthesis of adipic acid from glucose. The ability to express active ERs and significant acid tolerance of S. cerevisiae highlight the applicability of the developed yeast strain for the biocatalytic production of adipic acid from renewable feedstocks.

Biosynthesis and Activity of Prenylated FMN Cofactors.

Prenylated flavin mononucleotide (prFMN) is a recently discovered cofactor required by the UbiD family of reversible decarboxylases involved in ubiquinone biosynthesis, biological decomposition of lignin, and biotransformation of aromatic compounds. This cofactor is synthesized by UbiX-like prenyltransferases catalyzing the transfer of the dimethylallyl moiety of dimethylallyl-monophosphate (DMAP) to FMN. The origin of DMAP for prFMN biosynthesis and the biochemical properties of free prFMN are unknown. We show that in Escherichia coli cells, DMAP can be produced by phosphorylating prenol using ThiM or dephosphorylating DMAPP using Nudix hydrolases. We produced 14 active prenyltransferases whose properties enabled the purification and characterization of protein-free forms of prFMN. In vitro assays revealed that the UbiD-like ferulate decarboxylase (Fdc1) can be activated by free prFMNiminium or C2'-hydroxylated prFMNiminium under both oxidized and reduced conditions. These insights into the biosynthesis and properties of prFMN will facilitate further elucidation of the biochemical diversity of reversible UbiD (de)carboxylases.

Exploring Bacterial Carboxylate Reductases for the Reduction of Bifunctional Carboxylic Acids.

Carboxylic acid reductases (CARs) selectively reduce carboxylic acids to aldehydes using ATP and NADPH as cofactors under mild conditions. Although CARs attracts significant interest, only a few enzymes have been characterized to date, whereas the vast majority of CARs have yet to be examined. Herein the authors report that 12 bacterial CARs reduces a broad range of bifunctional carboxylic acids containing oxo-, hydroxy-, amino-, or second carboxyl groups with several enzymes showing activity toward 4-hydroxybutanoic (4-HB) and adipic acids. These CARs exhibits significant reductase activity against substrates whose second functional group is separated from the carboxylate by at least three carbons with both carboxylate groups being reduced in dicarboxylic acids. Purified CARs supplemented with cofactor regenerating systems (for ATP and NADPH), an inorganic pyrophosphatase, and an aldo-keto reductase catalyzes a high conversion (50-76%) of 4-HB to 1,4-butanediol (1,4-BDO) and adipic acid to 1,6-hexanediol (1,6-HDO). Likewise, Escherichia coli strains expressing eight different CARs efficiently reduces 4-HB to 1,4-BDO with 50-95% conversion, whereas adipic acid is reduced to a mixture of 6-hydroxyhexanoic acid (6-HHA) and 1,6-HDO. Thus, our results illustrate the broad biochemical diversity of bacterial CARs and their compatibility with other enzymes for applications in biocatalysis.

Alkene hydrogenation activity of enoate reductases for an environmentally benign biosynthesis of adipic acid.

Adipic acid, a precursor for Nylon-6,6 polymer, is one of the most important commodity chemicals, which is currently produced from petroleum. The biosynthesis of adipic acid from glucose still remains challenging due to the absence of biocatalysts required for the hydrogenation of unsaturated six-carbon dicarboxylic acids to adipic acid. Here, we demonstrate the first enzymatic hydrogenation of 2-hexenedioic acid and muconic acid to adipic acid using enoate reductases (ERs). ERs can hydrogenate 2-hexenedioic acid and muconic acid producing adipic acid with a high conversion rate and yield in vivo and in vitro. Purified ERs exhibit a broad substrate spectrum including aromatic and aliphatic 2-enoates and a significant oxygen tolerance. The discovery of the hydrogenation activity of ERs contributes to an understanding of the catalytic mechanism of these poorly characterized enzymes and enables the environmentally benign biosynthesis of adipic acid and other chemicals from renewable resources.

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families.

Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.