Highlight

Metabolic engineering, new antibiotics andbiofilm viscoelasticity

Craig Daniels,1 Manuel Espinosa-Urgel,1

José-Luis Niqui-Arroyo,2 Carmen Michán3 andJuan L. Ramos1*1Consejo Superior de Investigaciones Científicas,Estación Experimental del Zaidín, Dept. ofEnvironmental Protection, C/ Prof. Albareda, 1,E-18008 Granada, Spain.2Bio-Iliberis R&D, Edificio BIC, Lab. 203, Av. de laInnovación, 1, E-18100, Armilla, Granada, Spain.3Universidad de Córdoba, Campus de Rabanales,Dept. of Biochemistry and Molecular Biology, EdificioSevero Ochoa C-6, 2a Planta, 14071, Córdoba, Spain.

In the following highlight we refer to a number of newadvances in the field of Biotechnology that addressissues relating to the synthesis of new antibiotics,new biocatalysts and matrices in biofilms.

Biofilms and pathogens

In the article by DiStefano and colleagues (2009), biofilmsof Staphylococcus aureus and S. epidermidis strains (tworeference strains and two ocular isolates) were analysedin terms of their viscoelastic properties, along with thecharacterization of some virulence traits and biofilm struc-tures. These bacteria are relevant pathogens that cancause chronic infections, associated with their ability toestablish a biofilm on the surface of medical devices(implants, catheters) or other materials such as contactlenses. The results give an idea of the rheological prop-erties of these biofilms and their behaviour as viscoelasticmaterials, showing also how biofilms formed by closelyrelated strains may have significantly different character-istics and physical properties. The ultimate causes forthese differences offer a valuable focus for future work,since they may shed light on the various survival andadaptation strategies adopted by related microorganisms.

This work by DiStefano and colleagues (2009) opensthe door to transversal and multidisciplinary research inthe biofilm field, whereby the fields of physics, bacterialphysiology and genetics can join forces to provide noveland important information on the structure and stability ofmicrobial biofilms. As the authors point out, therapies

combining antimicrobial agents with compounds capableof altering the viscoelastic properties of the biofilm couldprove highly effective to combat biofilm-associatedchronic infections. Furthermore, biotechnological applica-tions involving biofilms, such as membrane or granularsludge bioreactors, could benefit from this knowledge.Being able to predict how the biofilm will behave under theflow conditions imposed in the bioreactor or when shearforces change might result in a significant improvement inperformance.

Moschioni and colleagues (2009) from Novartis Vaccineand Diagnostic have reviewed the field of adhesins withina collection of important human and animal pathogensgrouped under the genus Streptococcus. These micro-organisms cause pharyngitis, scarlet fever, as well assevere skin infections and invasive infections (endocardi-tis, necrotizing fasciitis etc.). They are also primary colo-nizers of the surface of teeth and are abundant in dentalplaque. The authors emphasize that bacterial adherenceto host tissues represents a critical step in pathogenicprocesses and is usually mediated by bacterial surface-exposed proteins. Adhesins can be either single mole-cules or ordered structures such as pili or fimbriae.Interactions often involve binding to host extracellularmatrix components and cell surfaces. Streptococcaladhesins have been grouped into families based on thesubstrate they bind to, although some redundancy tendsto occur. The authors discuss examples of adhesins thatbind to fibronectin, fibrinogen, lamini, AgI/II family proteinsand others such as choline-binding proteins. Fibronectin-binding proteins have a modular structure with aC-terminal cell-wall-anchoring motif and a short positivelycharged tail. In this C-terminal region short repeats of30–40 residues can be found. This type of repetitive motifhas also been found in some other adhesion familymembers and may represent a distinctive feature ofadhesins since these sequence repeats are often foundwithin Pseudomonas putida and P. fluorescens adhesins(Espinosa-Urgel and Ramos, 2004; Hinsa and O’Toole,2006; Yousef and Espinosa-Urgel, 2007). The authors goon to discuss the interest in identifying small moleculesthat may interfere with one or more of these adhesins andtheir potential to be incorporated into dentistry products as

Microbial Biotechnology (2010) 3(1), 10–14 doi:10.1111/j.1751-7915.2009.00157.x

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd

inhibitors of plaque formation. They also discuss thepotential of pili and fimbriae to generate vaccines andsuggest research on potential applications of adhesinsin biotechnological applications. The review also takesadvantage of the extensive knowledge of the genomics ofthese microorganisms and the previous characterizationof many different species in the genus, allowing insightinto the set of well-studied adhesins, which is comple-mented by a very exhaustive citation of previous work thatfacilitates further exploration by the reader.

New antibiotics

With the increased prevalence of multidrug-resistantstrains of bacteria, cutting edge research is required toenhance our understanding of antibiotics, as well as thedevelopment of new forms of antimicrobials. Threearticles that are featured in this issue provide timelyinsight into both of these topics. Grosse and colleagues(2009) identify two novel esterases that could be used tooptimize the biosynthesis of levofloxacin (LFC) – a third-generation antibiotic that is used clinically to treat anumber of infections, including multidrug-resistant tuber-culosis. The two esterases, BcEST and TtEST, act on akey intermediate in LFC biosynthesis in an enantioselec-tive manner, and may therefore be used to resolveracemic mixtures, providing a more green and chemo-enzymatic route to LFC production. With the aim ofimproving the stability of BcEST, the authors carried outdirected evolution. After a two-step mutagenesis screenfor mutants exhibiting intact activity at higher tempera-tures, and the analysis of close to 7000 strains, theyshowed that the apparent melting temperature (Tm) of theBcEST enzyme could be increased by up to five degrees.These results were then compared with the performanceof TtEST, which was isolated from a thermophilicmicroorganism, showing that the naturally occurringbiocatalyst outperformed the experimentally evolvedBcEST mutants. As such, their results also show thatgenome mining within thermophiles may provide a cost-effective approach for the discovery of new thermostablebiocatalysts.

The article by Deegan and colleagues (2009) also hasimportant implications in the fight against multidrug-resistant bacteria, although, rather than concentrating ontraditional antibiotics, the work is focused on an unusualtype of antibiotic called lantibiotics. These antimicrobialpeptides are of particular importance due to the fact thatthey exhibit activity against multidrug-resistant pathogensat minute concentrations. By creating a set of 16 variantsof a two-peptide lantibiotic, named lacticin 3147, the workclarifies a number of aspects about the determinants ofantibacterial activity. While previous work has suggestedthat the overall charge of a lantibiotic may be important to

activity, the current results show that changes made to thepeptides impact antimicrobial activity in a location-specificmanner. This is particularly apparent for one of the vari-ants of the lacticin 3147 peptides, which was made to beuncharged while still retaining bioactivity. Notably, whenassessing the activity of the variants, it was found that oneexhibits heightened antibacterial activity. This result issignificant in that it is the first reported case in which theactivity of a lantibiotic has been increased. The challengeraised by subsequent results is that, although the peptideis twice as active by itself, the normal synergy that occurswhen both peptides are present is greatly reduced.Despite this difficulty, the work calls for new researchfocused on the optimization of such peptides to furtherincrease their efficacy. Lantibiotics are particularly rel-evant to solving the problem of the rapid rise of multidrug-resistant bacteria, providing a quick and versatile toolfor the development of new and powerful antibacterialcompounds.

Another interesting group of molecules that are rel-evant for pharmaceutical research and drug develop-ment are b-peptides or mixed b,a-peptides that containb-amino acid residues – naturally occurring productsthat exhibit high biological activities. While commona-peptides are rapidly degraded in vivo and in vitro by amultitude of enzymes, peptides containing b-amino acidresidues show a high level of resistance to proteolyticattack, making them ideal candidates for new antibioticsand other pharmacologically active compounds. In thereport presented by Heyland and colleagues (2009),both factors are considered as they describe the devel-opment and characterization of whole-cell biocatalystsystems for obtaining b,a-peptide L-carnosine. The useof whole cells as industrial biocatalysers can consider-ably reduce time and material costs associated withalternative chemical synthesis procedures, althoughusually the technique may be limited due to reducedactivity or the generation of unwanted byproducts. Theauthors examine differences in the production of b,a-peptides (DmpA and 3-2W4 BapA) using enzymesversus the use of recombinant hosts (Escherichia coliand Pichia pastoris), thereby comparing the use of crudeextracts versus whole cells systems. They selected E.coli whole cells with recombinant DmpA as the mostpromising production method and, with this system, per-formed further optimization studies by varying the celldensity, temperature, pH, induction kinetics and sub-strate concentration. Furthermore, they also checked thestability and the efficacy of the process in both batchand fed-batch cultures, obtaining satisfactory rates inboth conditions, together with scarce production of unde-sirable secondary metabolites. These results could beapplied to the production of new peptides with usefulbiological activities although further efforts are required

Metabolic engineering 11

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 3, 10–14

in order to optimize the expensive substrate needs andto develop efficient new methods for product purification.

Metagenomics and biocatalysis

A recent Special Issue of Environmental Microbiology out-lined a number of approaches that have been developedfor the recovery of DNA from a given niche during metage-nomic analysis. Cloning of ‘environmental’ DNA withoutthe need to cultivate the source microorganism is a tre-mendous advantage in spite of the fact that a number ofbiases in cloning and/or expression have been identified(Aharoni, 2009; Ferrer, 2009). One of the most relevantadvantages of massive DNA cloning is the increased pos-sibility of discovering enzymes with novel properties. In thecurrent issue of Microbial Biotechnology, two researcharticles deal with the recovery of relevant new enzymesfrom the metagenomic point of view. In one of the articles,Vieites and colleagues (2009) screen a metagenomiclibrary constructed from the gut of the earthworm Aporrect-dea caliiginosa. During the experimental process, theearthworm was fed with cellulosic compounds in order tofavour the growth of microbes that are able to attackesters. Upon detailed screening, the authors describe anesterase, called 3A6, that exhibits unusual activity as acarboxyl esterase and a feruloyl esterase, which is signifi-cantly different from the known function of other proteinshomologous to 3A6. Using docking methods, the authorsconstruct a model of the enzyme based on the available3D structures of other carboxyl esterases. The modelidentifies three residues (Ser-143, Asp-273 and His-305)as potential catalytic sites, which are confirmed upon thegeneration of point mutants that lose activity. The model isthen examined to define ‘areas of influence’ regardingsubstrate specificity and substrate preference. Theseregions comprise triads or dyads made of residues 109–111, 315–317 and 281/282. The authors have performedexhaustive site-directed mutagenesis of these regionsfollowed by substrate discrimination assays. Mutants inthese residues are often associated with an increasedpreference in the mutant variant for carboxyl esteraseversus feruloyl esterase activity, while a correlationbetween substrate preference and activation energyexists. One of the most remarkable findings is that a34-amino-acid residue loop deletion leads to a variant thatretains carboxyl esterase activity while completely losingferuloyl esterase activity. Vieites et al. discuss how feruloylesterase activity may be the result of selective pressurethrough nutritional/environmental situations that demandfor a broader range of substrates at the expense of speci-ficity. In this regard the potential for regulators to interactwith a variety of effector molecules; i.e. XylR (Garmendiaet al. 2009) and XylS variants (Brautaset et al., 2008) havebeen described. Although the plasticity of the enzymes

and regulators reflects their evolutionary capacity, itseems unlikely that the changes can occur in such animmediate fashion that the response can account for thetemporary nutritional variation in the microbial status. Theresearch article by Vieites et al., together with the instruc-tive review on glutamate racemase by Fisher (2009),advocates the relevant role Microbial Biotechnology canplay in the diffusion of important biocatalysis information.

Fernández-Álvaro and colleagues (2009) also usesmetagenomic libraries to search for relevant new enzy-matic activities. In particular, the author screens one suchlibrary for the hydrolysis of ethyl esters within racemicphenylalkyl carboxylic acids. The elegant pH indicatorassay used was further refined to obtain enantioselectiveenzymes, which were then utilized to obtain excellentoptically pure chemicals. This work provides an exampleshowing how the search for specific activities provides anexcellent approach for the advancement of the use ofenzymes and microbes in the synthesis of chemicals.

Another innovative approach in the design of biocatalyticprocesses is the external control of the reactions. In thisissue, Kraus and colleagues (2009) present an attractivereview on the use of LOV (Light, Oxygen, Voltage) domainsfor the construction of photoactive-controlled chimeric pro-teins. Furthermore, the authors develop the use of thiswidely used mechanism for the construction of functionalhybrid regulators that can be easily controlled by light. Theadvantages of photoreceptors as external inducers includelimited cell damage and, most importantly, precise controlin the timing and the extent of the signal effect, whileadditionally, these new hybrid regulators could be poten-tially used within a broad range of organisms as photochro-mic proteins are present in numerous biological processes.In the past, this approach has been successfully used inthe construction of chimeras between structurally relatedproteins. For example, Stricland and colleagues (2008)placed the E. coli trp repressor domain under the control ofthe LOV domain from Avena sativa phototropin 1. The maintechnical difficulty associated with the use of these con-structs when connecting unrelated proteins is that it is oftendifficult to ensure that the domains are fused at the rightpositions. To solve this problem, the authors propose theuse of statistical coupling analysis (SCA) to identify net-works of co-evolving amino acids in different proteins,which allows the location of potential surface allostericsites connecting distant protein domains. Using thisapproach, they designed fusions between a LOV domainto the DHFR biocatalytic region, generating enzymes thatcatalyse the reduction of 5,6-dihydrofolate under lightcontrol. The mechanism proposed opens new avenues inthe research of effective and inexpensive regulators forbiocatalysis. Future research will be required to improvethe efficacy of the process and to discover potential usesfor modulating enzyme specificity.

12 C. Daniels et al.

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 3, 10–14

Enhanced vitamin production

Production of commercially significant metabolites thatare important human nutrients is a vital field of microbialbiotechnology (Sánchez and Demain, 2009). The amplifi-cation of commercial production processes is of funda-mental importance as increased yields translate intopositive public and financial outcomes. Recently in Micro-bial Biotechnology Biedendieck and colleagues (2009)published a fascinating research article which detailed themetabolic engineering of Bacillus megaterium to increasethe production of cobalamin (vitamin B12). They used thecobalamin production system of B. megaterium to assessvarious genetic optimization strategies including plasmidand genome-based overexpression of genes, enzymemodification for increased stability and manipulation ofregulatory genes. The authors report that all of the adjust-ments could lead to increased production of cobalamin,with some of the manipulations, such as the overexpres-sion of cobalamin pathways, leading to a near 40-foldincrease in B12 production. Follow-up experiments in-cluded the use of an antisense RNA strategy to reducethe flow of metabolites along an alternative route andremoval of the riboswitch inhibitory structure locatedupstream of the main B12 operon. Again, these latter modi-fications were shown to increase cobalamin production inB. megaterium when used individually. Interestingly andsomewhat unfortunately, experiments using combinationsof the above manipulations, while able to augment theintracellular cobalamin concentrations, showed compro-mised cell growth because the alterations placed the bac-teria under metabolic stress. This research emphasizesthe importance of a systematic approach in the implemen-tation of strategies to improve nutrient metabolite yield forlarge-scale production systems and suggests that a moresubtle approach may garner a better final result.

Biodegradation

Polycyclic aromatic hydrocarbons (PAHs), consideredcommon examples of persistent organic pollutants(POPs), have been reported to be present in many envi-ronments. As a result of their low water solubility andstrong sorption to soil particles (mainly to organic matterand clay fractions), these compounds can exhibitextremely long half-lives exceeding 1000 days for somePAHs, as benzo[a]pyrene in soils and sediments. SincePAHs occur in the environment as complex mixtures,behaviour and capabilities of bacterial consortia need tobe elucidated. Moreover, it is well known that in somecases, low-molecular-weight PAHs can inhibit or restrictthe biodegradation of heavier compounds (reviewed byRi-He et al., 2008). Despite the fact that PAHs are sub-jected to different abiotic processes, once they have beenreleased into the environment (volatilization, photo-

oxidation), it is assumed that biodegradation is the mainway that these compounds are removed (Lotfabat andGray, 2002). Thus the great interest that exists for theidentification of the mechanisms used by microorganismsto degrade these persistent chemicals is not surprising.Indeed, a considerable number of bacteria with PAH-degrading capabilities, either growth-linked reactions orcometabolic processes, have been isolated and charac-terized. Since the biodegradation of low-molecular-weightPAHs (especially naphthalene) is well documented, futureefforts must be aimed towards studying the biodegrada-tion of high-molecular-weight compounds (HMW), that is,those that contain more than three benzene rings. Alongthese lines, the current issue of Microbial Biotechnologyfeatures a article by Kanaly and Harayama (2009), inwhich the authors offer us a new and a comprehensivestudy of HMW-PAH biodegradation. Continuing along thelines of previous publications (Kanaly and Harayama,2000), these authors present us a magnificent over-view of the achievements attained during the past 20years, not only for pyrene and fluoranthene, but also forbenzo[a]anthracene and benzo[a]pyrene. Since the firstreports published in 1975 by Gibson and Barnsley aboutbiodegradation of the latter two compounds cited above,advances have been made due to the isolation of newPAH-degrading microorganisms as well as in-depth inves-tigations of previously known microorganisms, such asMycobacterium vanbaalenii PYR-1, which is one of themost relevant PAH degraders. In fact, the metabolicpathway used by this strain for the biodegradation offluoranthene has been recently elucidated. Novel metabo-lites and enzymes have been detected and identified, andnew pathways have been proposed in the last twodecades. In connection with this, methanogenic biodegra-dation of PAHs has been recently postulated (Dolfinget al., 2009). Furthermore, development of a number ofnew metabolomic, genomic and proteomic technologieshas contributed enormously to these findings. In summary,the article by Kanaly and Karayama is an invaluablesource of information for those who wish to deepen theirknowledge of HMW-PAH biodegradation.

References

Aharoni, A. (2009) Mining new enzymes. Microb Biotechnol2: 128–129.

Biedendieck, R., Malten, M., Barg, H., Bunk, B., Martens,H.-H., Deery, E., et al. (2009) Metabolic engineering ofcobalamin (vitamin B12) production in Bacillus megate-rium. Microb Biotechnol 3: 24–37.

Brautaset, T., Lale, R., and Valla, S. (2008) Positively regu-lated bacterial expression systems. Microb Biotechnol 2:15–30.

Deegan, L.H., Suda, S., Lawton, E.M., Draper, L.A., Hugen-holtz, F., Peschel, A., et al. (2009) Manipulation of charged

Metabolic engineering 13

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 3, 10–14

residues within the two-peptide lantibiotic lacticin 3147.Microb Biotechnol (in press): doi: 10.1111/j.1751-7915.2009.00145.x.

Di Stefano, A., D’Aurizio, E., Trubiani, O., Grande, R., DiCampli, E., Di Giulio, M., et al. (2009) Viscoelastic proper-ties of Staphylococcus aureus and Staphylococcus epider-midis mono-microbial biofilms. Microb Biotechnol 2: 634–641.

Dolfing, J., Xu, A., Gray, N.D., Larter, S.R., and Head, I.M.(2009) The thermodynamic landscape of methanogenicPAH degradation. Microb Biotechnol 2: 566–574.

Espinosa-Urgel, M., and Ramos, J.L. (2004) Cell density-dependent gene contributes to efficient root colonization byPseudomonas putida KT2440. Appl Environ Microbiol 70:5190–5198.

Fernández-Álvaro, E., Kourist, R., Winter, J., Böttcher, D.,Liebeton, K., Naumer, C., et al. (2009) Enantioselectivekinetic resolution of phenylalkyl carboxylic acids usingmetagenome-derived esterases. Microb Biotechnol 3:59–64.

Ferrer, M. (2009) The microbial reactoma. Microb Biotechnol2: 133–134.

Fisher, S.L. (2009) Glutamate reacemase as a target for drugdiscovery. Microb Biotechnol 2: 345–360.

Garmendia, J., de las Heras, A., Calgagno-Galvao, T., and deLorenzo, V. (2009) Tracing explosives in soil with transcrip-tional regulators of Pseudomonas putida evolved forresponding to nitrotoluenes. Microb Biotechnol 1: 236–246.

Grosse, S., Bergeron, H., Imura, A., Boyd, J., Wang, S.,Kubota, K., et al. (2009) Nature versus nurture in two highlyenantioselective esterases from Bacillus cereus and Ther-moanaerobacter tengcongensis. Microbial Biotechnol 3:65–73.

Heyland, J., Antweiler, N., Lutz, J., Heck, T., Geueke, B.,Kohler, H.-P. E., et al. (2009) Simple enzymatic procedurefor L-carnosine synthesis: whole-cell biocatalysis and effi-cient biocatalyst recycling. Microb Biotechnol 3: 74–83.

Hinsa, S.M., and O’Toole, G. (2006) Biofilm formation byPseudomonas fluorescens WCS365: a role for LapD.Microbiology 152: 1375–1383.

Kanaly, R.A., and Harayama, S. (2000) Biodegradation ofhigh-molecular-weight polycyclic aromatic hydrocarbonsby bacteria. J Bacteriol 182: 2059–2067.

Kanaly, R.A., and Harayama, S. (2009) Advances in the fieldof high-molecular weight polycyclic aromatic hydrocarbonbiodegradation by bacteria. Microb Biotecnol (in press):doi: 10.1111/j.1751-7915.2009.00130.x

Krauss, U., Lee, J., Benkovic, S.J., and Jaeger, K.-E. (2009)LOVely enzymes – towards engineering light-controllablebiocatalysts. Microb Biotecnol 3: 15–23.

Lotfabat, S.K., and Gray, M.R. (2002) Kinetics of biodegra-dation of mixtures of polycyclic aromatic hydrocarbons.Appl Microbiol Biotechnol 60: 361–365.

Moschioni, M., Pansegrau, W., and Barocchi, M.A. (2009)Adhesion determinants of the Streptococcus species.Microb Biotechnol (in press): doi: 10.1111/j.1751-7915.2009.00138.x

Ri-He, P., Ai-Sheng, X., Yong, X., Xiao-Yan, F., Feng, G., Wei,Z., et al. (2008) Microbial biodegradation of polyaromatichydrocarbons. FEMS Microbiol Rev 32: 927–955.

Sánchez, S., and Demain, A. (2009) Metabolic regulation andoverproduction of primary metabolites. Microb Biotechnol1: 283–319.

Stricland, D., Moffat, K., and Sosnik, T.R. (2009) Light-activated DNA binding in a designed allosteric protein. ProcNatl Acad Sci USA 105: 10709–10714.

Vieites, J.M., Ghazi, A., Beloqui, A., Polaina, J., Andreu, J.M.,Golyshina, O.V., et al. (2009) Inter-conversion of cataly-tic abilities in a bifunctional carboxyl/feruloyl-esterasefrom earthworm gut metagenome. Microb Biotechnol 3:48–58.

Yousef, F., and Espinosa-Urgel, M. (2007) In silico analysisof large microbial surface proteins. Res Microbiol 158:545–550.

14 C. Daniels et al.

© 2009 The AuthorsJournal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 3, 10–14