Multi-Enzymatic Cascades In Vitro

The combination of enzymatic reactions in a simultaneous or sequential fashion by designing artificial synthetic cascades allows for the synthesis of complex compounds from simple precursors. Such multi-catalytic cascade reactions not only bear a great potential to minimize downstream processing steps but can also lead to a drastic reduction of the produced waste. With the growing toolbox of biocatalysts, alternative routes employing enzymatic transformations towards manifold and diverse target molecules become accessible. In vitro cascade reactions open up new possibilities for efficient regeneration of the required cofactors such as nicotinamide cofactors or nucleoside triphosphates. They are represented by a vast array of two-enzyme cascades that have been designed by coupling the activity of a cofactor regenerating enzyme to the product generating enzyme. However, the implementation of cascade reactions requires careful consideration, particularly with respect to whether the pathway is constructed concurrently or sequentially. In this regard, this chapter describes how biocatalytic cascades are classified, and how such cascade reactions can be employed in order to solve synthetic problems. Recent developments in the area of dynamic kinetic resolution or cofactor regeneration and showcases are presented. We also highlight the factors that influence the design and implementation of purely enzymatic cascades in one-pot or multi-step pathways in an industrial setting

Enzyme engineering of a membrane-bound monooxygenase as key step of an artificial metabolic pathway towards Tulipalin A

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The Synthesis of Chiral Gamma-Lactones by Merging Decatungstate Photocatalysis with Biocatalysis

The implementation of light-driven catalytic processes in biocatalysis opens a golden window of opportunities. We hereby report the merging of photocatalytic C-C bond formation with enzymatic asymmetric reduction for the direct conversion of simple aldehydes and acrylates or unsaturated carboxylic acids into chiral ?-lactones. Tetrabutylammonium decatungstate (TBADT) is employed as the photocatalyst to trigger the hydroacylation of the starting olefins, yielding the corresponding keto esters/acids. Subsequently, an alcohol dehydrogenase converts the intermediate to the chiral alcohol, which undergoes lactonization to the desired ?-lactone. The photochemoenzymatic synthesis of aliphatic and aromatic ?-lactones was thereby achieved with up to >99 % ee  and >99 % yield. This synthesis highlights the power of building molecular complexity by merging photocatalysis with biocatalysis to access high-value added chiral compounds from simple, cheap and largely available starting materials

Исследование методов и реализация алгоритмов выявления скрытых закономерностей параметров при определении типа сахарного диабета на основе машинного обучения

Работа направлена на выявление скрытых закономерностей параметров при определении типа сахарного диабета на основе статистических методов. Полученные результаты будут положены в основу определения характеристических признаков (фичи) искусственных нейронных сетей. Планируется проведение сравнительного анализа результатов классификации, полученных на основе алгоритмов машинного обучения.The work is aimed at identifying hidden patterns of parameters in determining the type of diabetes mellitus based on statistical methods. The obtained results will be used as a basis for determining the characteristics (features) of artificial neural networks. It is planned to conduct a comparative analysis of the classification results obtained on the basis of machine learning algorithms

Internal Illumination to Overcome the Cell Density Limitation in the Scale-up of Whole-Cell Photobiocatalysis

Cyanobacteria have the capacity to use photosynthesis to fuel their metabolism, which makes them highly promising production systems for the sustainable production of chemicals. Yet, their dependency on visible light limits the cell‐density, which is a challenge for the scale‐up. Here, it was shown with the example of a light‐dependent biotransformation that internal illumination in a bubble column reactor equipped with wireless light emitters (WLEs) could overcome this limitation. Cells of the cyanobacterium Synechocystis sp. PCC 6803 expressing the gene of the ene‐reductase YqjM were used for the reduction of 2‐methylmaleimide to (R)‐2‐methylsuccinimide with high optical purity (>99 % ee). Compared to external source of light, illumination by floating wireless light emitters allowed a more than two‐fold rate increase. Under optimized conditions, product formation rates up to 3.7 mm h(−1) and specific activities of up to 65.5 U g(DCW) (−1) were obtained, allowing the reduction of 40 mm 2‐methylmaleimide with 650 mg isolated enantiopure product (73 % yield). The results demonstrate the principle of internal illumination as a means to overcome the intrinsic cell density limitation of cyanobacterial biotransformations, obtaining high reaction rates in a scalable photobioreactor

Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol’s enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.This research was funded by PROYECTO INTERDISCIPLINA-VRI-UC-II160020, number 3514-913, Pontificia Universidad Católica de Chile (to L.P.P. and A.S.) and BECA DE DOCTORADO NACIONAL 2016, number 21161084, National Agency for Research and Development (ANID), Chile (to D.P.H.), for which we are grateful. The APC was funded by Pontificia Universidad Católica de Chile

Photosynthetically produced sucrose by immobilized Synechocystis sp. PCC 6803 drives biotransformation in E. coli

Background: Whole-cell biotransformation is a promising emerging technology for the production of chemicals. When using heterotrophic organisms such as E. coli and yeast as biocatalysts, the dependence on organic carbon source impairs the sustainability and economic viability of the process. As a promising alternative, photosynthetic cyanobacteria with low nutrient requirements and versatile metabolism, could ofer a sustainable platform for the heterologous production of organic compounds directly from sunlight and CO2. This strategy has been applied for the photoautotrophic production of sucrose by a genetically engineered cyanobacterium, Synechocystis sp. PCC 6803 strain S02. As the key concept in the current work, this can be further used to generate organic carbon compounds for diferent heterotrophic applications, including for the whole-cell biotransformation by yeast and bacteria. Results: Entrapment of Synechocystis S02 cells in Ca2+-cross-linked alginate hydrogel beads improves the specifc sucrose productivity by 86% compared to suspension cultures during 7 days of cultivation under salt stress. The process was further prolonged by periodically changing the medium in the vials for up to 17 days of efcient production, giving the fnal sucrose yield slightly above 3000 mg l −1 . We successfully demonstrated that the medium enriched with photosynthetically produced sucrose by immobilized Synechocystis S02 cells supports the biotransformation of cyclohexanone to ε-caprolactone by the E. coli WΔcscR Inv:Parvi strain engineered to (i) utilize low concentrations of sucrose and (ii) perform biotransformation of cyclohexanone to ε-caprolactone. Conclusion: We conclude that cell entrapment in Ca2+-alginate beads is an efective method to prolong sucrose production by the engineered cyanobacteria, while allowing efcient separation of the cells from the medium. This advantage opens up novel possibilities to create advanced autotroph–heterotroph coupled cultivation systems for solar-driven production of chemicals via biotransformation, as demonstrated in this work by utilizing the photosynthetically produced sucrose to drive the conversion of cyclohexanone to ε-caprolactone by engineered E. coli.</p

Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer-Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMOXeno) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMOAcineto) in Synechocystis sp. PCC 6803 (25 vs 2.3 U g(DCW)(-1) at an optical density of OD750 = 10) and an initial rate of 3.7 +/- 0.2 mM h(-1). While the cells containing CHMOAcineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMOXeno, which was attributed to the much faster lactone formation and a 10-fold lower KM value of BVMOXeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h-1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygendependent enzymatic processes

Photosynthetic production of enantioselective biocatalysts

$\bf Background:$ Global resource depletion poses a dramatic threat to our society and creates a strong demand for alternative resources that do not compete with the production of food. Meeting this challenge requires a thorough rethinking of all steps of the value chain regarding their sustainability resource demand and the possibility to substitute current, petrol-based supply-chains with renewable resources. This regards also the production of catalysts for chemical synthesis. Phototrophic microorganisms have attracted considerable attention as a biomanufacturing platform for the sustainable production of chemicals and biofuels. They allow the direct utilization of carbon dioxide and do not compete with food production. Photosynthetic enzyme production of catalysts would be a sustainable supply of these important components of the biotechnological and chemical industries. This paper focuses on the usefulness of recombinant cyanobacteria for the photosynthetic expression of enantioselective catalysts. As a proof of concept, we used the cyanobacterium $\it Synechocystis$ sp. PCC 6803 for the heterologous expression of two highly enantioselective enzymes. $\bf Results:$ We investigated the expression yield and the usefulness of cyanobacterial cell extracts for conducting stereoselective reactions. The cyanobacterial enzyme expression achieved protein yields of 3% of total soluble protein (%TSP) while the expression in $\textit {E. coli}$ yielded 6-8% TSP. Cell-free extracts from a recombinant strain expressing the recombinant esterase ST0071 from the thermophilic organism $\textit {Sulfolobus tokodai}$ ST0071 and arylmalonate decarboxylase from $\textit {Bordetella bronchiseptica}$ showed excellent enantioselectivity (>99% ee) and yield (>91%) in the desymmetrisation of prochiral malonates. $\bf Conclusions:$ We were able to present the proof-of-concept of photoautotrophic enzyme expression as a viable alternative to heterotrophic expression hosts. Our results show that the introduction of foreign genes is straightforward. Cell components from $\it Synechocystis$ did not interfere with the stereoselective transformations, underlining the usability of photoautotrophic organisms for the production of enzymes. Given the considerable commercial value of recombinant biocatalysts, cyanobacterial enzyme expression has thus the potential to complement existing approaches to use phototrophic organisms for the production of chemicals and biofuels