Cell-free protein synthesis enables one-pot cascade biotransformation in an aqueous-organic biphasic system

Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueous-organic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents, and, importantly, the entire biocatalytic system can be consistently scaled up without a reduction of the substrate conversion rate. We, therefore, anticipate that our cell-free approach will make a possible cost-effective, high-yielding synthesis of valuable chemicals.     

Tools for Selective Enzyme Reaction Steps in the Synthesis of Laboratory Chemicals

The need for more selective reactions steps and the compatibility between process steps which follow on from each other has been a major driving force for organic synthesis. The synthesis of chiral compounds, metabolites, new chemical entities and natural products by a combination of chemical and enzyme reaction steps has become well established, due the existence of stable enzymes as selective catalysts which are inherently chiral by nature. Auxiliary tools such as suitable transfer reagents for reaching complete conversion, easy and robust reaction control as well as tools for straightforward workup and purification of the final product have been developed. Selective enzyme reaction steps in the area of hydrolyses, oxidation steps including hydroxylation and the Baeyer‐Villiger oxidation, carbon‐carbon bond formation and glycosylation reactions have compared favorably with existing methods of classical organic synthesis. The tools developed during optimization and scale‐up of these enzyme reaction steps have the potential to shorten development time. The introduction of selective enzyme reactions into an entire synthetic process has resulted in harmonization of improvements in economic efficiency with resultant solutions to health, safety and environment problems. This will become even more important in industrial synthetic chemistry in the future, for convenient solutions to certain intractable synthetic problems and for expanding the repertoire of chemistry by modular biocatalysts. Efficient isolation procedures for the final product are essential to take full advantage of the biocatalytic conversion to obtain high product yields.     

Perspectives and industrial potential of PGA selectivity and promiscuity

Penicillin G acylases (PGAs) are robust industrial catalysts used for biotransformation of β-lactams into key intermediates for chemical production of semi-synthetic β-lactam antibiotics by hydrolysis of natural penicillins. They are used also in reverse, kinetically controlled synthetic reactions for large-scale productions of these antibiotics from corresponding beta-lactam nuclei and activated acyl donors. Further biocatalytic applications of PGAs have recently been described: catalysis of peptide syntheses and the resolutions of racemic mixtures for the production of enantiopure active pharmaceutical ingredients that are based on enantioselective acylation or chiral hydrolysis. Moreover, PGAs rank among promiscuous enzymes because they also catalyze reactions such as trans-esterification, Markovnikov addition or Henry reaction. This particular biocatalytic versatility represents a driving force for the discovery of novel members of this enzyme family and further research into the catalytic potential of PGAs. This review deals with biocatalytic applications exploiting enantioselectivity and promiscuity of prokaryotic PGAs that have been recently reported. Biocatalytic applications are discussed and presented with reaction substrates converted into active compounds useful for the pharmaceutical industry.     

Engineered biosynthetic pathways and biocatalytic cascades for sustainable synthesis

Nature exploits biosynthetic cascades to construct numerous molecules from a limited set of starting materials. A deeper understanding of biosynthesis and extraordinary developments in gene technology has allowed the manipulation of natural pathways and construction of artificial cascades for the preparation of a range of molecules, which would be challenging to access using traditional synthetic chemical approaches. Alongside these metabolic engineering strategies, there has been continued interest in developing in vivo and in vitro biocatalytic cascades. Advancements in both metabolic engineering and biocatalysis are complementary, and this article aims to highlight some of the most exciting developments in these two areas with a particular focus on exploring those that have the potential to advance both pathway engineering and more traditional biocatalytic cascade development.     

A modular modulation method for achieving increases in metabolite production

Increasing the production of overproducing strains represents a great challenge. Here, we develop a modular modulation method to determine the key steps for genetic manipulation to increase metabolite production. The method consists of three steps: (i) modularization of the metabolic network into two modules connected by linking metabolites, (ii) change in the activity of the modules using auxiliary rates producing or consuming the linking metabolites in appropriate proportions and (iii) determination of the key modules and steps to increase production. The mathematical formulation of the method in matrix form shows that it may be applied to metabolic networks of any structure and size, with reactions showing any kind of rate laws. The results are valid for any type of conservation relationships in the metabolite concentrations or interactions between modules. The activity of the module may, in principle, be changed by any large factor. The method may be applied recursively or combined with other methods devised to perform fine searches in smaller regions. In practice, it is implemented by integrating to the producer strain heterologous reactions or synthetic pathways producing or consuming the linking metabolites. The new procedure may contribute to develop metabolic engineering into a more systematic practice. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:656–667, 2015     

The biocatalytic activity of bromelain in ester synthesis reaction: difference between the intrinsic lipase activity and thermal catalysis

Biocatalytic activities of bromelain preparations were carried out in proteolytic (4500 units g^–1), lipolytic (67 units g^–1) and, more particularly, in fatty acid ester synthetic reactions. The ester synthesis reactions were studied and several thermodynamic parameters and non-biological reference reactions were also investigated. Only temperature had a strong influence on the maximum reaction yield (30% after 10 days) and revealed that thermal catalysis, which exists in esterification, raises doubts concerning the real biocatalytic activity of the plant extract. When this thermal catalysis is taken into account, the intrinsic lipase activity of the bromelain preparations in esterification reactions is nil.     

Microbial systems for study of the biotransformations of drugs

The metabolic fate of drugs and other xenobiotics in mammalian organisms represents an area of intense contemporary interest. Traditionally, it is a difficult area of research becausethe biological systems which are used to study biotransformations are capable of yielding only minute quantities of metabolites. Recent developments in comparative biochemistry have made itpossible to link diverse metabolic systems through similarities in the pathways by which they alter foreign organic compounds. The potential thus exists for utilizing microbial metabolic systems to study and possibly predict the metabolic fate of a drug or other foreign compound in mammals. The ease with which microbial systems may be used to obtain large amounts of metabolites is an obvious Advantage. We havhe attemped to review the ways in which mammalian and microbialorganisms metabolize a variety of organic compounds. Attention has been focused on the similarities and differences in the mechanisms by which these living systems metabolize xenobiotics. Particular emphasis has been given to four types of reactions which are important in drug biotransformations: aromatic hydroxylationl; N- and O-dealkylations; and sulfur oxygenations.     

Whole cell biocatalysis in nonconventional media

Summary In this paper biocatalytic reactions carried out by whole cells in nonconventional media are reviewed. Similar relationships are observed between solvent hydrophobicity and catalytic activity in reactions carried out by isolated enzymes and whole cells. In addition to the effect of organic solvent on biocatalyst stability, microbial cells are susceptible to damaging effects caused by the organic phase. In general, more hydrophobic solvents manifest lower toxicity towards the cells. Whole cell biocatalysts require more water than isolated enzymes and two-phase systems have been most widely used to study whole cell biocatalysis. Immobilization makes cell biocatalysts more resistant to organic solvents and helps achieve homogeneous biocatalyst dispersion. Cell entrapment methods have been widely used with organic solvent systems and mixtures of natural and/or synthetic polymers allow adjustment of the hydrophobicity-hydrophilicity balance of the support matrix. Some examples of stereoselective catalysis using microbial cells in organic solvent media are presented.     

Modular and scalable biocatalytic tools for practical safety,health and environmental improvements in the production of speciality chemicals

Biocatalytic tools for both end-of-the-pipe solutions and direct reaction methodology have been developed for the improvement of practical oxidations. The identification of bottlenecks and limitations in biocatalytic Baeyer-Villiger oxidations, and the comparison of scalable process designs to overcome these limitations, have shown the direction for improvements. The first kilogram-scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity has been realized by the combination of a resin-based in-situ SFPR strategy together with micro-bubble aeration. Regioselective asymmetric dihydroxylation of aromatic nitriles has been achieved by recombinant chlorobenzenedioxygenase. The introduction of novel biocatalytic tools for key catalytic asymmetric transformations will change chemical manufacturing in the 21st century.     

Biocatalytic conversion of lignocellulose to platform chemicals

Naturally occurring lignocellulose can be used as a renewable resource for the sustainable production of platform chemicals that can in turn be converted to valuable fine chemicals, polymers, and fuels. The biocatalytic conversion of lignocellulose is a very promising approach due to its high selectivity, mild conditions, and low exergy loss. However, such biocatalytic processes are still seldom applied at the industrial scale since the single conversion steps (pretreatment, hydrolysis, and fermentation) may exhibit low conversion rates, low efficiencies, or high costs. The biocatalytic conversion of lignocellulose to platform chemicals is reviewed in this work. Structures and production rates of lignocellulose are described, and platform chemicals that may be produced from lignocellulose are summarized. Biocatalytic conversion of lignocellulose is distinguished from conventional non-selective approaches. All essential conversion steps used in biocatalytic approaches (pretreatment, hydrolysis, and fermentation) are reviewed in detail. Finally, potential interactions between these conversion steps are highlighted and the advantages as well as disadvantages of integrated process configurations are elucidated. In conclusion, a comprehensive understanding of the biocatalytic conversion of lignocellulose is provided in this review.     

Tools and ingredients for the biocatalytic synthesis of carbohydrates and glycoconjugates

The presence of multiple functional groups and stereocentres in carbohydrates and glycoconjugates make them challenging targets for synthesis. Although progress in chemical synthesis and engineering is impressive, there is still a need to selectively introduce and remove protecting groups in the total synthesis of target molecules of increasing complexity. Multiple hydroxyl-groups with similar reactivities have to be differentiated in order to form the desired glycosidic bonds in a regio- and stereospecific way. To complement the existing chemical tools and ingredients, biocatalysts for selective carbon–carbon bond formation and glycosylation reactions have been developed. The availability of auxiliary ingredients like transfer reagents is a prerequisite for the development of viable biocatalytic process steps. In the case of dihydroxyacetone-phosphate-dependent aldolases, e.g. fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), the large-scale availability of dihydroxyacetone-phosphate (DHAP) eliminates the need to synthesize the donor DHAP. For the pyruvate-dependent aldolases, e.g. the N-acetylneuraminic acid aldolase (EC 4.1.3.3) and acetaldehyde-dependent aldolases like the 2-deoxy-d-ribose-5-phosphate aldolase (4.2.1.4), the donors pyruvate and acetaldehyde are also available on a large scale. A broad range of natural and recombinant aldolases have been produced in stable lyophilized form. Recombinant transketolase together with a new synthesis of hydroxypyruvates has provided a platform technology for the preparation of monosaccharides, whereby the carbon backbone is extended by a two-carbon unit (C2-elongation). Natural and recombinant glycosyltransferases have been prepared on a large-scale to establish biocatalytic glycosylations in water as highly regio- and stereospecific reaction methodologies without the need for laborious protecting group manipulations, solubility adaptations and complex synthetic schemes. In order to simplify the synthetic manipulations for specific glycosylations, toolkits for β-1,4-galactosylations, α-1,3-galactosylations and α-1,3-fucosylations have been developed for rapid quantitative conversions. The introduction of matched pairs of biocatalysts and transfer reagents as ingredients together with the optimized reaction methodology as tool provide an important starting point for biocatalytic glycomics.     

Chemoenzymatic and microbial dynamic kinetic resolutions

This review tracks a decade of dynamic kinetic resolution developments with a biocatalytic inclination using enzymatic/microbial means for the resolution part followed by the racemization reactions either by means of enzymatic or chemocatalyst. These fast developments are due to the ability of the biocatalysts to significantly reduce the number of synthetic steps which are common for conventional synthesis. Future developments in novel reactions and products of dynamic kinetic resolutions should consider factors that are needed to be extracted at the early synthetic stage to avoid inhibition at scale-up stage have been highlighted.     

Microbial degradation of halogenated aromatics: molecular mechanisms and enzymatic reactions

Halogenated aromatics are used widely in various industrial, agricultural and household applications. However, due to their stability, most of these compounds persist for a long time, leading to accumulation in the environment. Biological degradation of halogenated aromatics provides sustainable, low-cost and environmentally friendly technologies for removing these toxicants from the environment. This minireview discusses the molecular mechanisms of the enzymatic reactions for degrading halogenated aromatics which naturally occur in various microorganisms. In general, the biodegradation process (especially for aerobic degradation) can be divided into three main steps: upper, middle and lower metabolic pathways which successively convert the toxic halogenated aromatics to common metabolites in cells. The most difficult step in the degradation of halogenated aromatics is the dehalogenation step in the middle pathway. Although a variety of enzymes are involved in the degradation of halogenated aromatics, these various pathways all share the common feature of eventually generating metabolites for utilizing in the energy-producing metabolic pathways in cells. An in-depth understanding of how microbes employ various enzymes in biodegradation can lead to the development of new biotechnologies via enzyme/cell/metabolic engineering or synthetic biology for sustainable biodegradation processes.     

Combinatorial biocatalysis

The published applications of combinatorial biocatalysis have continued to expand at a growing rate. This is exemplified by the variety of enzyme catalysts and whole-cell catalysts used for the creation of libraries through a wide range of biocatalytic reactions, including acylation, glycosylation, halogenation, oxidation and reduction. These biocatalytic methods add the capability to perform unique chemistries or selective reactions with complex or labile reagents when integrated with classical combinatorial synthesis methods. Thus, applications towards the production of libraries de novo, the expansion of chemically derived combinatorial libraries, and the generation of novel combinatorial reagents for library synthesis can be achieved. Theoretically, these results illustrate what is already evident from nature: that complex, biologically active, structurally diverse compound libraries can be generated through the application of biocatalysis alone or in combination with classical organic synthesis approaches.     

Advances in bio‐based production of dicarboxylic acids longer than C4

Growing concerns of environmental pollution and fossil resource shortage are major driving forces for bio‐based production of chemicals traditionally from petrochemical industry. Dicarboxylic acids (DCAs) are important platform chemicals with large market and wide applications, and here the recent advances in bio‐based production of straight‐chain DCAs longer than C4 from biological approaches, especially by synthetic biology, are reviewed. A couple of pathways were recently designed and demonstrated for producing DCAs, even those ranging from C5 to C15, by employing respective starting units, extending units, and appropriate enzymes. Furthermore, in order to achieve higher production of DCAs, enormous efforts were made in engineering microbial hosts that harbored the biosynthetic pathways and in improving properties of biocatalytic elements to enhance metabolic fluxes toward target DCAs. Here we summarize and discuss the current advantages and limitations of related pathways, and also provide perspectives on synthetic pathway design and optimization for hyper‐production of DCAs.     

Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

Background

The integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.

Results

A chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.

Conclusions

In this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.

     

Microbial P450 enzymes in biotechnology

Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes since they allow the introduction of oxygen into non-activated carbon atoms in a sterically and optically selective manner that is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. Surprisingly, cytochrome P450 monooxygenases have scarcely been studied for use in biooxidations, although they are one of the largest known superfamilies of enzyme proteins. Their gene sequences have been identified in various organisms such as humans, bacteria, algae, fungi, and plants. The reactions catalyzed by P450s are quite diverse and range from biosynthetic pathways (e.g. those of animal hormones and secondary plant metabolites) to the activation or biodegradation of hydrophobic xenobiotic compounds (e.g. those of various drugs in the liver of higher animals). From a practical point of view, the great potential of P450s is limited by their functional complexity, low activity, and limited stability. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways that could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes, and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are both covered in this review.     

The use of enzymes in the chemical industry in Europe

Many European chemical industries are in a phase of reorganization resulting in a general opening towards life sciences. Traditional chemical markets are served increasingly with products derived from bioprocesses or hybrid chemical/biocatalytic processes. Biocatalytic steps are already being used to produce a wide range of products, including agricultural chemicals, organics, drugs and plastic materials, to name but a few. Apart from the rapidly growing number of commercialized bioprocesses, a partial survey of exploratory activities points to future applications of enzymes in the European chemical industry, which will bring new products and technologies and, in some cases, replace traditional syntheses.     

Enzyme activation for organic solvents made easy

Enzymes are highly selective catalysts that perform intricate chemistries at ambient temperatures and pressures. Although water is the solvent of life, it is a poor solvent for most synthetic organic reactions and, therefore, most chemists avoid aqueous solutions for synthetic applications. However, when removed from the aqueous environment and placed in an organic solvent, enzyme activity is reduced greatly. Here, we present a general overview of recent efforts to activate enzymes for use in nonaqueous media, giving particular focus to the use of simple salts as additives that result in significant biocatalytic improvements.     

The imminent role of protein engineering in synthetic biology

Protein engineering has for decades been a powerful tool in biotechnology for generating vast numbers of useful enzymes for industrial applications. Today, protein engineering has a crucial role in advancing the emerging field of synthetic biology, where metabolic engineering efforts alone are insufficient to maximize the full potential of synthetic biology. This article reviews the advancements in protein engineering techniques for improving biocatalytic properties to optimize engineered pathways in host systems, which are instrumental to achieve high titer production of target molecules. We also discuss the specific means by which protein engineering has improved metabolic engineering efforts and provide our assessment on its potential to continue to advance biology engineering as a whole.