A multi-enzymatic cascade reaction for the stereoselective production of γ\gamma-oxyfunctionalyzed amino acids

A stereoselective three-enzyme cascade for synthesis of diasteromerically pure $\gamma$-oxyfunctionalized $\alpha$-amino acids was developed. By coupling a dynamic kinetic resolution (DKR) using an $\it N$-acylamino acid racemase (NAAAR) and an L-selective aminoacylase from $\textit {Geobacillus thermoglucosidasius}$ with a stereoselective isoleucine dioxygenase from $\textit {Bacillus thuringiensis,}$ diastereomerically pure oxidized amino acids were produced from racemic $\it N$-acetylamino acids. The three enzymes differed in their optimal temperature and pH-spectra. Their different metal cofactor dependencies led to inhibitory effects. Under optimized conditions, racemic $\it N$-acetylmethionine was quantitatively converted into L-methionine-$\textit {(S)}$-sulfoxide with 97% yield and 95% $\textit {de.}$ The combination of these three different biocatalysts allowed the direct synthesis of diastereopure oxyfunctionalized amino acids from inexpensive racemic starting material

A combined bioinformatics and functional metagenomics approach to discovering lipolytic biocatalysts

The majority of protein sequence data published today is of metagenomic origin. However, our ability to assign functions to these sequences is often hampered by our general inability to cultivate the larger part of microbial species and the sheer amount of sequence data generated in these projects. Here we present a combination of bioinformatics, synthetic biology, and $\textit {Escherichia coli}$ genetics to discover biocatalysts in metagenomic datasets. We created a subset of the Global Ocean Sampling dataset, the largest metagenomic project published to date, by removing all proteins that matched Hidden Markov Models of known protein families from PFAM and TIGRFAM with high confidence ($\it E$-value > 10$^{-5}$). This essentially left us with proteins with low or no homology to known protein families, still encompassing ~1.7 million different sequences. In this subset, we then identified protein families $\textit {de novo}$ with a Markov clustering algorithm. For each protein family, we defined a single representative based on its phylogenetic relationship to all other members in that family. This reduced the dataset to ~17,000 representatives of protein families with more than 10 members. Based on conserved regions typical for lipases and esterases, we selected a representative gene from a family of 27 members for synthesis. This protein, when expressed in $\textit {E. coli}$, showed lipolytic activity toward para-nitrophenyl (pNP) esters. The $\it K$$_{m}$-value of the enzyme was 66.68 $\mu$M for pNP-butyrate and 68.08 $\mu$M for pNP-palmitate with $\it k$$_{cat}$/$\it K$$_{m}$ values at 3.4 × 10$^{6}$ and 6.6 × 10$^{5}$ M$^{-1}$s$^{-1}$, respectively. Hydrolysis of model substrates showed enantiopreference for the R-form. Reactions yielded 43 and 61% enantiomeric excess of products with ibuprofen methyl ester and 2-phenylpropanoic acid ethyl ester, respectively. The enzyme retains 50% of its maximum activity at temperatures as low as 10°C, its activity is enhanced in artificial seawater and buffers with higher salt concentrations with an optimum osmolarity of 3,890 mosmol/l

Plasma‐driven in situ production of hydrogen peroxide for biocatalysis

Peroxidases and peroxygenases are promising classes of enzymes for biocatalysis because of their ability to carry out one‐electron oxidation reactions and stereoselective oxyfunctionalizations. However, industrial application is limited, as the major drawback is the sensitivity toward the required peroxide substrates. Herein, we report a novel biocatalysis approach to circumvent this shortcoming: in situ production of H$_2$O$_2$ by dielectric barrier discharge plasma. The discharge plasma can be controlled to produce hydrogen peroxide at desired rates, yielding desired concentrations. Using horseradish peroxidase, it is demonstrated that hydrogen peroxide produced by plasma treatment can drive the enzymatic oxidation of model substrates. Fungal peroxygenase is then employed to convert ethylbenzene to ($\it R$)‐1‐phenylethanol with an ee of >96 % using plasma‐generated hydrogen peroxide. As direct treatment of the reaction solution with plasma results in reduced enzyme activity, the use of plasma‐treated liquid and protection strategies are investigated to increase total turnover. Technical plasmas present a noninvasive means to drive peroxide‐based biotransformations

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

Enzymatic oxyfunctionalization driven by photosynthetic water-splitting in the Cyanobacterium Synechocystis\it Synechocystis sp. PCC 6803

Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts of reduced nicotinamide to a recombinant enoate reductase opened a new strategy for overcoming this hurdle. This paper presents the expansion of the photosynthetic regeneration system to a Baeyer-Villiger monooxygenase. Despite the potential of this strategy, this work also presents some of the encountered challenges as well as possible solutions, which will require further investigation. The successful enzymatic oxygenation shows that cyanobacterial whole-cell biocatalysis is an applicable approach that allows fuelling selective oxyfunctionalisation reactions at the expense of light and water. Yet, several hurdles such as side-reactions and the cell-density limitation, probably due to self-shading of the cells, will have to be overcome on the way to synthetic applications