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Oxyfunctionalization of fatty acids (FAs) is a key step in the design of novel synthetic pathways for biobased/biodegradable polymers, surfactants and fuels. Here, we show the isolation and characterization of a robust FA α-hydroxylase (P450Jα ) which catalyses the selective conversion of a broad range of FAs (C6:0-C16:0) and oleic acid (C18:1) with H2 O2 as oxidant. Under optimized reaction conditions P450Jα yields α-hydroxy acids all with >95 % regioselectivity, high specific activity (up to 15.2 U mg-1 ) and efficient coupling of oxidant to product (up to 85 %). Lauric acid (C12:0) turned out to be an excellent substrate with respect to productivity (TON=394 min-1 ). On preparative scale, conversion of C12:0 reached 83 % (0.9 g L-1 ) when supplementing H2 O2 in fed-batch mode. Under similar conditions P450Jα allowed further the first biocatalytic α-hydroxylation of oleic acid (88 % conversion on 100 mL scale) at high selectivity and in good yields (1.1 g L-1 ; 79 % isolated yield). Unexpectedly, P450Jα displayed also 1-alkene formation from shorter chain FAs (≤C10:0) showing that oxidative decarboxylation is more widely distributed across this enzyme family than reported previously.

UDP‐glucuronic acid 4‐epimerase is a short‐chain dehydrogenase/reductase that uses tightly bound NAD+ to catalyze interconversion of UDP‐glucuronic acid and UDP‐galacturonic acid. Here, the authors present a biochemical mechanistic study of the enzyme from Bacillus cereus. Epimerization involves oxidation at the substrate C4 and nonstereoselective reduction in a 4‐keto intermediate. Substrate positioning in a kinetically slow binding step can establish stereo‐electronic constraints to prevent decarboxylation of the intermediate., UDP‐glucuronic acid (UDP‐GlcA) is a central precursor in sugar nucleotide biosynthesis and common substrate for C4‐epimerases and decarboxylases releasing UDP‐galacturonic acid (UDP‐GalA) and UDP‐pentose products, respectively. Despite the different reactions catalyzed, the enzymes are believed to share mechanistic analogy rooted in their joint membership to the short‐chain dehydrogenase/reductase (SDR) protein superfamily: Oxidation at the substrate C4 by enzyme‐bound NAD+ initiates the catalytic pathway. Here, we present mechanistic characterization of the C4‐epimerization of UDP‐GlcA, which in comparison with the corresponding decarboxylation has been largely unexplored. The UDP‐GlcA 4‐epimerase from Bacillus cereus functions as a homodimer and contains one NAD+/subunit (k cat = 0.25 ± 0.01 s−1). The epimerization of UDP‐GlcA proceeds via hydride transfer from and to the substrate’s C4 while retaining the enzyme‐bound cofactor in its oxidized form (≥ 97%) at steady state and without trace of decarboxylation. The k cat for UDP‐GlcA conversion shows a kinetic isotope effect of 2.0 (±0.1) derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient UDP‐4‐keto‐hexose‐uronic acid intermediate whose formation is rate‐limiting overall, and is governed by a conformational step before hydride abstraction from UDP‐GlcA. Precise positioning of the substrate in a kinetically slow binding step may be important for the epimerase to establish stereo‐electronic constraints in which decarboxylation of the labile β‐keto acid species is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the catalytic base for substrate oxidation and show its involvement in the substrate positioning step. Collectively, this study suggests that based on overall mechanistic analogy, stereo‐electronic control may be a distinguishing feature of catalysis by SDR‐type epimerases and decarboxylases active on UDP‐GlcA.

Selective oxy‐functionalization of nonactivated C‐H bonds is a long‐standing “dream reaction” of organic synthesis for which chemical methodology is not well developed. Mono‐oxygenase enzymes are promising catalysts for such oxy‐functionalization to establish. Limitation on their applicability arises from low reaction output. Here, we showed an integrated approach of process engineering to the intensification of the cytochrome P450 BM3‐catalyzed hydroxylation of dodecanoic acid (C12:0). Using P450 BM3 together with glucose dehydrogenase for regeneration of nicotinamide adenine dinucleotide phosphate (NADPH), we compared soluble and co‐immobilized enzymes in O2‐gassed and pH‐controlled conversions at high final substrate concentrations (≥40mM). We identified the main engineering parameters of process output (i.e., O2 supply; mixing correlated with immobilized enzyme stability; foam control correlated with product isolation; substrate solubilization) and succeeded in disentangling their complex interrelationship for systematic process optimization. Running the reaction at O2‐limited conditions at up to 500‐ml scale (10% dimethyl sulfoxide; silicone antifoam), we developed a substrate feeding strategy based on O2 feedback control. Thus, we achieved high reaction rates of 1.86g·L−1·hr−1 and near complete conversion (≥90%) of 80mM (16g/L) C12:0 with good selectivity (≤5% overoxidation). We showed that “uncoupled reaction” of the P450 BM3 (~95% utilization of NADPH and O2 not leading to hydroxylation) with the C12:0 hydroxylated product limited the process efficiency at high product concentration. Hydroxylated product (~7g; ≥92% purity) was recovered from 500ml reaction in 82% yield using ethyl‐acetate extraction. Collectively, these results demonstrate key engineering parameters for the biocatalytic oxy‐functionalization and show their integration into a coherent strategy for process intensification., Cytochrome P450‐catalyzed oxy‐functionalization reactions of fatty acid substrates represent a class of synthetically important bio‐transformations. Here, the authors identified key engineering parameters for the hydroxylation of dodecanoic acid by soluble and immobilized forms of cytochrome P450 BM3 and demonstrated their integration into a coherent strategy for process intensification. Metrics of process performance (as shown in the figure) revealed a highly efficient enzymatic hydroxylation. Reaction scale up to 500 ml enabled production of gram amounts of purified product.

Surface modifications of nanoporous metals have become a highly attractive research field as they exhibit great potential for various applications, especially in biotechnology. Using self-assembled monolayers is one of the most promising approaches to modify a gold surface. However, only few techniques are capable of characterizing the formation of these monolayers on porous substrates. Here, we present a method to in situ monitor the adsorption and desorption of self-assembled monolayers on nanoporous gold by resistometry, using cysteine as example. During the adsorption an overall relative change in resistance of 18% is detected, which occurs in three distinct stages. First, the cysteine molecules are adsorbed on the outer surface. In the second stage, they are adsorbed on the internal surfaces and in the last stage the reordering accompanied by additional adsorption takes place. The successful binding of cysteine on the Au surface was confirmed by cyclic voltammetry, which showed a significant decrease of the double-layer capacitance. Also, the electrochemically controlled desorption of cysteine was monitored by concomitant in situ resistometry. From the desorption peak related to the (111) surface of the structure, which is associated with a resistance change of 4.8%, an initial surface coverage of 0.48 monolayers of cysteine could be estimated.

D-Apiose is a C-branched pentose sugar important for plant cell wall development. Its biosynthesis as UDP-D-apiose involves decarboxylation of the UDP-D-glucuronic acid precursor coupled to pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalyzed within a single active site by UDP-D-apiose/UDP-D-xylose synthase (UAXS). Here, we decipher the UAXS catalytic mechanism based on crystal structures of the enzyme from Arabidopsis thaliana, molecular dynamics simulations expanded by QM/MM calculations, and mutational-mechanistic analyses. Our studies show how UAXS uniquely integrates a classical catalytic cycle of oxidation and reduction by a tightly bound nicotinamide coenzyme with retro-aldol/aldol chemistry for the sugar ring contraction. They further demonstrate that decarboxylation occurs only after the sugar ring opening and identify the thiol group of Cys100 in steering the sugar skeleton rearrangement by proton transfer to and from the C3'. The mechanistic features of UAXS highlight the evolutionary expansion of the basic catalytic apparatus of short-chain dehydrogenases/reductases for functional versatility in sugar biosynthesis.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in BIOTEC, 322C (2020) 74-78, https://doi.org/10.1016/j.jbiotec.2020.06.023. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Sugar nucleotide-dependent (Leloir) glycosyltransferases are powerful catalysts for glycoside synthesis. Their applicability can be limited due to elaborate production of enzyme preparations deployable in biocatalytic processes. Here, we show that efficient enzyme formulation promotes glycosyltransferases for the synthesis of the natural C-glycoside nothofagin. Adding Brij-35 detergent (1 %, w/v) during sonication of the E. coli BL21-Gold (DE3) expression strain, recovery of Oryza sativa C-glycosyltransferase was enhanced by ∼3-fold, partly due to the release of enzyme activity trapped in insoluble pellet. Freeze drying of the resulting cell-free extract (∼17 U ml-1) reduced the volume ∼20-fold and gave ∼55 mg solids ml-1 liquid processed, with 83 % retention of the original activity and a specific activity of 0.20 U mg-1 solids. The Glycine max sucrose synthase was processed analogously, giving a solid enzyme preparation of 0.28 U mg-1 in 63 % yield. Both enzyme formulations were stable for several weeks. The glycosyltransferase cascade reaction for 3'-β-C-glucosylation of phloretin (60 mM; as inclusion complex with hydroxypropyl-β-cyclodextrin) from UDP-glucose (generated in situ by sucrose synthase from 500 mM sucrose and 0.5 mM UDP) showed excellent performance metrics (≥ 98 % yield; 3.2 g l-1 h-1 space-time yield; ∼90 regeneration cycles for UDP). Collectively, our study demonstrates a facile procedure for solid glycosyltransferase formulations practically usable in glycoside synthesis.

Cytochrome P450 monooxygenases (P450s) promote hydroxylations in a broad variety of substrates. Their prowess in C–H bond functionalization renders P450s promising catalysts for organic synthesis. However, operating P450 reactions involve complex management of the main substrates, O2 and nicotinamide adenine dinucleotide phosphate (NAD(P)H) reducing equivalents against an overall background of low operational stability. Whole‐cell biocatalysis, although often used, offers no general solution to these problems. Herein, we present the design of a tailor‐made, self‐sufficient, operationally stabilized and recyclable P450 catalyst on porous solid support. Using enzymes as fusion proteins with the polycationic binding module Zbasic2, the P450s BM3 (from Bacillus megaterium) was coimmobilized with glucose dehydrogenase (type IV; from B. megaterium) on anionic sulfopropyl‐activated carrier (ReliSorb SP). Immobilization via Zbasic2 enabled each enzyme to be loaded in controllable amount, thus maximizing the relative portion of the rate limiting P450 BM3 (up to 19.5 U/gcarrier) in total enzyme immobilized. Using lauric acid as a representative P450 substrate that is poorly accessible to whole‐cell catalysts, we demonstrate complete hydroxylation at low catalyst loading (≤0.1 mol%) and efficient electron coupling (74%), inside of the catalyst particle, to the regeneration of NADPH from glucose (27 cycles) was achieved. The immobilized P450 BM3 showed a total turnover number of ∼18,000, thus allowing active catalyst to be recycled up to 20 times. This study therefore supports the idea of practical heterogeneous catalysis by P450s systems immobilized on solid support., We report the design of a self‐sufficient, recyclable monooxygenase catalyst based on cytochrome P450 BM3 and glucose dehydrogenase coimmobilized on solid support. Fusion with the binding module Zbasic2 enabled convenient coimmobilization of the two enzymes in controllable activity ratio. Using lauric acid a substrate, we demonstrate robust and efficient hydroxylation combined with NADPH regeneration and catalyst reuse.

A three-step one-pot biocatalytic cascade was designed for the enantioselective formal α-amination of hexanoic acid to l-norleucine. Regioselective hydroxylation by P450CLA peroxygenase to 2-hydroxyhexanoic acid was followed by oxidation to the ketoacid by two stereocomplementary dehydrogenases. Combination with final stereoselective reductive amination by amino acid dehydrogenase furnished l-norleucine in >97% ee.

The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on large scale. As proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho-position using pressurized CO2 (~30-40 bar) catalyzed by ortho-benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants.

Oxyfunctionalization of fatty acids (FAs) is a key step in the design of novel synthetic pathways for biobased/biodegradable polymers, surfactants and fuels. Here, we show the isolation and characterization of a robust FA α-hydroxylase (P450

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Biocatalysis with cytochrome P450 enzymes are important for the industrial production of fine chemicals, pharmaceuticals, fragrance and flavor compounds since chemoselective hydroxylation of aromatics and terpenes are chemically difficult to achieve. A few P450 based industrial processes have been developed based on whole cell catalysis. However, the outer membrane of microbial cells forms an effective barrier, which reduces the uptake of hydrophobic substrates. The coexpression of outer membrane proteins in E. coli such as the ferric hydroxamate uptake protein (FhuA) can provide alternative solutions to chemical or physical methods for increasing compound flux through the outer membrane of E. coli and thereby to boost productivities. In this study we employed an engineered FhuA Δ1-160 variant in which the “cork domain” was removed (first 160 residues are deleted); FhuA Δ1-160 has a cross-section of 39–46 A with a “free” inner diameter of about 14 A that serves as passive diffusion channel. FhuA WT and Δ1-160 were coexpressed on a bicistronic system with two P450 BM3 variants for regiospecific hydroxylation of aromatic compounds toluene and anisole as well as for oxidation of two terpenes (α)-pinene and ( R )-(+)-limonene. The presence of FhuA Δ1‐160 resulted in a doubled product concentration for toluene (35 μ to 50 μM), anisole (25 μM to 45 μM), pinene (12 μM to 20 μM) and limonene (12 μM to 25 μM) and five times higher for the coumarin derivative BCCE. In order to characterizes and compensate for expression variations a quantification method based on Chromeo546-labled StrepTactinII was developed to quantify the number of FhuA Δ1-160 in the outer E. coli membrane (∼44000 of FhuA Δ1-160 per cell). Morphology studies showed that a 6% E. coli surface coverage can be achieved with FhuA Δ1‐160 without significantly influencing the E. coli rod shape. In summary, FhuA Δ1-160 efficiently increases uptake of hydrophobic aromatics and terpenes for whole-cell biotransformations and can likely be used for other enzymes and/or substrates.

The biocatalytic oxidative tandem decarboxylation of C7–C18 dicarboxylic acids to terminal C5–C16 dienes was catalyzed by the P450 monooxygenase OleT with conversions up to 29 % for 1,11-dodecadiene (0.49 g L–1). The sequential nature of the cascade was proven by the fact that decarboxylation of intermediate C6–C11 ω-alkenoic acids and heptanedioic acid exclusively gave nonconjugated 1,4-pentadiene; scale-up allowed the isolation of 1,15-hexadecadiene and 1,11-dodecadiene; the system represents a short and green route to terminal dienes from renewable dicarboxylic acids.

Utilization of gaseous carbon dioxide as a C1-building block in the biocatalytic ortho-carboxylation of a phenol., The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on a large scale. As a proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho-position using pressurized CO2 (∼30–40 bar) catalyzed by ortho-benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants.

l-Tyrosine derivatives were obtained in >97% ee via a biocatalytic one-pot two-step cascade using substituted benzenes, pyruvate, and NH3 as starting materials. In the first step, monosubstituted arenes were regioselectively hydroxylated in the o-position by monooxygenase P450 BM3 (using O2 as oxidant with NADPH-recycling) to yield the corresponding phenols, which subsequently underwent C–C coupling and simultaneous asymmetric amination with pyruvate and NH3 using tyrosine phenol lyase to furnish l-DOPA surrogates in up to 5.2 g L–1. Instead of analytically pure arenes, crude aromatic gasoline blends containing toluene were used to yield 3-methyl-l-tyrosine in excellent yield (2 g L–1) and >97% ee.

Die enzymatische oxidative Decarboxylierung von linearen kurzkettigen Fettsauren (C4:0–C9:0) unter Verwendung der P450-Monooxygenase OleT, O2 und NAD(P)H als Elektronendonor ermoglicht die Synthese terminaler C3- bis C8-Alkene mit Produkttitern von bis zu 0.93 g L−1 und TTNs >2000. Essentiell fur diesen Prozess war die Entwicklung einer effizienten Elektronentransferkette unter Verwendung von Putidaredoxin (CamAB) und NAD(P)H-Regeneration basierend auf Glukose, Ameisensaure oder Phosphonat. Mithilfe dieses Reaktionssystems lassen sich auf biokatalytischem Weg industriell wichtige Alkene, wie z. B. Propen und 1-Octen zum ersten Mal vollstandig aus nachwachsenden Rohstoffen herstellen.

1-Alkenes represent primary building blocks for the chemical industry. Here we show the development of a novel biosynthetic redox-cascade involving the P450 monooxygenase OleT and electron transfer proteins CamA and CamB to allow the biocatalytic synthesis of short chain alkenes such as propene or 1,4-pentadiene from fatty acids. The cascade is fully based on renewables and can be operated under mild reaction conditions as opposed to industrial alkene synthesis.

The functionalization of bio‐based chemicals is essential to allow valorization of natural carbon sources. An atom‐efficient biocatalytic oxidative cascade was developed for the conversion of saturated fatty acids to α‐ketoacids. Employment of P450 monooxygenase in the peroxygenase mode for regioselective α‐hydroxylation of fatty acids combined with enantioselective oxidation by α‐hydroxyacid oxidase(s) resulted in internal recycling of the oxidant H2O2, thus minimizing degradation of ketoacid product and maximizing biocatalyst lifetime. The O2‐dependent cascade relies on catalytic amounts of H2O2 and releases water as sole by‐product. Octanoic acid was converted under mild conditions in aqueous buffer to 2‐oxooctanoic acid in a simultaneous one‐pot two‐step cascade in up to >99 % conversion without accumulation of hydroxyacid intermediate. Scale‐up allowed isolation of final product in 91 % yield and the cascade was applied to fatty acids of various chain lengths (C6:0 to C10:0).

One of the most important obstacles to overcome in biocatalysis with monooxygenases is the enzyme's dependency on the costly redox cofactor NAD(P)H. Electrochemical regeneration systems, in which an electrode serves as electron donor, provide an alternative route to enzymatic redox reactions. Mediators are often used to accelerate electron transfer between electrode and enzyme. We investigated the mediated bioelectrochemical conversion of p-xylene to 2,5-dimethylphenol (2,5-DMP) by a P450 BM3 variant and were able to produce 2,5-DMP electrochemically. Due to the fact that mediator reduction is limited by the electrode surface a scale-up was performed. However, increasing the electrode surface area to reactor volume ratio led to a drastic increase in cathodic oxygen reduction, causing a drop in product formation. It was shown that reduced cobalt sepulchrate reacts with the co-substrate oxygen. Furthermore, the reportedly oxygen stable mediator [Cp*Rh(I)(bpy)H]+ was compared to cobalt sepulchrate. While its turnover frequency is of comparable magnitude to cobalt sepulchrate when transferring the electrons between electrode and enzyme, using NADP+ as intermediary between the mediator and the enzyme significantly increased the mediator's turnover frequency. The rhodium mediator [Cp*Rh(I)(bpy)H]+ does not appear to be significantly more oxygen stable.

Baeyer-Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer-Villiger monooxygenases with new specificities.

Directed evolution of Yersinia mollaretii phytase (Ymphytase) yielded an improved variant SM2P3E4 (also named M1; D52N, T77K, K139E, G187S, V298M) in our previous study. Variant M1 retained high specific activity (993U/mg; equivalent to 93% of wild-type activity) and improved thermal resistance (T50 improved by 1.5°C compared to wild-type at 58°C; 20min incubation time), making variant M1 an attractive enzyme for industrial applications. Recently, the OmniChange method was developed for multi-site saturation mutagenesis. The five sites identified in variant M1 were subjected to OmniChange saturation in order to explore whether a variant with higher activity, higher thermal resistance, and higher resistance at low pH (2-3h incubation was performed to mimic the gastric residence time of phytase) could be identified. Screening of a small library of 1100 clones, covering

A novel whole cell cascade for double oxidation of cyclooctane to cyclooctanone was developed. The one-pot oxidation cascade requires only a minimum of reaction components: resting E. coli cells in aqueous buffered medium (=catalyst), the target substrate and oxygen as environmental friendly oxidant. Conversion of cyclooctane was catalysed with high efficiency (50% yield) and excellent selectivity (>94%) to cyclooctanone. The reported oxidation cascade represents a novel whole cell system for double oxidation of non-activated alkanes including an integrated cofactor regeneration. Notably, two alcohol dehydrogenases from Lactobacillus brevis and from Rhodococcus erythropolis with opposite cofactor selectivities and one monooxygenase P450 BM3 were produced in a coexpression system in one single host. The system represents the most efficient route with a TTN of up to 24363 being a promising process in terms of sustainability as well.

Flow cytometry-based screening systems have successfully been used in directed evolution experiments. Herein, we report the first whole-cell, high-throughput screening platform for P450 monooxygenases based on flow cytometry. O-dealkylation of 7-benzoxy-3-carboxycoumarin ethyl ester (BCCE) by P450 BM3 generates a fluorescence coumarin derivative. After one round of directed evolution, P450 BM3 variants with up to 7-fold increased activity (P450 M3 DM-1: R255H) could be identified at a sampling rate of 500 events s–1. The reported screening platform can likely be applied to directed evolution campaigns of any P450 monooxygenase that catalyzes the O-dealkylation of BCCE.

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is ∼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a phytase variant with ∼20% improved thermostability (58°C for 20 min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).

Simultaneous multi site-saturation mutagenesis enables to reshape binding pockets especially when cooperative amino acids are targeted, which affect activity and/or selectivity of enzymes. Simultaneous saturation of five positions with OmniChange generates up to 3.2 million different variants in one experiment in a robust and technically simple protocol. OmniChange generates a diversity, which is challenging to be screened by current screening technologies.

The enzymatic oxidative decarboxylation of linear short-chain fatty acids (C4:0-C9:0) employing the P450 monooxygenase OleT, O2 as the oxidant, and NAD(P)H as the electron donor gave the corresponding terminal C3 to C8 alkenes with product titers of up to 0.93 g L(-1) and TTNs of2000. Key to this process was the construction of an efficient electron-transfer chain employing putidaredoxin CamAB in combination with NAD(P)H recycling at the expense of glucose, formate, or phosphite. This system allows for the biocatalytic production of industrially important 1-alkenes, such as propene and 1-octene, from renewable resources for the first time.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method developed to overcome the limitations of existing error-prone PCR (epPCR) protocols. SeSaM is advantageous with respect to (1) elimination of mutagenic "hot spots", (2) increase in frequency of subsequent nucleotide substitutions, (3) control over the mutational bias through the utilization of universal base analogs, and, consequently, (4) the prospect of generating transversion-enriched mutant libraries. These advanced features lead to chemically diverse mutant libraries on the protein level, essentially making SeSaM a complementary technology to transition biased epPCR mutagenesis methods.

Multi-site-saturation mutagenesis allows altering of "localizable" properties such as activity and selectivity and enables the discovery of cooperative amino acid substitutions which are unlikely to be discovered by saturating single codons individually or iteratively. The herein presented method "OmniChange" does not require any DNA modifying enzyme (e.g., endonucleases or ligases), and diverse mutant libraries with up to five simultaneously saturated positions are generated in a robust and technically simple manner in four steps. The key feature of the OmniChange method is a highly efficient chemical cleavage of phosphorothiolated nucleotides by ethanol-iodine to generate 12-nucleotide-long 5' overhangs in double-stranded DNA. The generated vector and inserts can be hybridized in a one-pot assembly leading to fully functional mutagenic plasmids, and the employed E. coli host can easily ligate up to 10 DNA nicks without any further enzymatic treatment. OmniChange is furthermore a reliable and general tool for multi-DNA fragment assembly which is DNA sequence independent.

Biocascades allow one-pot synthesis of chemical building blocks omitting purification of reaction intermediates and expenses for downstream processing. Here we show the first whole cell double oxidation of n-heptane to produce chiral alcohols and heptanones. The concept of an artificial operon for co-expression of a monooxygenase from Bacillus megaterium (P450 BM3) and an alcohol dehydrogenase (RE-ADH) from Rhodococcus erythropolis is reported and compared to the widely used two-plasmid or Duet-vector expression systems. Both catalysts are co-expressed on a polycistronic constructs (single mRNA) that reduces recombinant DNA content and metabolic burden for the host cell, therefore increasing growth rate and expression level. Using the artificial operon system, the expression of P450 BM3 reached 81mgg(-1) cell dry weight. In addition, in situ cofactor regeneration through the P450 BM3/RE-ADH couple was enhanced by coupling to glucose oxidation by E. coli. Under optimized reaction conditions the artificial operon system displayed a product formation of 656mgL(-1) (5.7mM) of reaction products (heptanols+heptanones), which is 3-fold higher than the previously reported values for an in vitro oxidation cascade. In conjunction with the high product concentrations it was possible to obtain ee values of >99% for (S)-3-heptanol. Coexpression of a third alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) in the same host yielded complete oxidation of all heptanol isomers. Introduction of a second ADH enabled further to utilize both cofactors in the host cell (NADH and NADPH) which illustrates the simplicity and modular character of the whole cell oxidation concept employing an artificial operon system.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method developed to overcome the limitations of existing error-prone PCR (epPCR) protocols. SeSaM is advantageous with respect to (1) elimination of mutagenic "hot spots", (2) increase in frequency of subsequent nucleotide substitutions, (3) control over the mutational bias through the utilization of universal base analogs, and, consequently, (4) the prospect of generating transversion-enriched mutant libraries. These advanced features lead to chemically diverse mutant libraries on the protein level, essentially making SeSaM a complementary technology to transition biased epPCR mutagenesis methods.

Multi-site-saturation mutagenesis allows altering of "localizable" properties such as activity and selectivity and enables the discovery of cooperative amino acid substitutions which are unlikely to be discovered by saturating single codons individually or iteratively. The herein presented method "OmniChange" does not require any DNA modifying enzyme (e.g., endonucleases or ligases), and diverse mutant libraries with up to five simultaneously saturated positions are generated in a robust and technically simple manner in four steps. The key feature of the OmniChange method is a highly efficient chemical cleavage of phosphorothiolated nucleotides by ethanol-iodine to generate 12-nucleotide-long 5' overhangs in double-stranded DNA. The generated vector and inserts can be hybridized in a one-pot assembly leading to fully functional mutagenic plasmids, and the employed E. coli host can easily ligate up to 10 DNA nicks without any further enzymatic treatment. OmniChange is furthermore a reliable and general tool for multi-DNA fragment assembly which is DNA sequence independent.

Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes in many fields of science and industry. Advances in screening formats and screening systems are fueling progress and enabling novel directed evolution strategies, despite the fact that the quality of mutant libraries can still be improved significantly. Diversity generation strategies in directed enzyme evolution comprise three options: (a) focused mutagenesis (selected residues are randomized); (b) random mutagenesis (mutations are randomly introduced over the whole gene); and (c) gene recombination (stretches of genes are mixed to chimeras in a random or rational manner). Either format has both advantages and limitations depending on the targeted enzyme and property. The quality of diverse mutant libraries plays a key role in finding improved mutants. In this review, we summarize methodological advancements and novel concepts (since 2009) in diversity generation for all three formats. Advancements are discussed with respect to the state of the art in diversity generation and high-throughput screening capabilities, as well as robustness and simplicity in use. Furthermore, limitations and remaining challenges are emphasized 'to get what we aim for' through 'optimal diversity' generation.

Rational guided generation of protein chimeras has developed into an attractive approach in protein engineering for tailoring catalytic and biophysical properties of enzymes. Combinatorial recombination of structural elements or whole protein domains is still technically challenging due to sequence dependent biases diminishing the overall quality of resulting chimeric libraries. Since methods for generating such libraries are often limited by a low frequency of crossover points and suffer from challenges in handling, we developed the phosphorothioate-based DNA recombination method (PTRec). PTRec is an enzyme-free method and only requires a short stretch of four amino acids that is identical among the proteins to be recombined in order to define a single crossover point. In a PTRec-generated chimeric library that shuffled five domains of phytase using genes from three different species, 88% of 42 randomly picked and sequenced genes were efficiently recombined. Furthermore, PTRec is a technically simple, fast, and reliable method that can be used for domain-and exon-shuffling or recombination of DNA fragments in general.

Multiphoton microscopy is a promising technique to detect spatially and temporally resolved concentration gradients of chemical compounds, e.g., reactants in hydrogel-encapsulated biocatalysts. In contrast to current techniques, the improved spatial and temporal resolution of this method in data acquisition and its ability to measure hydrogel beads facilitates the identification of various kinetic phenomena. To our knowledge, multiphoton microscopy is used here for the first time to examine diffusion, mass transfer, and reaction in immobilized hydrogel systems. In a first step, the phenomena of diffusion and diffusion-coupled mass transfer through the phase interface are investigated in the bead center. Finally, the complete system—consisting of diffusion, mass transfer, and enzymatic reaction—is observed by measuring concentration gradients along the bead radius with temporal and spatial resolution. This metrology enables a subsequent mechanistic model identification, which in turn leads to an enhanced knowledge of reaction kinetics and supports the design of biotechnological processes. This task was only possible due to excellent spatial (25 μm) and temporal (5 s) resolution and the accuracy (±1%) achieved by using a multiphoton microscopy setup.