Modern industrial microbiologyhas a history going back at most

half a century, so it was fitting thatthe most recent of the quadrennialGenetics of Industrial Microorgan-isms conferences was held in Israel*,a country celebrating its 50th birth-day in 1998. Not surprisingly, theimpact of genomics on the rate atwhich information on microbialdiversity, genetics and metabolism isgenerated featured strongly, and theknowledge gained is rapidly beingintegrated into therapeutic discoveryand development strategies.

After the previous GIM confer-ence (Montreal, 1994), there was areturn to emphasizing genetics, withStreptomyces and other actinomycetesholding centre stage, illustrating themolecular biology of growth, differ-entiation and secondary metabolism.Genomics is also finding its way intosome less-obvious areas of industrialR&D, enhancing host-strain produc-tivity and product quality, and themajority of microorganisms of indus-trial interest – lactic-acid bacteria,yeasts, bacilli, clostridia, filamentousfungi and Archaea – had dedicatedsymposia.

Bacterial genomicsThe rise of microbial genetics over

the past four decades has followedthe development of technologies forgene transfer and analysis to the cur-rent capabilities for complete genomesequencing, global functional analy-sis of the genome and comparativegenomics. With the Sanger Centre(Hinxton, UK) initiating sequencingof the Streptomyces coelicolor genome on 1 August 1997, the past year hasalready yielded many genetic noveltiesin the >2 Mb so far sequenced.

In the first plenary lecture, DavidHopwood (John Innes Centre, Norwich, UK) noted that approxi-mately 27% of the open readingframes (ORFs) identified were pre-

viously unknown, a finding commonto that for completed genomes ofother bacteria. However, assuming asimilar high gene density of approxi-mately 1.2 kb per ORF around its8 Mb genome, S. coelicolor shouldpossess nearly 7000 genes, almosttwice as many as Escherichia coli orBacillus subtilis, and some 20% morethan yeast.

Why should this be? Streptomycetesshow complex morphological differ-entiation throughout their life cycle,coupled with the production of abouttwo-thirds of the known naturallyoccurring antibiotics, as well anti-helminthic, antitumour and antifungalagents, and herbicides. Many of thesecomplex secondary metabolites areproduced by large multifunctional ormultienzymic proteins.

Modular polyketide synthases haveevolved ‘like a conveyor belt’ to makemacrolide antibiotics: for example,candicidin D biosynthesis requires 21 condensations, and approximately50 kb are required to handle this bio-chemically. However, it appears thatdifferentiation and secondary metabo-lism account for only a few hundreddedicated genes; many of the remain-der are probably involved in theorganism’s sensing and responding tochanges in its environment (the soil).So far, nine ABC transporters havebeen found; again extrapolating, thiswould predict approximately 180 forS. coelicolor, compared with 80 forE. coli and 77 for B. subtilis.

Genomic sequencing and func-tional analysis should reveal answersto many other key questions, such aswhy the G�C content of Strepto-myces is so high and why its chromo-some is linear. There is circumstan-tial evidence that, in contrast to otherbacteria, RecA is indispensable inStreptomyces (at least in Streptomyceslividans), where it has been suggestedto be involved in recombinationalrepair of single-stranded breaks in thelinear chromosome (Gunter Muth,University of Tubingen, Tubingen,Germany).

Comparative genomics may answersome questions but it will pose others.

Carton Chen (Institute of Genetics,Taipei, Taiwan) noted the surprisingsimilarity between the secondarystructure (hairpin structures, withidentical loop sequences) of the 3�ends of the Streptomyces chromosomesand of the autonomous parvoviruses.These hairpin loops consist of purine–purine sheared base pairs and a singleunpaired nucleotide, and are highlyresistant to (single-strand-specific) nu-cleases. Why this similarity betweenthe telomeres of bacteria and eukary-otic viruses? Have they been selectedindependently during evolution forstructural stability, or have they some-how crossed paths during evolution?

Assessing and exploitingmetabolic diversity

In another plenary lecture, Julian Davies (TerraGen Diversity,Vancouver, Canada) illustrated howthe assessment of biodiversity is lead-ing to the concept of a phylogenetic‘bush’, whereby there was no singleprogenitor: horizontal gene transfer,probably between all species and allgenera at some time, is leading to achange in the way we think aboutevolutionary relationships.

The therapeutic bioactives that arecurrently available originate from the�1% of microorganisms that are culturable – what might the other99% yield? By direct PCR cloningfrom ‘environmental DNA’ into sur-rogate host libraries, it is possible tocapture novel chemical diversity:30–40 kb in a novel host will yieldnovel products, lead to gene-clusterexpression and the activation of cryp-tic genes, and allow the study of mol-ecular decoration and the generationof random and directed hybrids.

Symbiotic relationships are also arich source of secondary metabo-lites: among the ascomycetes (29 000species in 37 orders), 46% formlichens; lichenized forms of cyano-bacteria are a distinct evolutionarygrouping. Lichens are rich sources ofnew polyketide synthase (PKS) genes,which are involved in the synthesisof linear and aromatic (both simpleand complex) polyketides. Studies ofmicrobial-community interactionsand evolution, and changes in diver-sity patterns could be applied to moni-toring and predicting environmentalchanges.

The search for new secondarymetabolites is expanding to includeboth slow-growing and unculturableorganisms such as endophytic fungi

FORUM

178 0167-7799/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0167-7799(98)01285-2 TIBTECH MAY 1999 (VOL 17)

*The 8th International Symposium on the Genet-ics of Industrial Microorganisms (GIM ’98), orga-nized by G. Cohen and Y. Aharonowitz, washeld in Jerusalem, Israel, 28 June – 2 July, 1998.

The genetics of industrial microorganisms: the first half

centuryMeetingreport

and actinomycetes (Stefano Donadio,BioSearch Italia, Gerenzano, Italy).Michael Young (University ofWales, Aberystwyth, UK) reportedthe first ‘bacterial cytokine’, a 17 kDaproteinaceous pheromone (Rpf)secreted by Micrococcus luteus, whichis active at picomolar concentrations.Similar genes are found in other highG�C bacteria, including S. coelicolor,several mycobacteria and coryne-bacteria. By stimulating the growthof recalcitrant microorganisms inculture, Rpf will enable untappedbiodiversity to be accessed andshould also facilitate the diagnosis ofmycobacterial infections.

Genomics in therapeuticR&D

Yigal Koltin (Millenium Pharma-ceuticals, Cambridge, MA, USA)emphasized the need to improve theproductivity of drug discovery. Gen-etic and genomic technologies (e.g.transcription profiling and proteom-ics) offer an opportunity to exploitthe new chemical diversity withhigh-throughput technologies to linklead compounds with their cognatetargets, and to reverse the discoveryprocess from target identification,validation and screening to a processdriven by chemistry that dispenseswith the slow and expensive validationprocess.

Experimentation in therapeuticR&D is increasingly driven by bio-infomatics. Mark Egerton (IncytePharmaceuticals, Palo Alto, CA, USA)indicated how genomics is affectingantifungal R&D in both target identi-fication and the downstream phases.Primary sequence homology can beused to evaluate target selectivity:minimal homology with human se-quences indicates a greater chance ofsuccess. Candidate targets must have aknown, essential function: the modeof action of lead compounds identifiedthrough high-throughput screeningcan be examined through transcriptimaging, which monitors genome-wide changes in gene expression.

The search for new antibacterialdrugs is expanding to include viru-lence genes and genes required formaintenance of the infection as tar-gets (Philip Youngman, Millenium Pharmaceuticals). Complete bacterialgenomes can be surveyed for suchgenes by comparing the input ofmarked genes to the output from aninfected model or by identifying pro-moters induced during infection.

Microbial models in drugdiscovery

Julian Davies’ observations on therelatedness of all organisms are beingput to good use in developing drug-discovery strategies. Yeasts are valu-able for expressing and studyingmany human proteins, which oftenexhibit normal processing and func-tion in this host. One example of thisis the engineering of Saccharomycescerevisiae strains to produce activehuman oestrogen receptors, allowinghigh-throughput screening of candi-date inhibitor molecules (ChuckHershberger, Eli Lilly, Indianapolis,IN, USA).

Drugs targeting G-protein-signalling pathways already pro-vide global sales of approximatelyUS$85 billion (1996 data), but anestimated 2000–5000 G-protein-coupled receptors (GPCRs) are yetto be identified. The development ofsurrogate agonists can be used to studyreceptor activation in cultured cells:human GPCRs were linked to themating-factor response of S. cerevisiaeby deletion of the endogenouspheromone receptor and substitutionof the yeast G� subunit with a mam-malian G� protein (James Broach,Cadus Pharmaceutical, Tarrytown,NY, USA).

Hilgund Schrempf (University ofOsnabruck, Osnabruck, Germany)reported the discovery of the firstprokaryotic potassium channel (KcsA)in S. lividans. It is a small protein,unrelated to other bacterial potas-sium transporters, and possesses twopotential membrane-spanning heliceslinked by a central domain, resem-bling the H5 segment of eukaryoticion channels. Homologues have beenidentified in other streptomycetesand, based on sequence homology,are also likely to be found in Clostrid-ium, Mycobacterium, Methanococcus andSynechocystis.

Genome to transcriptome toproteome

The development of sensitive tech-nologies for ‘global analysis’ of geneexpresssion is permitting the entireprogramme of a bacterial cell’s re-sponse to environmental changes tobe deciphered. As Richard Losick(Harvard University, Cambridge,MA, USA) noted in his plenary ‘four-dimensional view’ of a bacterial cell,it is important to know not only whata protein or DNA segment does butwhen and where it does it. Cytology

and time-lapse microscopy are re-vealing much about chromosomesegregation and architecture, cyto-kinesis and morphogenesis, and thatproteins have addresses – even multi-ple subcellular addresses – and thatthese addresses can change.

Several speakers emphasized that,even though the genome provides theblueprint, the proteome (recentlyredefined as the ‘protein complementexpressed by a genome at a giventime’) provides a more-accurate pic-ture of life. From studies of the stressresponse in B. subtilis, Michael Hecker(Institute of Microbiology and Mol-ecular Biology, Greifswald, Germany)illustrated how the function ofunknown regulons can be uncoveredby examining induction patterns inresponse to particular stimuli.

Simultaneous whole-transcriptomeanalysis is being facilitated by DNA-chip technology, whereby functionalanalysis of whole gene sets [peptidenucleic acid (PNA) oligomers beingpreferable to DNA as PNA–RNA-probe hybrids are more stable] canprecede and direct sequencing togenomic regions of interest (JörgHoheisel, DKFZ, Heidelberg, Germany). In examining regulatorynetworks induced by antibiotics,Chris Gray (Hoffman La Roche,Basel, Switzerland) indicated thatcare is needed in interpreting tran-script analysis, as mRNA levels mayreflect a qualitative but not a quanti-tative relationship with protein syn-thesis for particular proteins. Theworking theory is that pulse-labelledproteins are analysing only a smallsubpopulation of cells (continuouslabelling shows a less dramaticchange), while mRNA levels reflectthe whole population – this mightindicate that transcriptional analysis isnot adequate to detect small but sig-nificant changes, such as occur forregulatory proteins.

Metabolic engineeringThe production of secondary

metabolites is dependent on inter-actions between primary and sec-ondary metabolites, and nowhere isthis better illustrated than in thecoordination of the growth, differ-entiation and antibiotic productionof streptomycetes. S. coelicolor pro-duces at least four chemically diverseantibiotics of known structure,whose production is controlled byboth global and pathway-specificregulatory genes.

FORUM

TIBTECH MAY 1999 (VOL 17) 179

One pleiotropic regulatory gene,bldA, encodes a leucyl tRNA; bldAmutants are defective in the produc-tion of aerial hyphae and of all fourantibiotics. The interaction of severalgenes (relA, bldA, afsR and afsB) controls the production of the pig-mented antibiotics actinorhodin andundecylprodigiosin, and a pathway-specific regulatory cascade for undecylprodigiosin has been charac-terized (Mervyn Bibb, John InnesCentre). In addition, an unusual �-butyrolactone has been identifiedas a signalling molecule that elicitsthe production of both these antibiotics.

Multiphasic growth is associatedwith changes in the levels of cAMP,which appears to function as a sig-nalling molecule, mediating switchesin carbon metabolism required forgermination, antibiotic synthesis andaerial mycelium formation (CharlesThompson, Biozentrum, Universityof Basel, Switzerland). The first Strep-tomyces adenylate cyclase (cya) mutantto be isolated cannot neutralizemedium acidified during myceliumformation. Such acid toxicity can bealleviated by the addition of ex-ogenous cAMP, which also increasesthe accumulation of actinorhodinbut decreases undecylprodigiosinproduction.

The production of the microbialhormone A-factor is growthdependent and, by binding to thespecific receptor ArpA, switches onboth streptomycin production andsporulation in Streptomyces griseus.Sites involved in DNA binding andA-factor recognition were revealedby site-directed mutagenesis of ArpA(Sueharu Horinouchi, University ofTokyo, Tokyo, Japan). The targetsequence of ArpA was found up-stream from adpA, whose producthad previously been identified as anactivator of strR, which encodes apathway-specific transcriptional acti-vator for streptomycin biosynthesis.

The �R subunit of RNA polym-erase is encoded by the sigR gene.This subunit is responsible for theresponse to oxidative stress in S. coeli-color A3(2) (Mark Paget, John InnesCentre). The tyxBA operon, encod-ing thioredoxin reductase and thio-redoxin, is under the control of �R. RsaA was identified as a sensorof the oxidative stress in this system,which resembles that found ineukaryotes in that it operates via signal transduction.

Where to startUnderstanding the pathways of

secondary metabolism is a prerequi-site for their manipulation. HeinzFloss (University of Washington,Seattle, WA, USA) showed that, in the riflamycin biosynthetic path-way, the aminohydroxybenzoic acid(AHBA) starter unit is formed via anamino shikimate pathway, and thatthe completed polyketide is releasedfrom the enzyme and cyclized by anamide synthase rather than by athioesterase. William Strohl (MerckResearch Laboratories, Rahway, NJ,USA) studied the PKS-gene clusterinvolved in daunorubicin produc-tion. Although both the dpsC anddpsD genes are involved in selectionof the propionyl starter unit, it islikely to be the dpsC gene that controls starter-unit selection.

Until now, only the type-I fungaland type-II bacterial PKSs had beenreported to synthesize aromatic com-pounds. However, Stephen Gould(Oregon State University, Cornvallis,OR, USA) reported that Pseudo-monas fluorescens PFj-5 uses modulartype-I PKS subunits to producepyoluteorin, an aromatic polyketide,by assembling the benzene ring froma proline-derived starter unit andthree malonyl extender units.

The mithramycin gene clusterspecifies the formation of an anti-tumour polyketide with an aromaticthree-ring core decorated with severaldeoxy sugars. It contains two of thethree genes encoding the enzymesdirecting synthesis of S-adenosyl-L-methionine (José Salas, University ofOviedo, Oviedo, Spain). Presumably,this cofactor is needed for the O- andC-methylation steps of the mithra-mycin pathway.

Enhancing host productivityrDNA technology can be used to

optimize host performance by modi-fying basic metabolic capabilities. Adoubling of erythromycin yield bybatch culture was achieved by stableexpression of Vitreoscilla haemo-globin (VHb) in the producer strainSaccharopolyspora erythrae (Jay Bailey,ETH Zurich, Zurich, Switzerland).The mechanism by which VHbovercomes oxygen limitation mightbe an upregulation of oxygen-response-gene expression because, inoxygen-limited E. coli, intracellularVHb expression increases ATP pro-duction, membrane �pH, growthrate and protein production.

Lactic-acid bacteriaExopolysaccharides (EPSs) pro-

duced by lactic-acid bacteria are usedextensively as thickeners in the foodindustry and also show potential foruse as immunostimulants. The gen-etic organization of several eps geneclusters has been reported, and thatof Lactococcus lactis NIZO B40 (14genes) has been functionally analysed.Engineering of EPS production hasmade use of a nisin-inducible expres-sion system to enhance expression,and plasmid-based systems conferredEPS synthesis on nonproducingL. lactis strains (Willem de Vos, NIZO,Wageningen, The Netherlands).

The productivity of lactic-acidbacteria is severely affected by phageattack and Doug van Sinderen (Uni-versity College, Cork, Ireland) out-lined strategies based on a naturallactococcal defence system to inter-fere with phage amplification or toblock phage adsorption or injection.

In addition to their traditionalfood-industry use, L. lactis expressionsystems can yield high levels of het-erologous products such as Bacillusthuringiensis insect toxin, vaccineantigens, mammalian cytokines andnovel EPSs (Jeremy Wells, Univer-sity of Cambridge, Cambridge, UK).With GRAS (generally regarded assafe) status and as a noninvasive, non-pathogenic, ‘particulate’ (i.e. whole-cell) system, L. lactis is also of inter-est for delivering protective antigensfor mucosal immunization: oral orintranasal immunization of micewith recombinant L. lactis expressingtetanus-toxin fragment C elicited agood protective response.

ClostridiumDavid Jones (University of Otago,

Dunedin, New Zealand) discussed thepotential impact of the completion ofthe genome sequence of Clostridiumacetobutylicum 824 (expected by the endof 1998) on renewed interest for its usein industrial butanol production.

As with lactic-acid bacteria, bac-teriophage infection and strain de-generation lead to problems of poor productivity, but the discoveryof dormant or defective bacterio-phage sequences in the genome couldprove valuable – harbouring suchsequences makes a strain immune toother phages. So far, as with otherbacterial genomes, approximately20–30% of the ORFs have no knownfunction, although many degradativeenzymes have been found (11 for

FORUM

180 TIBTECH MAY 1999 (VOL 17)

polysaccharide degradation, 16 forhemicellulose and 24 for cellulose).

BacillusThree presentations by members

of the European Bacillus SecretionGroup (EBSG) (Sierd Bron, Depart-ment of Genetics, University ofGröningen, Haren, The Nether-lands; Colin Harwood, NewcastleUniversity, Newcastle, UK; WimQuax, University Centre of Phar-macy, Gröningen, The Netherlands)reviewed the Sec-dependent secre-tion pathway in B. subtilis.

Most components of the B. subtilissecretion apparatus have now beenidentified – an achievement greatlyaided by the recently completedB. subtilis genome project. B. subtiliscontains five related signal-peptidasegenes, which vary in their patterns oftemporal gene expression and sub-strate preference, presumably toenable the host to adapt to changingsoil conditions. Modification of cell-wall-associated proteases andother components of the secretionpathway should permit improvedsecretion of heterologous proteins:preliminary results, using humaninterleukin 3, are encouraging.

The groups of Lubbert Dijkhuizenand Wim Dijkstra (University of Gröningen, Gröningen, TheNetherlands) have crystallized thecyclodextrin glycosyltransferase(CGTase) from Bacillus circulans.Modification of specific amino acidresidues has generated CGTasemutants with improved specificity,and a study of these mutants showedthat the C-terminal raw-starch-binding motif is largely responsiblefor strong product inhibition.

Redirecting evolutionIn the final plenary session, Willem

Stemmer (Maxygen, Santa Clara,CA, USA) illustrated the successes of molecular breeding, a concept that combines the exploitation ofnatural diversity and recombination(as in classical plant breeding) withmolecular biological techniques such as directed mutagenesis. Theminimum inhibitory concentration(MIC) of -lactamase was improved32 000-fold by three cycles of DNAshuffling followed by two rounds ofwild-type back-crossing to flush outany mutations not contributing to an increased MIC. The molecularbreeding of green fluorescent protein(GFP) for enhanced fluorescence and

enzymes for altered specificity (e.g.converting glucosidase to fucosidase)have been reported over the past yearor so. T. Borchert (Novo Nordisk,Bagsvaerd, Denmark) also describeda method based on the in vivo re-combination of mutated DNA se-quences that significantly increasesthe ability to use directed evolutionin S. cerevisiae.

By recombining conservativeblocks of DNA (homologues fromdifferent species or related genesfrom one species), ‘family shuffling’allows sparse sampling of a broadersequence space and gives better opti-mization than using single-genemutations. For cephalosporinase, forexample, using a family-shufflinglibrary generated a 270–540-foldincrease in resistance, compared withan eightfold increase obtained with apoint-mutation library.

Multipurpose enzyme libraries arenow being developed; the parentalenzymes are added in and the desiredspecificities then evolved. If closelyrelated genes and comprehensivelibraries are used, it is possible to getsignificant improvements from arelatively small number (100–1000)of mutants: a recombinant humaninterferon with a 10 000-fold increasein activity was generated using six seg-ments from five ‘parental’ molecules.

The future – GIM 2002In the final round-up of the first

GIM conference with a majorgenomics input, panellists consideredthe impact of genomics on therapeu-tics discovery. There are three chal-lenges – generating targets, generatingassays and generating compounds forscreening. Genomics can provide alarge number of targets and a ‘genomeon a chip’ will allow parallel andreproducible analysis of all targets.Major breakthroughs will come fromthe ability to assay proteins ofunknown function. Assay develop-ment will include ‘genetic’ assaysbased on phenotype, binding assays inwhich proteins are bound to chips andscreened with analytes, and enzymaticassays (which, however, requireknowledge of protein function).

Functional analysis will be facili-tated by comparative genomics, butthis requires data from many wholegenomes. However, genomics andmethods for accessing diversity aretools, not solutions. Moreover,genome projects and most bindingassays ignore sequences of less than

50 amino acids, thus excluding manyregulatory molecules and low mol-ecular weight inhibitors (e.g.microcins). Both Leonard Katz(Abbott Labs, Abbott Park, IN,USA) and Chris Gray indicated thatknown targets should be revisited;for example, there are ten well-characterized targets in the cell wallbut very few drugs.

Using environmental DNArequires an understanding of regu-lation and expression, and expressionhosts need to be optimized – throughthe discovery of regulatory networks,for example – to express knowngenes. Stephen Gould noted thatgenomes are likely to prove a morereliable source of chemical diversitythan environmental biodiversity.

There is certainly now a strongemphasis on hybrid molecules andnovel synthetic pathways, but onekey question is whether microbialgenomics can regenerate industrialinterest in discovery programmes forantibiotics and other drugs. The drastic reduction in research fundingfollowing the discovery of vanco-mycin in the 1980s, coupled with thelag phase between lead discovery andmarket approval, means that the nextcouple of years will see only newderivatives of known therapeutics,before the genomics-era drugsemerge.

Over the past 50 years, the studyof microbial genetics has movedfrom in vivo to in vitro to in silico. Howrapidly can the exponentially ex-panding knowledge be translatedback into developing new orimproved industrial products andprocesses? The next GIM confer-ence, to be held in Kyongju, Korea(10–14 June, 2002) will undoubtedlyshow both unexpected progress andunexpected hurdles in achieving this.

AcknowledgmentsReporting highlights from con-

current sessions at GIM ’98 wouldnot have been possible without theassistance of the following sessionconvenors: S. Jensen, E. Vandamme,W. Wohlleben, S. Bron, Y. Koltin,C. Hershberger, H. Schrempf,T. Beppu, C. R. Hutchinson andC. Chen. Their contributions aregreatly appreciated.

Clare RobinsonJohn Innes Centre, Norwich Research Park,

Norwich, UK NR4 7UH. (Email: clare.robinson@bbsrc.ac.uk)

FORUM

TIBTECH MAY 1999 (VOL 17) 181