•We compared an enzyme cascade in vivo as well as in vitro.•Mechanistic insights were gained for the presented cascade.•Monomer production for biorenewable polyesters was investigated.•Pathway construction with non-related enzymes was performed.
An artificial enzyme cascade composed of an alcohol dehydrogenase, an enoate reductase and a Baeyer–Villiger monooxygenase was investigated in vitro to gain deeper mechanistic insights and understand the assets and drawbacks of this multi-step biocatalysis. Several substrates composed of different structural motifs were examined and provided access to functionalized chiral compounds in high yields (up to >99%) and optical purities (up to >99%). Hence, the applicability of the presented enzymatic cascade was exploited for the synthesis of biorenewable polyesters.
ADH, alcohol dehydrogenase; BVMO, Baeyer–Villiger monooxygenase; CFE, cell free extract; CHMO, cyclohexanone monooxygenase; ERED, enoate reductase; G6P, glucose 6-phosphate; G6P-DH, glucose 6-phosphate dehydrogenase; GC, gas chromatography; IPTG, isopropyl-β-d-thiogalactopyranoside; LB, Lysogeny broth; NAD+, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced); NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); OD, optical density; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TB, terrific broth; Multi-step biocatalysis; Baeyer–Villiger monooxygenase; Biorenewable polyesters; Enoate reductase; Alcohol dehydrogenase
Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known. In this study we show the degradation of the aliphatic ketone dodecane-2-one by Candida maltosa and the related yeasts Candida tropicalis, Candida catenulata and Candida albicans as well as Trichosporon asahii and Yarrowia lipolytica. One pathway is initiated by the formation of decyl acetate, resulting from a Baeyer-Villiger-oxidation of this ketone. Beyond this, an initial reduction to dodecane-2-ol by a keto reductase was clearly shown. In addition, two different ways to metabolize dodec-1-ene were proposed. One involved the formation of dodecane-2-one and the other one a conversion leading to carboxylic and dicarboxylic acids. Furthermore the induction of ketone degrading enzymes by dodecane-2-one and dodec-1-ene was shown. Interestingly, with dodecane no subterminal degradation products were detected and it did not induce any enzymes to convert dodecane-2-one.
Hydrocarbon; alkene; ketone; Candida; yeast; biotransformation
Triacylglycerol lipases (EC 126.96.36.199) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.
The crystallization and preliminary X-ray diffraction studies of DppA from P. pacifica SIR-I are reported.
DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 188.8.131.52) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P21212. The DppA crystal diffracted X-rays to 1.9 Å resolution using an in-house X-ray generator.
haloalkane dehalogenases; Plesiocystis pacifica SIR-I
Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit.
Baeyer-Villiger monooxygenases; camphor; Pseudomonas putida NCIMB 10007; 2,5-diketocamphane 1,2-monooxygenase; bicyclic ketones
Enantiomerically pure β‐arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. A library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter plate‐based screening using a pH‐indicator assay. Out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. The most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. Thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields.
While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E ≫100).
A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.
To date, expression of the lipase from Rhizopus oryzae (ROL) in Escherichia coli always led to the formation of inclusion bodies and inactive protein. However, the production of active ROL and its precursor ProROL in soluble form was achieved when E. coli Origami(DE3) and pET-11d were used as expression systems.