BUI LDING BETTER SCIENCE & TECHNOLOGY BACTERIA Synthetic biologists turn toa MICR OBI D SHEWIEto ‘wire’ microorganisms for fuel production, energy storage LAUREN K. WOLF, BIOLOGISTS PLAY with ratsand mice, and microbiologists tinker with Eckerihia coli Researchers ely onthe well-known genetic codesand physiologies ofthese ‘model onganismsto advance scientific ‘knowledge and even make new products Butthere'sa new abrat on the block. Shewanelconidesis a ram-negativebae- terium capable of breathing” metals na ‘way similar tohow humans respie oxygen, hasbecomes species popular with environ- ‘ental microbiologists ecent years, sys Kenneth Nealon, whodiscovered the conganismin New York's Oneida Lake, near Syracuse, imi987, Shewic, ast isafectionatly aed by some of the scientists who studyit,con tainsa chain ofheme-bearing proteins thatallow the microorganism to shutle electrons rom ts insdeto its outside dur- inganserobie respiration. nother words, feeditandS.meidensscan directly transfer changetoand reduce external aqueous met also solid metal oxides. ‘Thisinherent capability makes Shewie ateraciveto scientists developing miro bial fuctcells, with biofins ofelectrode- reducing bacteria consumingeomponents of wastewater and generatingelectrc (CREN WASHINGTON ity. Others want to use Shewie for soil remediation a sites contaminated with heavy metals or radioactive materials. For instance, researchers have shown that ‘the icroorganism can convert soluble ‘uranium( (I) ions into insoluble, isolaable ‘uranium dioxide nanoparticles (C&EN, Aug 21,2006, page 44). But what really makes Shewiea model ‘organism, explains Nealson, a University of Southern California geobiologist who has been studying metal-reducing bacteria for decades, is that itis armless and bas fully sequenced genome, And although icthrives ‘nan anaerobic environment, italsolikes ‘oxygen, makingiteasy to grow and usein the lab. These qualities have scientists— particularly synthetic biologists—work- {ng hardto understand the bacterium’s clectron-transfer pathway and taking advantage ofits metal-breathing capability toconstruct microorganisms that are even ‘more useful Caroline M, Ajo-Prankli, forone,bas her ‘eyeon Shewie's respi ratory pathwayasaway to"wire” other ypes of ‘mierobesand cllsfor electronic communica tion. By enablingin- tracellolar electrons to cross what in most cells areinsulating outer ‘membranes, says Ajo Franklin, asynthetic biologist at Lawrence Berkeley National Laboratory scientists could createliving biosensors that givean electrical readout ‘when exposed to small molecules. Similarly, ‘researchers could transform light sensitive ‘organisms into photocatalysts,riggingthem toroute the lectrons they generate into batteries for storage, AS AFIRST STEPtoward making these cell-electrode systemsa reality, Ajo: Pranklin and her team took portion of ‘Shewie'selectron-transfer pathway and, via genetic engineering, inserted it into E.coli (Proc. Natl. Aad. Sci. USA, DOL: 10.1073/pnas-1009645107). This synthett- cally “tricky” feat the LBNLscientsr says, involved adding tree S. oneidensispro- teins—MurA, MerB,and MurC—tothe outer membrane reyion of Ecol. “MirA isa decaheme protein that a ‘cepts electrons from the inner workings ‘ofS. onedensis cells. tesides in the space ‘between the two cell membranes charac- teristic of gram-negative bacteria suchas B, coliand Shewie, This protein passes along. its charged cargo through the pore of MtrB, nintegral protein embedded in the out- cermost of Shewie's two membranes. MirB facilitates the transport of the electrons to -MtrC, another decaheme protein that sts ‘outside the cell and can move the chargeto electrodes or other aqueous species. ‘ter some trials and tribulations, and ‘ith help from chemical biologists YuriY. Londer and Peter Weigele of New tingland Biolabs in Ipswich, Mass. Ajo-Franklin's ‘teameventually inserted the Mie pathway into. colt. Asa result, the F.colfwasable toreduce iron oxide nanoparticles in slu- tion. Although the engineered bacterium reduces the particles an order of magni tude more slowly than does natives. ont 34densi, “we've made B.clfthatcan breathe ust,” Ajo-Franklin emphasizes. The ac- ccomplishment provides a “blueprint” for adding the electron-transfer functionality toother cells, she says,and leaves cell-bound strands, bringing thecells close tothe surface for electron transfer. ‘Buc he amine linker needed for mam. malian-cell attachment worrt work for thacthis method will more efficiently route electrons tothe surface because the DNA linkers should coax the engineered cells. tobind otheelectrode mare tightly and densely than they would otherwise Sore faeacme | | Sotneee er eee ers, Soraoanres | Mantportietev ys ‘ofthebenefiial capabilities ofather bacteria “Using native organism that already does something unique” has advantages, says Gralnick,amicrobi- ologistat the University of Minnesota, "Twin Cities, because “jou don'thave E-eolitoan electrode, Herteam snow ‘exploring both physisorption—letting thebacteriaattach themselves othe surface—and controlled immobiliea tion toachieve that gol Forthelacter scheme, Ajo-Franklin innards to the Mtr protein pathway and beyond via CymA, a tetraheme protein ‘that sits ints inner Iscllabrating ith chemist Mat- colmembrane —/ sotechit thet hater thew Bs rancs Caroya- Ber, | AFrankinand | erestedin”—inthicase Stewie andRlcard A, Mathes othe Unie Gyms soworers at NB6C. | Shitytomovecleron ound, Styof California Berkey oof) rx Ssimlartetrahome |” Grlickhas been ng “Theresearchereceiy devel- ey | edonvateinste plot opedawayto ringtheeells coset Titties Improving een ofa reactions. Current bioconversion pro- cesses, in which microbes catalytically convert feedstocks into chemical products orfuels often create unwanted side prod: uuctsbecause the cellsdon'tachieveredox balance, Gralnick says. But with bacteria ike Shewie that can transfer electrons from the an electrode surface by chemically attaching single-stranded DNA tothe ‘outside of maramaliancels via naturally ‘occurringamine groups on their mem- branes (Lab Chip, DOl:10.1030/b821690h).. Complementary single strands of DNA chored toan electrodehybridize with the gram-negative bacteria because ofthelt double cell membranes, Francis says. 80 the Berkeley researchersare concocting nev type of chemistry tolink the DNA their engineered E col. jo-Franklin hopes 15" Annual Green Chemistry & Engineering Conference in partnership with the 5" International Conference on Green and Sustainable Chemistry Advanced registration ends April 29th gton, D.C. | June 21-23, 2011 | www.geande.org Chemistry for tie™Innovation at the Interface of elias and Biology STi -e pubs.acs.org/r/biomed NG lo) fer1ule gr SCIENCE & TECHNOLOGY Insideout, excess charge could be removed withan external electrode actingasasink, “That way, you could stoichiometrcaly convert your substrate into your product” hesays Along with Daniel 8. Bond, another mi- crobiologist and acollaborator at Minne sota, Gralnick has recently demonstrated the concepi, using electrode adsorbed S. onelensis to transform glycerol into etha- nol (mBio, DOL: 10.128/mBlo.c0190-10). ‘Through genetic engi- neering, the researchers added to Shewie proteins and enzymes from other bacteria hat would al- low. oncidensis to ingest slycerol, shuttle leftover electronstoits native clectron-transfer path way, and eject ethanol ‘When theelectrode thacheld che engineered S.oneidensis was poised atavoltage toaccept electrons, thenew-and- PR Improved Shewie sucked in almost 33-mM glycerol and pumped out about 28:mMethanol—ans9% carbon conversion. The engineered mieroorgan- ismgenerated onlya small amount of acetate by-product (45%), rather than the multipleby-products E.coli might yield froma similar bioconversion REACTIONS OF THIS sort with Shewie could “in principle open up the landscape for new types of industrial bioreactions thacmaybe weren't considered before ‘because of redox imbalance,” Gralnick says, additionally citing glycerol-t0-dihy droxyacetone conversion as an example. ‘And although it's not yet clear howto scale upthistype of reaction for industrial size fermenters,he saysthat the team heleads ‘with Bond isworking with engineers to fig. ure out how to make ithappen, Despitehisinterest in applied work, Gralnickadvises that researchers not for setthe true beauty of engineering Shewie's pathways and proteins into nonnative or sganisms: That tinkering an “help find out about how the bacterium actually works,” he says. Many thingsare stillnot known. about Shewie, Gralnickadds.Forinstance, “tere have been rumblings in the past year orso of X-ray crystal structures" ofthe Mtr 36 Arai 2s.2on proteins, he says, but nothinghas been published yet. Soresearchersaren’t sure hhowthe hemes in the electron-transfer pathway are oriented Inaddition, researchers are tryingto ‘understand how Shewie rouces electrons from MtrC tometal species or electrodes ‘over large distances (greater than 101m), "Transfer tll oceurs when direct Mu ‘metal contacts obstructed, Some scien- tists including Gralnick, think that flavins, Which S oneidnsi secretes arean electron shuttle bevween bacte- umand metal insolution (Proc tl Acad. Sc, USA, DOI: 10073) ppnas.o710525105).Others, inclu ing Nealson and physicist Mo- hhamed El-Naggar,alsoat USC, have observed conductive nanowires ‘growing rom the outside of Shewie thavcould function like extendable arms, reaching out to transfer charge to other materials (Pr, Nal Acad. Sc USA, DOI: 10.1073/pnas.1004880107). ‘Ajo-Franklin mightbe able to answer some of these questions with her work, Gralnick ays, because B, col doesn’tin hherently secrete flavins and presumably doesn't grow nanowices. In optimizing clectron-transfer efficiency in E.coli she right also discover further details about the Mer pathway, he adds with excitement, ‘Being able to show that the components that you're lookingat are not only neces- saryforaprocessbut arealso sufficient to reconstruct itisan important scientific achievement,” Gralnick contends. ‘Nealsonisalso confident that some of these unknowns will become clearin the next generation ofstudies with Shewie, the “bug” he discovered, "Itisabsohite fan towatchall the research activity," he says. Young scientists such as jo- Franklin reso good at what they are 'maybavetolook or an-