Equipping Saccharomyces cerevisiae with an Additional Redox Cofactor Allows F₄₂₀-Dependent Bioconversions in Yeast

Industrial application of the natural deazaflavin cofactor F420 has high potential for the enzymatic synthesis of high value compounds. It can offer an additional range of chemistry to the use of well-explored redox cofactors such as FAD and their respective enzymes. Its limited access through organisms that are rather difficult to grow has urged research on the heterologous production of F420 using more industrially relevant microorganisms such as Escherichia coli. In this study, we demonstrate the possibility of producing this cofactor in a robust and widely used industrial organism, Saccharomyces cerevisiae, by the heterologous expression of the F420 pathway. Through careful selection of involved enzymes and some optimization, we achieved an F420 yield of ∼1.3 μmol/L, which is comparable to the yield of natural F420 producers. Furthermore, we showed the potential use of F420-producing S. cerevisiae for F420-dependent bioconversions by carrying out the whole-cell conversion of tetracycline. As the first demonstration of F420 synthesis and use for bioconversion in a eukaryotic organism, this study contributes to the development of versatile bioconversion platforms

The Biocatalytic Synthesis of Syringaresinol from 2,6-Dimethoxy-4-allylphenol in One-Pot Using a Tailored Oxidase/Peroxidase System

Syringaresinol was synthesized in a one-pot conversion containing eugenol oxidase (EUGO) and horseradish peroxidase (HRP) using the relatively cheap 2,6-dimethoxy-4-allylphenol as a substrate. This conversion is fully coupled as the hydrogen peroxide generated from the reaction of EUGO with the substrate is utilized by the HRP to convert the formed sinapyl alcohol into syringaresinol. To improve the performance of EUGO on 2,6-dimethoxy-4-allylphenol, structure-inspired enzyme engineering was performed. This yielded the I427A EUGO mutant that is significantly more efficient with 2,6-dimethoxy-4-allylphenol. The I427A EUGO mutant together with HRP were capable of efficiently producing syringaresinol as a major product. After optimization and upscaling the conversion to a semipreparative scale (1 gr), syringaresinol was obtained in 81% yield

Systematic Assessment of Uncoupling in Flavoprotein Oxidases and Monooxygenases

Flavin as a cofactor is an extremely versatile molecule that participates in a wide range of biochemical reactions. A special characteristic of the flavin cofactor, unique for a metal free cofactor, is its ability to react in its reduced form with molecular oxygen. This is exploited in flavoprotein oxidases and monooxygenases. The flavin-mediated reduction of dioxygen can lead to various reactive oxygen species (ROS), primarily hydrogen peroxide and superoxide. No systematic analysis of the formation of such reduced oxygen species produced by flavoprotein oxidases and monooxygenases had been performed before. In this work, we investigated which ROS are formed by several prototype flavoprotein oxidases and monooxygenases: phenylacetone monooxygenase (TfPAMO), an engineered TfPAMO variant that acts as an NADPH oxidase (TfPAMO C65D), eugenol oxidase (RjEUGO), and 5-hydroxymethyl furfural oxidase (MHMFO). The formed amounts of superoxide and hydrogen peroxide were measured under various conditions (different substrate concentrations, pH values and cosolvents). Interestingly, all flavoenzymes were found to produce, except for hydrogen peroxide, significant amounts of superoxide. Moreover, increased superoxide levels were measured at higher pH, which could be indicative for a pH-sensitive caged radical pair dissociation. To probe the effect of ROS formation on biocatalytic performance, conversions catalyzed by TfPAMO or RjEUGO, with or without catalase, were monitored. This revealed that catalase has a beneficial effect. No detrimental effect of the accumulation of superoxide on biocatalysis could be demonstrated. The results reveal that formation of ROS by flavoenzymes is highly dependent on the experimental conditions used. The results provide a better insight into the mechanism by which ROS is formed in flavoenzymes and may help studies or applications in which ROS formation should be promoted or minimized for industrial biocatalysis

Reduction of Carbon−Carbon Double Bonds Using Organocatalytically Generated Diimide

An efficient method has been developed for the reduction of carbon−carbon double bonds with diimide, catalytically generated in situ from hydrazine hydrate. The employed catalyst is prepared in one step from riboflavin (vitamin B2). Reactions are carried out in air and are a valuable alternative when metal-catalyzed hydrogenations are problematic

Mechanistic and Crystallographic Studies of Azoreductase AzoA from <i>Bacillus wakoensis</i> A01

The azoreductase AzoA from the alkali-tolerant Bacillus wakoensis A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates (kcat values of 100–400 s–1 for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases

Structure-Based Enzyme Tailoring of 5‑Hydroxymethylfurfural Oxidase

5-Hydroxymethylfurfural oxidase (HMFO) is a flavin-dependent enzyme that catalyzes the oxidation of many aldehydes, primary alcohols, and thiols. The three-step conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid is relevant for the industrial production of biobased polymers. The remarkable wide substrate scope of HMFO contrasts with the enzyme’s precision in positioning the substrate to perform catalysis. We have solved the crystal structure of HMFO at 1.6 Å resolution, which guided mutagenesis experiments to probe the role of the active-site residues in catalysis. Mutations targeting two active-site residues generated engineered forms of HMFO with promising catalytic features, namely enantioselective activities on secondary alcohols and improved 2,5-furandicarboxylic acid yields

Data_Sheet_1_Mining the Genome of Streptomyces leeuwenhoekii: Two New Type I Baeyer–Villiger Monooxygenases From Atacama Desert.docx

<p>Actinobacteria are an important source of commercial (bio)compounds for the biotechnological and pharmaceutical industry. They have also been successfully exploited in the search of novel biocatalysts. We set out to explore a recently identified actinomycete, Streptomyces leeuwenhoekii C34, isolated from a hyper-arid region, the Atacama desert, for Baeyer–Villiger monooxygenases (BVMOs). Such oxidative enzymes are known for their broad applicability as biocatalysts by being able to perform various chemical reactions with high chemo-, regio-, and/or enantioselectivity. By choosing this specific Actinobacterium, which comes from an extreme environment, the respective enzymes are also expected to display attractive features by tolerating harsh conditions. In this work, we identified two genes in the genome of S. leeuwenhoekii (sle_13190 and sle_62070) that were predicted to encode for Type I BVMOs, the respective flavoproteins share 49% sequence identity. The two genes were cloned, overexpressed in E. coli with phosphite dehydrogenase (PTDH) as fusion partner and successfully purified. Both flavin-containing proteins showed NADPH-dependent Baeyer–Villiger oxidation activity for various ketones and sulfoxidation activity with some sulfides. Gratifyingly, both enzymes were found to be rather robust by displaying a relatively high apparent melting temperature (45°C) and tolerating water-miscible cosolvents. Specifically, Sle_62070 was found to be highly active with cyclic ketones and displayed a high regioselectivity by producing only one lactone from 2-phenylcyclohexanone, and high enantioselectivity by producing only normal (-)-1S,5R and abnormal (-)-1R,5S lactones (ee > 99%) from bicyclo[3.2.0]hept-2-en-6-one. These two newly discovered BVMOs add two new potent biocatalysts to the known collection of BVMOs.</p

A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics

Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated form NADP are crucial cofactors for a large array of biocatalytically important redox enzymes. Their high cost and relatively poor stability, however, make them less attractive electron mediators for industrial processes. Nicotinamide cofactor biomimetics (NCBs) are easily synthesized, are inexpensive, and are also generally more stable than their natural counterparts. A bottleneck for the application of these artificial hydride carriers is the lack of efficient cofactor recycling methods. Therefore, we engineered the thermostable F420:NADPH oxidoreductase from Thermobifida fusca (Tfu-FNO), by structure-inspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor F420H2 was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with Km values in the millimolar range and kcat values ranging from 0.09 to 1.4 min–1. Saturation mutagenesis at positions Gly-29 and Pro-89 resulted in mutants with up to 139 times higher catalytic efficiencies. Mutant G29W showed a kcat value of 4.2 s–1 toward 1-benzyl-3-acetylpyridine (BAP+), which is similar to the kcat value for the natural substrate NADP+. The best Tfu-FNO variants for a specific NCB were then used for the recycling of catalytic amounts of these nicotinamides in conversion experiments with the thermostable ene-reductase from Thermus scotoductus (TsOYE). We were able to fully convert 10 mM ketoisophorone with BAP+ within 16 h, using F420 or its artificial biomimetic FOP (FO-2′-phosphate) as an efficient electron mediator and glucose-6-phosphate as an electron donor. The generated toolbox of thermostable and NCB-dependent Tfu-FNO variants offers powerful cofactor regeneration biocatalysts for the reduction of several artificial nicotinamide biomimetics at both ambient and high temperatures. In fact, to our knowledge, this enzymatic method seems to be the best-performing NCB-recycling system for BNAH and BAPH thus far

Kinetic Mechanism of Phenylacetone Monooxygenase from <i>Thermobifida fusca</i>

Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle (kcat = 3.1 s−1), rapid binding of NADPH (Kd = 0.7 µM) is followed by a transfer of the 4(R)-hydride from NADPH to the FAD cofactor (kred = 12 s−1). The reduced PAMO is rapidly oxygenated by molecular oxygen (kox = 870 mM−1 s−1), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate (k1 = 73 s−1). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s−1) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP+ release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions

Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts

Pollution, accidents, and misinformation have earned the pharmaceutical and chemical industry a poor public reputation, despite their undisputable importance to society. Biotechnological advances hold the promise to enable a future of drastically reduced environmental impact and rigorously more efficient production routes at the same time. This is exemplified in the Baeyer–Villiger reaction, which offers a simple synthetic route to oxidize ketones to esters, but application is hampered by the requirement of hazardous and dangerous reagents. As an attractive alternative, flavin-containing Baeyer–Villiger monooxygenases (BVMOs) have been investigated for their potential as biocatalysts for a long time, and many variants have been characterized. After a general look at the state of biotechnology, we here summarize the literature on biochemical characterizations, mechanistic and structural investigations, as well as enzyme engineering efforts in BVMOs. With a focus on recent developments, we critically outline the advances toward tuning these enzymes suitable for industrial applications