Panta rei: everything is changing!

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    Current Opinion in Biotechnology 2002, 13:533535

    0958-1669/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved

    Panta rei: everything is changing!Editorial overviewAndreas Kiener

    Andreas Kiener

    Biotechnology Research and Development,Lonza AG, CH-3930 Visp, Switzerland; e-mail:

    Dr Andreas Kiener studiedmicrobiology/biochemistry at the ETHin Zrich from 1982 to 1986. From1986 to 1988 he worked as aPostdoctoral researcher at theMassachusetts Institute of Technologyand Harvard Medical School and iscurrently senior scientist at Lonza inVisp, Switzerland.

    The Greek philosopher Heraclitus lived from 536 to 470 BC. He captured hisphilosophy in just two words: panta rei, literally everything flows or every-thing is changing. The philosopher argued that everything is constantlychanging, from the smallest grain of sand to the stars in the sky. Only changeitself is real, every object ultimately is a figment of peoples imagination.

    Let us now take a huge jump into biocatalysis and start thinking about theconstant changes that have occurred here. Early this century, as a piece ofgrain, it started with the formation and analysis of natural products by fer-mentation (let us forget about biological alcohol formation from centuriesago) mainly by chemists, and then constantly moved through space reachingfrom star to star by major scientific achievements. But, where are we now?And how do we move from the current star to the next one?

    Let us look at some of the possibilities we have to move forward in space. Thepapers published in this Chemical Biotechnology section of Current Opinion inBiotechnology present a summary of the current status of bioorganic chemistryand the moves it is making into the biology arena.

    Substrate engineering biohydroxylation Specific oxidation to provide products with high enantiomeric excess (ee) values still remains a problem in classical organic chemistry. The selective oxidation of non-activated carbon atoms is discussed in the article of de Raatand Griengl (pp 537542). The selectivity and mildness of biohydroxylationscatalysed by monooxygenases and the application of substrate engineering tothese reactions provide the focus of this review.

    The use of substrate engineering is demonstrated by the use of docking/protection (d/p) groups. The introduction of these groups can help promotecatalysis and prevent unwanted side reactions. In an ideal case, these d/p groupscan be removed after the biotransformation leading to the desired compoundwith a high ee value. In some cases it has also been possible to control the stereochemistry of the end product by using an appropriate chiral d/p group.

    Whole cells have also been used as biocatalysts for biohydroxylations. Theseinclude fungi such as Beauveria bassina and Cunninghamella blakesleeana andmicroorganisms like Sphingomonas species. The application of whole cells to biohydroxylations are of special interest, because it might be possible to use themfor the large-scale production of certain products the growth of these micro-organisms is very easy to perform on a multilitre scale. Furthermore, the isolationof the desired oxidation products from bioreactors is facilitated by their hydrophobicstructures. The most convenient biohydroxylation systems depend to a largeextent on the exact isolation procedure. Such hydroxylation systems can be further improved by site-directed mutagenesis, a task for molecular biologists.

    Various oxidation reactions have been shown for ketones, fluorinated alcoholsand cyclic amines or lactams. All of these reactions lead to hydroxylated

  • product in high quantities with good yields and high eevalues. Biohydroxylations using Sphingomonas species arealso described, the use of this strain is advantageous as it iseasy to grow.

    Methods to increase the enantioselectivity oflipases and esterasesIsolated enzymes used under different reaction conditionsare important for the synthesis of optically pure compounds.Bornscheuer (pp 543547) gives a summary of the investi-gations of his group as well as the achievements of otherlaboratories. Over 50 enzymes are currently used underdifferent conditions for the isolation of chiral molecules.The use of organic solvents still remains a useful approachto enhance enantioselectivity, but their effects are difficultto predict. The author examines three other strategies thathave been used to increase enantioselectivity: the func-tional expression of enzymes, rational protein design, anddirected evolution.

    Most enzymes are sold as crude products with no attemptsmade to isolate a distinct biocatalyst. The result of this isthat reactions are often catalysed by a mixture of isozymes.The functional expression of recombinant isozymes hasbeen investigated as a way to increase enantioselectivity.Such enzymes would be free of other interfering isozymesand could be produced at high levels. The authors discusssome recent successful examples of this approach, includingthe overexpression of pig liver esterase and the expressionof the lipase from the yeast Candida rugosa in Pichia pastoris.

    As shown by crystallographers, many lipase structures havepaved the way for the use of computer modelling to rationalise the substrate specificity and enantioselectivityof these enzymes. For instance, the enantioselectivity of the Candida antarctica B lipase-catalysed resolution of1-chloro-2-octanol was improved by a factor of two by asingle amino acid substitution. It was also shown that otheresterases contain GGGX motifs for stereoselective activity.

    Directed evolution involves the generation of large mutantlibraries using various methods for random mutagenesis andrecombination, followed by high-throughput screening.This methodology has been used to identify a lipase fromPseudomonas aeruginosa with increased enantioselectivity.

    The production of fine chemicals bybiotransformationsBiocatalysis today is an important technology for the syn-thesis of a wide variety of compounds used in theproduction of food-stuffs, pharmaceutical compounds andeven the chemical building blocks for synthetic reactions.The article by Straathof, Panke and Schmid (pp 548556)provides a summary of the latest developments for the production of fine chemicals by biotransformations. Themain focus is on the production of compounds on a multi-gram per litre scale or less. Some of these compounds haveimportant clinical applications.

    In general, industrial biotransformations describe a reactionor a set of simultaneous reactions in which a pre-formedprecursor molecule is converted in to product. Obviously,chirality is a key issue in these bioconversions, but for mostof the compounds the configuration originates from theprecursor. Whole cells are often used in many non-redoxbiotransformations, the cells do not metabolise any startingmaterials themselves and only one or two enzymes areactive for the biotransformation. Aqueous suspension reactions with organic solvents are already employed in13 industrial processes.

    The most predominant biotransformation used in thechemical industry is for the production of acrylamide fromacrylonitrile with a nitrilehydratase from Rhodococcus. Theauthors also discuss the recent use of several other biotransformations by the chemical industry, including theproduction of -lactam antibiotics, and consider the criteriathat need to be met for these processes to be adopted.

    Compared with biotransformations for the production ofchemicals, the productivity of biotransformations in thepharmaceutical industry is generally low. The success ofsuch processes is, however, owned by pharmaceuticalcompanies. In an ideal situation the producers of pharma-ceuticals, both the pharmaceutical and chemical industries,should have a joint goal to reach an efficient productionmethod rather than to develop individual methods for target compounds.

    Biotransformations using prokaryoticP450 monooxygenasesUrlacher and Schmid (pp 557564) consider the use ofcytochrome P450 monooxygenases (CYPs) for biotransfor-mations employing the isolated enzyme. These arepotentially a very useful class of enzyme for oxidation, asthey are able to introduce oxygen at non-activated carbonhydrogen bonds to yield sterically and opticallypure compounds. In addition, they represent one of the largestknown superfamilies of proteins, being found in a widerange of organisms. Mammalian CYPs, located in the livermicrosomes, have also been widely studied in the contextof drug activation and the livers response to toxic chemicals,illustrating the broader interest of these enzymes.

    The development of CYPs as efficient biocatalysts hasbeen investigated by several groups. Their work hasfocused on three key areas: structurefunction analyses;the development of artificial electron-transfer systems; andprotein engineering. The high-resolution crystal structuresof eight CYPs have been determined and provide valuableinformation. The structural data have facilitated elucidationof the enzyme mechanism and helped to identify with certainty the positions and mechanistic role of solvent molecules. One drawback of CYP-catalysed reactions istheir requirement for the expensive cofactor NAD(P)H,which is consumed in the reaction. Thus, there has beenconsiderable interest in the development of a technical

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  • process to supply or regenerate this cofactor. The authorsdiscuss several advances in the development of artificialelectron-transfer systems that aim to address this issue.Finally, site-directed mutagenesis and directed evolutiontechniques have been used to re-engineer CYPs withdesired properties.

    Taken together these three approaches to the study ofCYPs have yielded exciting results that are likely to promote the use of these enzymes in the future.

    Enzyme catalysis in ionic liquids Kragl, Eckstein and Kaftzik (pp 565571) look at thepotential of using ionic liquids for solvent engineering inbiocatalytic reactions. Over the past decade, ionic liquidshave gained increasing attention for performing all types ofreactions with sometimes remarkable results. Ionic liquidsoffer several advantages over other solvents: they possessno vapor pressure, are able to dissolve many compounds,and can be used to form two-phase systems with many solvents. The authors consider these properties in relationto their use for a range of biocatalytic reactions.

    The majority of enzymes reported so far to be active inionic liquids belong to the class of lipases. Lipases andesterases are often used for the kinetic resolution of racemates either by the hydrolysis, esterification or trans-esterification of suitable precursors. The authors alsodiscuss the use of ionic liquids to carry out proteolytic andgalactosylation reactions and review recent work to charac-terise the properties of this important class of solvents.

    Screening for novel enzymes for biocatalyticprocessesLorenz and colleagues (pp 572577) summarise novelscreening techniques for the isolation of biocatalysts, whichcan be used in industry for the production of compounds forgeneral use. Obviously, the range of organic reactions performed by enzymes is enormous and finding a suitablebiocatalyst within a reasonable time scale requires efficientand sensitive screening strategies. In this article the authorsfocus on the recognition of non-cultivated and, in particular,prokaryotic microorganisms as the source of novel biocata-lysts. Traditional methods for obtaining such biocatalysts,through the cultivation and screening of pure strains, hasproved to be a powerful technique. Nevertheless, thisapproach is not straightforward and molecular ecologicalstudies have shown that only a fraction of the microbial

    diversity can be retrieved using this route. These limitationsprompted researchers to investigate the direct isolation ofmicrobial DNA from the environment, so giving a completerepresentation of the genome. The collective genomes of allliving organisms in a given habitat have been termed themetagenome. Analysis of the metagenome allows the maximum amount of sequence space to be accessed and is likely to aid the discovery of new enzymes. In this article,the authors discuss the use of both traditional cultivation methods and new direct cloning strategies and consider theroles they can play in identifying novel biocatalysts.

    Asymmetric conversion of ketones and alcoholsby enzymes coupled with metal catalysisThe article of Kim, Ahn and Park (pp 578587) reviewsrecent advances in the asymmetric conversion of ketonesand alcohols by enzymes coupled with metal catalysis. Theauthors describe a new practical application of enzymescoupled with palladium or ruthenium complexes for thesynthesis of asymmetric compounds. In the first section,the use of dynamic kinetic enzymemetal combo catalysis(where combo refers to a combination of differentenzymes and metals) is considered. This approach hasbeen shown to provide high overall yields and increasedenantiospecificity. For example, several racemic secondaryalcohols were converted to enantiomerically pure acylatedcompounds using a lipaseruthenium reduction systemwith very high yield and ee values generally greater than90%. Likewise, the authors discuss the resolution ofracemic hydroxyl acids, diols and hydroxy aldehydes tooptically pure molecules. Similar reactions with secondaryamines using lipasepalladium complexes are also presented.

    The second part of the review concentrates on asymmetrictransformations using enzymemetal combo catalysis.Examples of the reduction of many ketones using alipaseruthenium combination are given; the rutheniumcomplex catalyses the asymmetric reductive hydrogenationof the ketones. The asymmetric hydrogenation of enolacetates by a lipaseruthenium combo was also investigatedand again provided high yields and ee values.

    Again, where are the stars? We can see from the reviews published in this issue thatthey follow different pathways; however, all travel in thesame direction of the natural sciences. Where will they goto now? Maybe a cluster of new independent stars will beformed with a close relationship to each other.

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