Green Chemistry, Biocatalysis, and the Chemical Industry of the Future.

In the movement to decarbonize our economy and move away from fossil fuels we will need to harness the waste products of our activities, such as waste lignocellulose, methane, and carbon dioxide. Our wastes need to be integrated into a circular economy where used products are recycled into a manufacturing carbon cycle. Key to this will be the recycling of plastics at the resin and monomer levels. Biotechnology is well suited to a future chemical industry that must adapt to widely distributed and diverse biological chemical feedstocks. Our increasing mastery of biotechnology is allowing us to develop enzymes and organisms that can synthesize a widening selection of desirable bulk chemicals, including plastics, at commercially viable productivities. Integration of bioreactors with electrochemical systems will permit new production opportunities with enhanced productivities and the advantage of using a low-carbon electricity from renewable and sustainable sources.

The Application of Biocatalysis in the Preparation and Resolution of Morita-Baylis-Hillman Adducts and Their Derivatives.

The Morita-Baylis-Hillman (MBH) reaction affords highly functionalised allylic alcohols containing a new stereogenic centre. These MBH adducts are very versatile and have been transformed into a large range of products, some of which have medicinal potential. Several examples of asymmetric syntheses of MBH adducts have been reported, although a generally applicable method remains to be developed. Biocatalytic approaches for the synthesis and enzymatic kinetic resolution of MBH adducts have been reported, and are discussed in detail in this review. Enzymes able to catalyse the asymmetric MBH reaction have been identified, but selectivity and efficiency have generally been low. Lipases, esterases and nitrile-converting enzymes have all been successfully applied in the resolution of MBH adducts, with excellent selectivity being realised in most cases.

New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy.

This tutorial review focuses on recent advances in technologies for enzyme immobilisation, enabling their cost-effective use in the bio-based economy and continuous processing in general. The application of enzymes, particularly in aqueous media, is generally on a single use, throw-away basis which is neither cost-effective nor compatible with a circular economy concept. This shortcoming can be overcome by immobilising the enzyme as an insoluble recyclable solid, that is as a heterogeneous catalyst.

Development of fructose-1,6-bisphosphate aldolase enzyme peptide mimics as biocatalysts in direct asymmetric aldol reactions.

This study describes the design and synthesis of mimetic peptides modelled on the catalytic active site of the fructose-1,6-bisphosphate aldolase (FBPA) enzyme. The synthesized peptides consisting of the turn motifs and catalytic site amino acids of FBPA enzyme were evaluated for catalytic activity in direct asymmetric aldol reactions of ketones and aldehydes. The influence of substrate scope, catalyst loading and solvents including water, on the reaction were also investigated. Nuclear magnetic resonance (NMR) and circular dichroism (CD) were used to determine the secondary structure of the peptides to provide an understanding of the structure-activity relationship. The peptides showed catalytic activity and the aldol products were obtained in low yields (up to 44%), but excellent enantioselectivity (up to 93%) and moderate diastereoselectivity (65 : 35).

Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases.

Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were-in addition-able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.

The Hitchhiker's guide to biocatalysis: recent advances in the use of enzymes in organic synthesis.

Enzymes are excellent catalysts that are increasingly being used in industry and academia. This perspective is primarily aimed at synthetic organic chemists with limited experience using enzymes and provides a general and practical guide to enzymes and their synthetic potential, with particular focus on recent applications.

Substrate Profiling of the Cobalt Nitrile Hydratase from Rhodococcus rhodochrous ATCC BAA 870.

The aromatic substrate profile of the cobalt nitrile hydratase from Rhodococcus rhodochrous ATCC BAA 870 was evaluated against a wide range of nitrile containing compounds (>60). To determine the substrate limits of this enzyme, compounds ranging in size from small (90 Da) to large (325 Da) were evaluated. Larger compounds included those with a bi-aryl axis, prepared by the Suzuki coupling reaction, Morita-Baylis-Hillman adducts, heteroatom-linked diarylpyridines prepared by Buchwald-Hartwig cross-coupling reactions and imidazo[1,2-a]pyridines prepared by the Groebke-Blackburn-Bienaymé multicomponent reaction. The enzyme active site was moderately accommodating, accepting almost all of the small aromatic nitriles, the diarylpyridines and most of the bi-aryl compounds and Morita-Baylis-Hillman products but not the Groebke-Blackburn-Bienaymé products. Nitrile conversion was influenced by steric hindrance around the cyano group, the presence of electron donating groups (e.g., methoxy) on the aromatic ring, and the overall size of the compound.

The complete genome sequence of the nitrile biocatalyst Rhodocccus rhodochrous ATCC BAA-870.

BACKGROUND: Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. RESULTS: The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. CONCLUSIONS: The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.

Efficient one-pot synthesis of functionalised imidazo[1,2-a]pyridines and unexpected synthesis of novel tetracyclic derivatives by nucleophilic aromatic substitution.

Novel tetracyclic imidazo[1,2-a]pyridine derivatives have been prepared by intramolecular nucleophilic aromatic substitution of 5-fluoroimidazo[1,2-a]pyridines under basic conditions. Use of the non-nucleophilic alcoholic solvent tert-butanol, rather than methanol, increased the yield of the tetracycles by reducing the competing intermolecular reaction observed for methanol. In addition, a modified protocol for the dehydration of formamides to isocyanides has been found to be tolerant of an unprotected hydroxyl functional group and one-pot conversion to imidazo[1,2-a]pyridyl-aminocyclohexanol analogues is reported.

Identification and characterisation of a fluorinase from Actinopolyspora mzabensis.

The incorporation of fluorine has been shown to improve the biophysical and bioactive properties of several organic compounds. However, sustainable strategies of fluorination are needed. Fluorinases have the unique ability to catalyse a C-F bond, hence, have vast potential to be applied as biocatalysts in the preparation of fine chemicals. But fluorinases are extremely rare in nature with only five representatives isolated thus far. Moreover, the heterologous expression of fluorinases is challenged by low yields of soluble protein. This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8 M urea, and successfully refolded into a biologically active form. Following hydrophobic interaction chromatography, >80 mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An additional gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temperature was 65 °C. At these conditions, the enzyme exhibited an activity of 0.44 ±â€¯0.03 µM min-1 mg-1. Furthermore, the Am-fluorinase showed exceptional stability at 25 °C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.

A green, economical synthesis of ß-ketonitriles and trifunctionalized building blocks from esters and lactones.

The acylation of the acetonitrile anion with lactones and esters in ethereal solvents was successfully exploited using inexpensive KOt-Bu to obtain a variety of ß-ketonitriles and trifunctionalized building blocks, including useful O-unprotected diols. It was discovered that lactones react to produce the corresponding derivatized cyclic hemiketals. Furthermore, the addition of a catalytic amount of isopropanol, or 18-crown-6, was necessary to facilitate the reaction and to reduce side-product formation under ambient conditions.

Biodiesel's trash is a biorefineries' treasure: the use of "dirty" glycerol as an industrial fermentation substrate.

"Dirty" glycerol from biodiesel production is having a considerable environmental impact since its disposal is expensive and difficult. The increased biodiesel production in the last two decades has forced glycerol prices down, thereby making it now unprofitable for chemical companies to produce. The problem lies with the impurities of the biodiesel conversion process usually ending up within the crude glycerol fraction. These impurities are often too costly to purify with current processes, particularly for small scale producers. A wide variety of industries, including the paint, tobacco, food and pharmaceutical industries, utilize glycerol as part of their technology or products. However, the crude glycerol from biodiesel production is not of a high enough grade to be used in these industries. Biodiesel-produced crude glycerol is therefore cheap, readily available and presents itself as an attractive carbon source for industrial microbial production systems synthesizing value-added products. This mini-review will look at (a) microbial production processes which use crude glycerol to produce high-value products (product-driven research) and (b) genetic engineering of microbes which is aimed at improving microbial "dirty" glycerol utilization (substrate driven research).

Bacterial nitrilases and their regulation.

Commercially, nitrilases are valuable biocatalysts capable of converting a diverse range of nitriles to carboxylic acids for the greener synthesis of chemicals and pharmaceuticals. Nitrilases are widespread in nature and are both important components of metabolic pathways and a response to environmental factors such as natural or manmade nitriles. Nitrilases are often grouped together on a genome in specific gene clusters that reflect these metabolic functions. Although nitrilase induction systems are still poorly understood, it is known that a powerful Rhodococcal transcription regulator system permits accumulation of intracellular nitrilase of up to 30-40% of total soluble protein in wild type Rhodococcous rhodochrous and host Streptomyces strains. Nitrilase expression inducer molecules encompass a broad range of aliphatic, aromatic and heteroaromatic nitriles, as well as some secondary and tertiary amides that are resistant to nitrilase degradation.

Broadening the Scope of Biocatalysis in Sustainable Organic Synthesis.

This Review is aimed at synthetic organic chemists who may be familiar with organometallic catalysis but have no experience with biocatalysis, and seeks to provide an answer to the perennial question: if it is so attractive, why wasn't it extensively used in the past? The development of biocatalysis in industrial organic synthesis is traced from the middle of the last century. Advances in molecular biology in the last two decades, in particular genome sequencing, gene synthesis and directed evolution of proteins, have enabled remarkable improvements in scope and substantially reduced biocatalyst development times and cost contributions. Additionally, improvements in biocatalyst recovery and reuse have been facilitated by developments in enzyme immobilization technologies. Biocatalysis has become eminently competitive with chemocatalysis and the biocatalytic production of important pharmaceutical intermediates, such as enantiopure alcohols and amines, has become mainstream organic synthesis. The synthetic space of biocatalysis has significantly expanded and is currently being extended even further to include new-to-nature biocatalytic reactions.

Streptomyces albulus yields ε-poly-L-lysine and other products from salt-contaminated glycerol waste.

Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-L-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1 g ε-poly-L-lysine per 1 g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.

The limits to biocatalysis: pushing the envelope.

In the period 1985 to 1995 applications of biocatalysis, driven by the need for more sustainable manufacture of chemicals and catalytic, (enantio)selective methods for the synthesis of pharmaceutical intermediates, largely involved the available hydrolases. This was followed, in the next two decades, by revolutionary developments in protein engineering and directed evolution for the optimisation of enzyme function and performance that totally changed the biocatalysis landscape. In the same period, metabolic engineering and synthetic biology revolutionised the use of whole cell biocatalysis in the synthesis of commodity chemicals by fermentation. In particular, developments in the enzymatic enantioselective synthesis of chiral alcohols and amines are highlighted. Progress in enzyme immobilisation facilitated applications under harsh industrial conditions, such as in organic solvents. The emergence of biocatalytic or chemoenzymatic cascade processes, often with co-immobilised enzymes, has enabled telescoping of multi-step processes. Discovering and inventing new biocatalytic processes, based on (meta)genomic sequencing, evolving enzyme promiscuity, chemomimetic biocatalysis, artificial metalloenzymes, and the introduction of non-canonical amino acids into proteins, are pushing back the limits of biocatalysis function. Finally, the integral role of biocatalysis in developing a biobased carbon-neutral economy is discussed.

A modern and practical laccase-catalysed route suitable for the synthesis of 2-arylbenzimidazoles and 2-arylbenzothiazoles.

Heterocyclic aromatic compounds containing an imine (C[double bond, length as m-dash]N) bond such as benzimidazoles and benzothiazoles are important active pharmaceutical ingredients. The synthesis of 2-aryl-1H-benzimidazoles and 2-arylbenzothiazoles in good to excellent yields was achieved by reacting 2-aminoaromatics with various benzaldehyde derivatives catalysed by the commercial laccases Novoprime and Suberase® at room temperature and in the presence of atmospheric oxygen.

A one-pot laccase-catalysed synthesis of coumestan derivatives and their anticancer activity.

Suberase®, a commercial laccase from Novozymes, was used to catalyse the synthesis of coumestans. The yields, in some cases, were similar to or better than that obtained by other enzymatic, chemical or electrochemical syntheses. The compounds were screened against renal TK10, melanoma UACC62 and breast MCF7 cancer cell-lines and the GI50, TGI and LC50 values determined. Anticancer screening showed that the cytostatic effects of the coumestans were most effective against the melanoma UACC62 and breast MCF7 cancer cell-lines exhibiting potent activities, GI50=5.35 and 7.96µM respectively. Moderate activity was obtained against the renal TK10 cancer cell-line. The total growth inhibition, based on the TGI values, of several of the compounds was better than that of etoposide against the melanoma UACC62 and the breast MCF7 cancer cell lines. Several compounds, based on the LC50 values, were also more lethal than etoposide against the same cancer cell lines. The SAR for the coumestans is similar against the melanoma UACC62 and breast MCF7 cell lines. The compound having potent activity against both breast MCF7 and melanoma UACC62 cell lines has a methyl group on the benzene ring (ring A) as well as on the catechol ring (ring B). Anticancer activity decreases when methoxy and halogen substituents are inserted on rings A and B.

Metagenomic mining of glycoside hydrolases from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) and the characterization of a multimodular ß-1,4-xylanase (GH11).

In recent years, there have been particular emphases worldwide on the development and optimization of bioprocesses for the utilization of biomass. An essential component of the biomass processing conduit has been the need for robust biocatalysts as high-performance tools for both the depolymerization of lignocellulosic biomass and synthesis of new high-value bio-based chemical entities. Through functional screening of the metagenome of the hindgut bacterial symbionts of a termite, Trinervitermes trinervoides, we discovered open reading frames for 25 cellulases and hemicellulases. These were classified into 14 different glycoside hydrolase (GH) families: eight GH family 5; four GH9, two GH13, and one each in GH2, GH10, GH11, GH26, GH29, GH43, GH44, GH45, GH67, and GH94 families. Of these, eight were overexpressed and partially characterized to be shown to be endocellulases (GH5C, GH5E, GH5F, and GH5G), an exocellulase (GH5D), endoxylanases (GH5H and GH11), and an α-fucosidase (GH29). The GH11 (Xyl1) was of particular interest as it was discovered to be a multimodular ß-1,4-xylanase, consisting of a catalytic domain and two carbohydrate-binding modules (CBMs). The CBM functions to selectively bind insoluble xylan and increases the rate of hydrolysis. The primary structure of GH11 showed a classical catalytic dyad of glutamic acid residues that generally forms part of the active site in GH11 enzyme family. This endoxylanase was optimal at pH 6 and 50 °C, and generated xylobiose and xylotriose from various xylan sources, including beechwood, birchwood, and wheat arabinoxylan. The catalytic ability of GH11 against natural substrate (e.g., wheat arabinoxylan) renders GH11 as a potential useful biocatalyst in the effective dismantling of complex plant biomass architecture.

Tools for metabolic engineering in Streptomyces.

During the last few decades, Streptomycetes have shown to be a very important and adaptable group of bacteria for the production of various beneficial secondary metabolites. These secondary metabolites have been of great interest in academia and the pharmaceutical industries. To date, a vast variety of techniques and tools for metabolic engineering of relevant structural biosynthetic gene clusters have been developed. The main aim of this review is to summarize and discuss the published literature on tools for metabolic engineering of Streptomyces over the last decade. These strategies involve precursor engineering, structural and regulatory gene engineering, and the up or downregulation of genes, as well as genome shuffling and the use of genome scale metabolic models, which can reconstruct bacterial metabolic pathways to predict phenotypic changes and hence rationalize engineering strategies. These tools are continuously being developed to simplify the engineering strategies for this vital group of bacteria.