New types of asymmetric functionalizations of alkenes are highly desirable for chemical synthesis. Here, we develop three novel types of regio- and enantioselective multiple oxy- and amino-functionalizations of terminal alkenes via cascade biocatalysis to produce chiral α-hydroxy acids, 1,2-amino alcohols and α-amino acids, respectively. Basic enzyme modules 1–4 are developed to convert alkenes to (S)-1,2-diols, (S)-1,2-diols to (S)-α-hydroxyacids, (S)-1,2-diols to (S)-aminoalcohols and (S)-α-hydroxyacids to (S)-α-aminoacids, respectively. Engineering of enzyme modules 1 & 2, 1 & 3 and 1, 2 & 4 in Escherichia coli affords three biocatalysts over-expressing 4–8 enzymes for one-pot conversion of styrenes to the corresponding (S)-α-hydroxyacids, (S)-aminoalcohols and (S)-α-aminoacids in high e.e. and high yields, respectively. The new types of asymmetric alkene functionalizations provide green, safe and useful alternatives to the chemical syntheses of these compounds. The modular approach for engineering multi-step cascade biocatalysis is useful for developing other new types of one-pot biotransformations for chemical synthesis.

Green and selective oxidation methodsa re highly desired in chemical synthesisa nd manufacturing. In this work, we have developedabiocatalytic method for the regio-and stereoselective oxidation of styrene derivatives into arylacetic and( S)-2-arylpropionic acids via ao ne-pot epoxidation-isomerization-oxidation sequence.T his was done via the engineering of Escherichia coli (StyABC-EcALDH)c oexpressing styrene monooxygenase (SMO), styrene oxide isomerase (SOI) and aldehyde dehydrogenase (EcALDH)a sa na ctive and easily available wholecell catalyst. Regioselective oxidation of styrene and 11 substituted styrenes using the E. coli cells wasperformed in ao ne-pot set-up,p roducing 12 phenylacetic acids in bothh igh conversion and high yield. Engineering of E. coli (StyABC-ADH9v1) coexpressing SMO,S OI andA DH9v1 (a mutated alcohol dehydrogenase) led to biocatalystsc apable of regio-and stereoselective oxidation of a-methylstyrene derivatives to the corresponding chiral acids.O ne-pot asymmetric synthesis of 4( S)-2-arylpropionic acids was achieved in good conversion and excellent ee with the E. coli cells.T his is an ew type of asymmetric alkene oxidation to give chiral acids with no chemical counterpart thus far. Thec ascade bio-oxidation operates under mild conditions,u ses molecular oxygen, exhibitsv ery high regio-and enantioselectivity,a nd gives high conversion, thus providing ag reena nd efficientm ethod for the synthesiso fa rylacetic acids and (S)-2-arylpropionic acids directly from easily available styrenes.

Sustainable synthesis of useful and valuable chiral fine chemicals from renewable feedstocks is highly desirable but remains challenging. Reported herein is a designed and engineered set of unique non-natural biocatalytic cascades to achieve the asymmetric synthesis of chiral epoxide, diols, hydroxy acid, and amino acid in high yield and with excellent ee values from the easily available biobased l-phenylalanine. Each of the cascades was efficiently performed in one pot by using the cells of a single recombinant strain over-expressing 4-10 different enzymes. The cascade biocatalysis approach is promising for upgrading biobased bulk chemicals to high-value chiral chemicals. In addition, combining the non-natural enzyme cascades with the natural metabolic pathway of the host strain enabled the fermentative production of the chiral fine chemicals from glucose.

Engineered enzyme cascades offer powerful tools to convert renewable resources into valueadded products. Man-made catalysts give access to new-to-nature reactivities that may complement the enzyme's repertoire. Their mutual incompatibility, however, challenges their integration into concurrent chemo-enzymatic cascades. Herein we show that compartmentalization of complex enzyme cascades within E. coli whole cells enables the simultaneous use of a metathesis catalyst, thus allowing the sustainable one-pot production of cycloalkenes from oleic acid. Cycloheptene is produced from oleic acid via a concurrent enzymatic oxidative decarboxylation and ring-closing metathesis. Cyclohexene and cyclopentene are produced from oleic acid via either a six-or eight-step enzyme cascade involving hydration, oxidation, hydrolysis and decarboxylation, followed by ring-closing metathesis. Integration of an upstream hydrolase enables the usage of olive oil as the substrate for the production of cycloalkenes. This work highlights the potential of integrating organometallic catalysis with whole-cell enzyme cascades of high complexity to enable sustainable chemistry.

This study investigated the human alveolar osteoblasts (AOs) proliferation and extracellular matrix formation at seeding density of 0.05, 0.1, 0.2, 0.4, and 0.8 million (M) per 3x4x4 mm3 on medical grade polycaprolactone-tricalcium phosphate (mPCL-TCP) scaffolds designed for bone regeneration. Over 80-90% of the initial seeded cells were retained in the scaffolds after 24 h. AOs bridged over pores at density of 0.2M/scaffold and below, but formed cell balls at density of 0.4M/scaffold and above. At seeding density of 0.2M and below, cell proliferation increased with time having DNA content peaked to 1600 ng/scaffold at day 21 and 28, respectively, whereas at 0.4 and 0.8M, the corresponding DNA content decreased to 1600 ng in 28 days. At day 7, higher alkaline phosphatase (ALP) activity and higher osteocalcin (OCN) secretion were detected at 0.2M/scaffold and below. After 28 days, multilayered cell-sheet formation and collagen fibers were observed at all densities. ALP and OCN in matrix and mineral nodules were found mainly at the border of AOs-scaffold construct. These findings demonstrated that the density of 0.2M and below per 3 x 4 x 4 mm(3) scaffold resulted in better cell proliferation and extracellular matrix synthesis, potentially resulting in better mineralized tissue formation.

The biotin–streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17 000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.

Discussions about the nature of the charge carriers in the scandium tungstate and other isostructural tungstates and molybdates have persisted in the literature since a variety of experimental indications pointed toward trivalent cations as the mobile species. Here variations of the structure over a wide temperature range are analyzed by XRD and computational methods, demonstrating that the negative thermal expansion persists throughout the range of 11-1300 K. Over a limited temperature range (<500 K) molecular dynamics simulations with an optimized forcefield reproduce this negative thermal expansion. Likewise, charge transport is monitored both experimentally by impedance spectroscopy and Tubandt experiments and computationally based on the molecular dynamics simulation trajectories. Extended isothermal-isobaric simulations suggest a complex migration of polyatomic tungstate anions as the energetically most favorable transport mechanism in Sc 2 (WO 4 ) 3 . A bond valence analysis depicts possible diffusion pathways for WO 4 2-, although there is no indication of a pathway for Sc 3+ . The hopping mechanism of tungstate ions from one equilibrium site to another one follows the instantaneous diffusion pathways. A long-range transport still requires the rare formation of WO 4 2-Frenkel defects limiting the accuracy of the simulated absolute conductivity. Both MD simulations and bond valence analysis suggest WO 42be the mobile species, which follow the interstitialcy diffusion mechanism. Our 3-section Tubandttype experiments qualitatively show that the transfer of W occurs in the form of a negatively charged complex. This should be the first example of polyatomic diffusion species and opens a new field in the search for new ionic conductors.

Enantiopure d-phenylglycine and its derivatives are an importantg roup of chiral amino acids with broad applications in thepharmaceutical industry.H owever, the existing synthetic methods for dphenylglycine mainly rely on toxic cyanide chemistry and multistep processes.T op rovide green and safe alternatives,w ee nvisaged cascade biocatalysis for the one-pot synthesis of d-phenylglycine from racemic mandelic acid, styrene,a nd biobased l-phenylalanine,r espectively.R ecombinant Escherichia coli (LZ110) was engineered to coexpress four enzymes to catalyze a3 -step reactioni no ne pot, transforming mandelic acid (210 mM) to give enantiopure d-phenylglycine in 29.5 gL À1 (195 mM) with 93% conversion. Using the same whole-cell catalyst, twelve other d-phenylglycine derivatives were also produced from the corresponding mandelic acid derivatives in high conversion (58-94%) andv ery high ee (93-99%). E. coli (LZ116) expressing seven enzymes was constructed for the transformation of styrene to enantiopure d-phenylglycine in 80% conversion via ao ne-pot 6-step cascade biotransformation. Tw elve substituted d-phenylglycines were also produced from the corresponding styrene derivatives in high conversion (45-90%) and very high ee (92-99%) via the same cascade reactions.An ine-enzymeexpressing E. coli (LZ143) was engineered to transform biobased l-phenylalanine to enantiopure d-phenylglycine in 83% conversion via ao ne-pot 8-step transformation. Preparative biotransformations were also demonstrated. Theh igh-yielding synthetic methods use cheap and green reagents (ammonia, glucose, and/or oxygen), and E. coli whole-cell catalysts,t hus providing green and useful alternative methodsf or manufacturing d-phenylglycine.