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Biological Chemistry: Engineering New Functions For Natural Systems Table of ConTenTs I. Executive Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 II. Tools for Genetic and Genome Engineering . . . . . . . . . . . . . . . . . . 3 III. Tools for Protein Engineering . . . . . . . . . . . . . . . . . . . . . . . 5 IV. Microbial Metabolic Engineering . . . . . . . . . . . . . . . . . . . . . 14 V. Synthetic Biology: The Sum of the Parts . . . . . . . . . . . . . . . . . . 19 VI. Regulating Synthetic Biology Research . . . . . . . . . . . . . . . . . . 21 VII. Materials and Sensors Made From Biomolecules . . . . . . . . . . . . . . 23 VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 IX. Works Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 abouT This RepoRT This special report is for exclusive use by members of the American Chemical Society . It is not intended for sale or distribution by any persons or entities . Nor is it intended to endorse any product, process, or course of action . This report is for information purposes only . Biological Chemistry: Engineering New Functions For Natural Systems 1 I . EXECUTIVE SUMMARY The languages of DNA and proteins are buried in the sequence of building blocks in the biomolecules . When scientists decoded these languages, they learned clues to how a biomolecule’s structure influences its function . Modern genetic and protein engineering allows researchers to adapt the language for redesigning and improving existing proteins . Another biological language is that of chemical circuits in cells, the relationship between proteins and small molecules that make up the metabolic pathways of a cell . Understanding this language, scientists can also manipulate these pathways to create cellular chemical factories . Biomolecular engineering has practical benefits . Engineered enzymes are important catalysts in the pharmaceutical industry, which needs specific reactions that leave few impurities . Engineered microbes already make some commodity chemicals, like lactic acid for plastics . And with future development, these mighty microbes might be able to move chemical production away from petroleum-derived chemicals as a feedstock . Instead, sugar-eating microbes could provide the same precursors to plastic and rubber . No longer content with merely improving natural molecules, scientists now are testing their knowledge of biological principles by recreating biomolecules and organisms from only their building blocks . Some researchers use computers to help design proteins with unnatural functions . Others develop standardized biological parts, like genes and metabolic control molecules, that one day could be clicked together to create a synthetic living organism . From this idea of recreating nature emerges the new field of synthetic biology, however you define it . Some scientists describe the field as an extension of genetic and metabolic engineering . Others say it is building biological circuits using standardized genes, proteins and metabolic controls . While some scientists dream of creating new organisms, others work to use biomolecules like DNA and proteins for entirely new purposes: to make materials and sensors . Recently, an electronics company even built a prototype cell phone using protein-containing plastic . And with that demonstration, the world of biotechnology begins to leave the lab and further enters our daily lives . 2 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms II . Tools for Genetic and Genome Engineering In genetic engineering, scientists take genes from one organism and insert them into another . Sometimes that engineered organism benefits from the addition . For example, corn carrying a gene for herbicide resistance will survive a dose of weed killer, unlike weeds growing nearby . Other times, the new gene turns the new organism into a protein-making factory, like the bacteria that churn out human insulin for the pharmaceutical industry . Manipulating DNA is essentially cutting and pasting nucleotide sequences . Scientists first design and build short loops of DNA, called plasmids or vectors . Cutting enzymes chop open a plasmid, the new gene slides into the gap in the ring, and other enzymes paste the DNA together . This recombined vector is then inserted into bacterial or yeast cells . Sequences in the DNA vectors force the host cell to activate the new gene, transcribing it to messenger RNA (mRNA) and then translating the mRNA into a protein . By combining genetic engineering techniques, scientists can coax simple organisms like bacteria, as well as more complex plants, to produce foreign proteins . But these changes are only done with a few genes at a time . These tools have changed little since the dawn of genetic engineering in the 1970s . Some of the cutting enzymes are so important that the researchers who discovered them were awarded a Nobel Prize in 1978 . But the next generation of genetic manipulation is being developed in research labs now . Scientists are now tinkering with the whole collection of an organism’s genes — its genome .[1] On such a large scale, the traditional tools of genetic engineering no longer apply . So scientists are developing other tools to engineer genomes, starting with molecular scissors that snip out unwanted genes . The cutting enzymes used for genetic engineering would chop a genome to bits . Each enzyme recognizes and cleaves a particular DNA sequence . While scientists can design a 3000-base plasmid to contain only one cutting site, that special sequence might appear many times in a 3-billion base genome . Thus, scientists need cutting enzymes with more specificity . Synthetic genome-cutting proteins contain two pieces . One section, the zinc finger domain, binds specific sequences of DNA while the other section snips the DNA nearby . The resulting combined protein is called a zinc finger nuclease .[2] These proteins work inside cells, compared to the molecular manipulation of nucleic acids in some genetic engineering . Scientists use these proteins to knock out genes in laboratory mice, Biological Chemistry: Engineering New Functions For Natural Systems 3 simulating biochemical effects of some diseases . A zinc finger nuclease is even in clinical trials for HIV gene therapy, as of May 2012 .[3] Scientists engineer the binding domain on these zinc finger proteins so they can control where to insert a new gene in a chromosome . But the nucleases tend to cut other places in the genome besides the desired location . A nuclease variant, the zinc finger nickase, could solve the problem . These proteins only cut one of the two DNA strands . And they are more selective, though less efficient, than their nuclease cousins .[3] Both zinc finger nucleases and nickases insert a new gene using a cellular repair process called homologous recombination . The cut chromosome swaps its damaged section for a piece of synthetic DNA inserted with the nuclease . In 2008, scientists at the J . Craig Venter Institute took advantage of this process to synthesize a bacterial genome in a yeast cell .[4] First they inserted short segments of synthesized DNA into bacteria, which combined the pieces into artificial chromosomes . Then the researchers combined those larger segments in yeast to create the 583-kilobase genome of the bacteria Mycoplasma genitalium . These scientists dream of creating a “minimal” cell with only the basic genetic instructions necessary to start metabolism . Synthesizing a genome is one step towards that goal . Knowing which gene combinations create a functional organism is another challenge . A new automated machine, custom-built by researchers at Harvard Medical School, speeds the process of identifying how genetic changes affect cellular function .[5] The instrument allows researchers to make multiple changes to a genome and then lets evolution decide which changes perform the best . George Church, at Harvard Medical School, and colleagues mixed 50 different DNA segments with some Escherichia coli bacteria . These DNA sequences code for improved versions of genes known to influence production of the antioxident lycopene . The scientists used their custom machine to carry the cells through successive temperature and chemical cycles, coaxing the bacteria to incorporate the DNA segments into their genome . The bacteria also generate new mutations as they divide . After three days of cycles and cell division, the scientists had created E . coli that produced five times more lycopene than usual . That increased lycopene production corresponded to 24 changes in the engineered cell’s genome . Church hopes to build a commercial version of the machine to help other scientists quickly find the right mix of genome-wide changes that makes microbes produce useful products like medicine or biofuels .[6] 4 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms III . Tools for Protein Engineering Natural proteins are collections of 20 different building blocks called amino acids . Amino acids in the binding pocket, or active site, of a protein help confer an enzyme’s exquisite recognition for molecular shape and functionality . Other amino acid sequences force a protein to fold into ridged β-sheets or twisted α-helices . Scientists build proteins in the lab for several reasons . Sometimes they alter amino acids in a binding site to see if the changes affect a protein’s activity . Other scientists prefer to let evolution guide changes to a protein’s structure while they tailor a protein for a new function . Both of these types of studies help researchers learn how a protein’s structure influences its function . These same techniques are also used to create new enzyme catalysts for the pharmaceutical industry . Other scientists combine their knowledge of protein structure with information from these structure-function studies to build computer models that simulate protein folding . With these models, scientists have even designed proteins with structures or functions not seen in nature starting only with a collection of building blocks . And some scientists work on the organic chemistry of biochemistry — changing the atoms in the amino acids of a protein . They’ve altered a cell’s protein-making machinery so a cell will add these unnatural amino acids to a growing peptide chain . The unnatural amino acids provide unique chemical functionality so scientists can add a molecule, like a polymer chain, to a protein drug . Scientists understand proteins well enough to create ones that work in organic solvents instead of water or contain folds not found in nature . Still, these engineered proteins are orders of magnitude less efficient than natural proteins . The future of engineered proteins will combine several of these engineering strategies to boost the activity and efficiency of these creations . RaTional design Scientists change individual amino acids in a protein using the tools of modern genetic engineering . Such purposeful changes to alter a protein’s function or structure are called rational design . Sometimes the changes improve existing function . Other times, they alter it completely . Biological Chemistry: Engineering New Functions For Natural Systems 5 In 2001, Hagan Bayley, then at Texas A&M University’s Health Science Center, and colleagues used rational design to change a pore protein into a cavity that captured small molecules . [7] Ordinarily, charged ions pass through the channel in a protein called α-hemolysin . The scientists altered amino acids in the protein’s pore so that it captured two sugar rings in its channel . These rings held organic molecules inside the protein for hundreds of milliseconds . Other rational designs alter a protein’s function by changing the reaction that it catalyzes . Scientists inserted an unnatural active site into myoglobin, transforming the oxygen-carrying protein into one that reduces nitric oxide .[8] The shape and size of the iron-binding site in myoglobin partially resembled the active site of an enzyme called nitric oxide reductase . So the scientists added three more amino acids to the binding pocket of myoglobin to complete the functional transformation . Thus, they created an enzyme with the activity of the reductase in the body of a gas-carrying protein . Nitric oxide reductase was a difficult protein to create in the lab . So mimicking it in readily-available myoglobin could provide a model system to start studying the reductase activity — with one caveat . Like most engineered enzymes, the altered myoglobin was less active than the native protein . Metalloproteins like myoglobin are notoriously difficult to engineer . But recently, Yi Lu, at the University of Illinois Urbana-Champaign, and co-workers improved the efficiency of designed metalloproteins that reduce oxygen to water . The scientists built two coppercontaining proteins with high efficiencies and turnovers (which reflects how long they work) . [9] Surprisingly, the copper was not crucial to their activity, but some amino acid positions and connections were . These engineered proteins might help scientists understand some elusive details about the inner workings of natural oxidases . The activities of these two new proteins were about 0 .7% that of the natural enzymes, which are comparable to another recently engineered protein . And the artificial catalysts cycled through more than 1,000 turnovers, compared to the thousands to millions of reactions of a natural enzyme . Specific alterations to purposefully change a protein’s function demonstrates a careful knowledge of protein structure-function relationships . Scientists’ inability to match the activity of natural proteins in their designs reveals the fact that they still have more to learn . So another way to engineer proteins takes cues from natural evolution: In a large pool of proteins, only the strongest survive . 6 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms diReCTed evoluTion In directed evolution of proteins, scientists create a large pool of randomly mutated genes, express the proteins encoded by those genes, and select the best-performing proteins for their desired task — for example, a protein that works in organic solvent .[10] The researchers copy the DNA sequences that created the best proteins and feed those genes through another round of mutation and selection . Over several cycles, the most beneficial mutations survive and the protein now has a new function . In 2000, researchers in England combined rational design and directed evolution to change an enzyme’s function . Alan Fersht and co-workers replaced the substrate binding and catalytic loops of one protein with randomly mutated sections from another protein, phosphoribosylanthranilate isomerase, nicknamed PRAI .[11] The engineered protein that survived selection had an activity and catalytic efficiency similar to PRAI . The two proteins share a structure called an α/β-barrel . Altering this scaffold to change a protein’s function was a clue that scientists might be able to design any function for that scaffold . “I was in the first wave of protein engineers and ended my first review of the subject in 1984, in Angewandte Chemie, with this sentence: ‘The ultimate goal is to design a tailor-made enzyme for every reaction .’ Fersht told Chemical & Engineering News at the time . “Some of my colleagues thought I was rather overoptimistic . So this result is of great emotional significance for me since, after waiting 15 years, I can see light at the end of the tunnel .” Since then, directed evolution has been used to improve and alter the functions of other natural proteins, like ketoreductases used widely in pharmaceutical industry as catalysts during drug synthesis . CompuTeR-aided pRoTein design Designing proteins through directed evolution requires sifting through large collections of mutated proteins . Building 100-residue-long peptides using the 20 natural amino acids creates more than 10130 different peptides .[10] Many of those peptides won’t have the function desired by the scientists . Chemical screens can identify functional mutants, thus narrowing down a library for further tests . Computers can help, too . Protein structure programs perform directed evolution through calculations that maximize the stability of a protein structure . The programs only carry the most stable (lowest energy) structures forward to the next calculation . Thus, these programs can help researchers design new proteins, while identifying and eliminating potential non-functioning proteins in a library .[12] Biological Chemistry: Engineering New Functions For Natural Systems 7 Computer models can help redesign and improve existing enzymes .[13] But often the tight packing of amino acids inside a redesigned protein resembles that of its parent protein . That means some programs have a biased head start on their calculations: They can use the backbone structure of a natural protein to help it solve the structure of the new protein . The true test of a model’s power to predict protein structure comes when it’s challenged to design a protein with a novel structure or function . In 1998, researchers in Massachusetts designed a helical protein with a right-handed twist not seen in nature . The scientists input a string of amino acids that they thought would form such a shape into their model and asked the program to identify amino acids that stabilized the coil . Then the researchers synthesized the calculated protein and found that its structure matched the predicted twist .[14] A few years later, David Baker, at the University of Washington, and colleagues designed a different unnatural protein fold completely from scratch, without knowing a sequence in advance .[15] The scientists drew a combination of α-helices and β-sheets . Then they challenged the computer to find an amino acid sequence that might fold into that structure . After it identified a sequence, the computer predicted the structure of a protein with that sequence . Ten rounds of sequence and structure optimization yielded the engineered protein, called Top7 . The crystal structure of Top7 matched computer predictions . Besides new structures, Baker and colleagues also design proteins with new functions . One such engineered protein catalyzes a retro-aldol reaction, like some natural proteins, but it uses an unnatural substrate .[16] The team has also designed two proteins with functions never seen in nature . One protein catalyzes a Kemp elimination[17], which models deprotonation in an enzyme active site . Another triggers a Diels-Alder reaction, forming two bonds instead of breaking them .[18] Not Seen in Nature: Baker’s team envisioned an enzyme that could catalyze the intermolecular Diels-Alder reaction of the diene 4-carboxybenzyl trans-1,3-butadiene1-carbamate and the dienophile N,N-dimethylacrylamide 8 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms To create an enzyme with a new function, the researchers first engineer an active site with residues necessary for catalysis and a proper shape to hold the substrate . Then they insert that active site into a protein backbone and challenge the computer to find the most stable structures . For the Diels-Alderase, the researchers first identified 1019 possible active sites . Further calculations including stabilization from the protein backbone winnowed potential candidates down 106 . Of that group, the scientists synthesized and purified 84 sequences . Only 50 synthesized proteins were water soluble, and only two of those catalyzed the Diels-Alder reaction . While computer models can help reduce the number of potential proteins, significant laboratory work is still needed to confirm an engineered protein’s function . Baker wanted to reduce the number of potential proteins before confirming the structures in the lab . To that end, he reexamined structures of active proteins from the 2008 Kemp elimination study, comparing them to some inactive proteins generated by the models .[19] From this comparison, Baker developed a ranking system for the structures based on molecular dynamics methods, which could have reduced the researchers’ pool of synthesized proteins from 120 to 24 . Researchers may increasingly be able to build their dream catalysts using engineered proteins . But that doesn’t mean those proteins will work quickly or efficiently . Both of these engineered enzymes speed their particular reaction about a million times (106) faster than it would go on its own . Natural enzymes speed reactions up to 1019 times . This reduced efficiency is one of the big challenges facing computational protein design .[20] Did a protein design fail because it was flawed from the start? Perhaps the computer partially completed an active site . Or did protein structure far away from the binding pocket influence the activity? Answering these questions requires combining computational design with other protein engineering tools, like directed evolution, and structural characterization of the engineered proteins . unnaTuRal amino aCids Another way to alter proteins involves changing their internal chemical functionality using unnatural amino acids . The functionalities in these synthetic amino acids are useful chemical handles to attach small molecules to a protein . They can also serve as additional probes of structure and function, just like rationally designed mutants made with the 20 natural residues . Engineered protein-making machinery can accommodate these new building blocks, too . Now bacteria, yeast, and mammalian cells can create proteins containing about 70 functionalities not found in nature, including azides, halogens, and boronic acids .[21] Biological Chemistry: Engineering New Functions For Natural Systems 9 Here’s how it works: Building a protein starts with the genetic instructions . Cells first transcribe DNA into messenger RNA (mRNA) . Cellular machinery translates the mRNA, building a string of amino acids based on codes in the mRNA . Those codes are groups of three RNA bases called codons . Each codon is unique for a particular amino acid, though this code has some degeneracy: Several codons may correspond to one amino acid . For example, two codons, UUU and UUC, represent the amino acid phenylalanine . During protein synthesis, another molecule of RNA called aminoacyl transfer RNA (tRNA) translates the language of mRNA into a protein sequence . One end of a tRNA matches a specific codon on the mRNA . The other end carries the amino acid corresponding to that codon . As the cellular machinery reads the mRNA, various tRNAs match the codons and bring each amino acid to the growing peptide . To coax a cell to incorporate unnatural amino acids into a peptide, scientists engineer tRNAs to recognize specific codons and to carry specific altered amino acids . Scientists find specific codons for these unnatural amino acids in duplicate instructions for the natural amino acids . In 2003, David Tirrell, at the California Institute of Technology, and co-workers broke the degeneracy of mRNA codons using two different engineered tRNAs .[22] Remember that two mRNA sequences, UUU and UUC, code for the amino acid phenylalanine . The scientists designed a mutant tRNA from yeast to carry a phenylalanine analog to UUU, but not to UUC . Another engineered mutant tRNA from a bacterium carried phenylalanine only to UUC . Using a nonsense codon, or other unused codon, scientists can use engineered tRNAs to precisely place unnatural amino acids in a protein . To ensure specificity, those engineered tRNAs are often from a different organism than the cell that’s building the protein . And scientists have built enough tRNAs that bacteria, yeast, and mammalian cells can synthensize proteins with unnatural amino acids . Unnatural amino acids can serve as probes of structure and function, like rationally designed mutant proteins using the natural 20 residues . But they might also improve protein drugs, too . Peter Schultz, at The Scripps Research Institute, has co-founded a pharmaceutical company, Ambrx, to use non-natural amino acids in protein drugs . It’s common to attach small molecules like cancer drugs to antibodies to help direct the proteins to a tumor . Unnatural amino acids help scientists tailor the location of the small molecule to minimize its interference with the drug . Ambrx added a chain of polyethylene glycol to human growth hormone to extend the hormone’s lifetime in the body .[23] Other companies, like one using Tirrell’s unnatural amino acid technology, are looking into modifying current drugs with unnatural amino acids, though these companies have different methods for introducing the modifications . 10 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms The fuTuRe of engineeRing new pRoTeins Using rational design, directed evolution, and computer modeling, scientists can now design and build enzymes with new functions from scratch . They can also introduce unnatural amino acids with unique chemical functionalities . But many of these changes also create proteins with reduced activity or efficiency . Thus, the next step for protein engineering is to improve these proteins through directed evolution .[24] Directed evolution can add unnatural amino acids into a protein . Vaughn Smider, Peter Schultz, and co-workers introduced partially randomized antibody genes into cells containing the unnatural amino acid p-boronophenylalanine . The boronate group on this amino acid can bind two hydroxyl groups . So the scientists expressed the antibody genes and tested the resulting proteins to see if they could capture some hydroxyls on a sugar . After three rounds of evolution and selection, more than 80% of the DNA sequences encoded the unnatural amino acid .[25] Such boronate-containing proteins might be useful inhibitors that bind to sugars on a protein or stick to serine residues on an enzyme . Combining computer models with enzyme evolution helped David Baker and colleagues improve three of their designs for a novel Kemp eliminase . Most recently, the scientists introduced stabilizing mutations into one of their original designs and then carried the protein through 16 rounds of directed evolution . The efficiency of the optimized enzyme was more than 2000 times greater than the original design .[26] Baker also has another method for protein optimization: video games . He added protein design optimization into a computer game normally used to solve structures of natural proteins .[27] Gamers changed residues and tugged at the backbone of various loops in Diels-Alderase created in Baker’s lab . The final optimized structure, containing an additional amino acid sequence, improved the activity of the enzyme more than 18-fold . paThways To gReaTeR poTenCy Several trends in drug development to improve upon the impact of established compounds, or increase a drug’s potential targets, are finding traction in small molecule and protein-based drug discovery . The renewed interest in covalent drugs [33], which form an irreversible bond with their protein targets, has resulted in some candidates for clinical trial . Although some researchers have been reluctant to pursue covalent drug candidates, fearing their permanent bond might be too toxic, this fear has been calmed in some more recent candidate drugs that are weakly reactive . The covalent drug AVL-292, which blocks an enzyme involved in lymphoma, was one such candidate of interest for Celgene when it acquired its developer Avila Biological Chemistry: Engineering New Functions For Natural Systems 11 Therapeutics in early 2012 . Covalent compounds are being considered for treatments for other cancers, hepatitis C and obesity, among other conditions . The current generation of potential covalent compounds are very selective, suggesting smaller doses than with usual drugs are needed to see clinical effects . Interest is also growing in developing multivalent drugs, which use multiple copies of their bioactive chemical group to inhibit multiple targets at once . Multivalency can significantly increase a drug’s potency, specificity and duration of action . One route to multivalency in future drug design may include techniques for adding short peptide nucleic acids to DNA strands . [34] The artificially synthesized polymers have already shown some promise in anticancer, antiviral and gene silencing applications . As the crystal structure and molecular details of more proteins are revealed, drug designers are exploring the possibilities of therapies that address protein-protein interactions . The multiple targets involved in these interactions were previously thought to be intractable .[35] But researchers now see the variety and versatility in protein-protein interactions as offering a wealth of targets that can be addressed by drugs that offer subtle alteration—rather than blunt inhibition—of protein activity . Protein profiling and screening for drug candidates and targets remains a key challenge in drug discovery, one that researchers are finding new ways to accelerate and fine-tune . Competitive activity-based protein profiling, for instance, could make it possible to identify small molecule inhibitors that work across multiple enzymes with similar functions . In early 2012, the technique was used to identify potential inhibitors for serine hydrolases, one of the largest and most diverse classes of enzymes .[36] Other screening techniques under development include new ways to profile protein oxidative stress and to create new networked maps of similar enzymes .[37] indusTRial bioTeChnology Enzymes with altered functions are common catalysts in the chemical industry, including pharmaceuticals .[28] Proteins are a quick way to make chiral molecules and separate mixtures of enantiomers . Their preferred reaction conditions — neutral pH, ambient pressure and temperature, and often in water — are ideal for large-scale reactions . Enzymes also produce clean products, without trace impurities possible with metal or organocatalysts . Therefore, biocatalysts are one way for chemical companies to make process chemistry more environmentally friendly .[29] 12 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms Industrially relevant enzymes - such as lipases, ketoreductases, and transaminases - have already been tweaked to work with unnatural substrates . Now companies are refining their catalysts through directed evolution, developing proteins that are stable at 60° Celsius in organic solvents or tailoring proteins to work at conditions where their substrates are soluble . Previously, process chemists designed a reaction around an enzyme’s limitations, researchers wrote in a recent review of enzyme biocatalysts .[30] Now scientists design an enzyme for a particular process, they say . Enzymes help make the cholesterol drug Lipitor, new medicine for hepatitis C, and chiral building blocks for many pharmaceuticals . Despite the dearth of catalysts used in the industry, companies making the biocatalysts still struggle to find a long-term business model .[31] Some process chemists regard enzymes as a catalyst of last resort, preferring tried and true reactions instead . Even if chemists try enzymes, they must still optimize the reaction as they would for a chemical catalyst . Biocatalysis companies struggle to anticipate their clients’ needs, as pharmaceutical companies closely guard information about Biological Chemistry: Engineering New Functions For Natural Systems 13 their current lead compounds . So companies new to the enzyme market may look for clients outside the sphere of pharma . Some search for novel enzymes . Other companies think the key to success in biocatalysis includes protein engineering for optimizing enzymes and discovering new ones . Consequently, there are many ways to run a business selling enzyme catalysts . While every company may not succeed, those in the industry remain optimistic that potential customers, especially in pharmaceuticals, exist . IV: Microbial Metabolic Engineering Microbes are part of some synthetic chemistry toolkits in academic and industrial labs around the world . Bacteria and yeast build biofuels and precursors to medicine and plastics . Scientists rely on metabolic engineering to create microbes with these unusual functions . At first glance, the technique may seem like basic genetic engineering: Insert a gene, say, for a protein that reduces a ketone, feed the bacteria a ketone-containing substrate, and collect the reduced substrate to use as part of a medicine . But transforming bacteria into industrial workers is more difficult than that .[32] If the foreign protein doesn’t shut down cellular functions, then perhaps the molecule it makes will . Metabolic engineering requires scientists to understand and control the cellular networks that link genetics, protein synthesis, and metabolism . That can take some meticulous biomolecular manipulation . But once large tanks of bacteria or yeast are cranking out molecules, these microbial workers are cheap . They’ll work for sugar and produce fragrances and fine chemicals in return . Creating microbes with new functions might be considered synthetic biology, but some scientists say there is a difference between metabolic engineering and synthetic biology . The former is focused on practical applications, while the latter studies the fundamental science of these altered cellular networks .[32] This section will focus on microbes engineered for industrial applications . phaRmaCeuTiCal synThesis Many medicines have complicated structures that make them difficult to synthesize in the lab . Trees, plants, and corals produce some of these medicines naturally, though not in high enough concentrations to make these organisms efficient sources of medicine . Plus, relying on natural sources can leave our medicine supply vulnerable to destruction by natural disasters . So scientists use metabolic engineering to create microbes that build portions of a 14 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms desired drug, like the cancer drug paclitaxel (Taxol) .[33] The researchers identified genes involved in the biosynthesis of the drug and inserted these genes into bacteria or yeast . Then they altered the microbe’s metabolism to funnel substrates to the new proteins . In 2006, Jay Keasling and co-workers used this trick to engineer yeast that produced artemisinic acid, a precursor to artemisinin .[34] Artemisinin is an antimalaria drug produced by the plant wormwood . But the plant produces too little of the drug to meet worldwide demand . The researchers identified two proteins in the biosynthetic pathway for artemisinic acid and inserted the genes for those proteins into yeast . The first enzyme in that sequence uses a molecule called farnesyl pyrophosphate (FPP) that yeast produces during normal metabolism . The researchers boosted FPP production in the yeast with two types of changes: turning on some FPP production genes and turning off other pathways Taxadine is a key intermediate in the biosynthesis of Taxol that consume FPP . With those changes, the wormwood enzymes had enough FPP to make artemisinic acid . As a fraction of biomass, the engineered cells produced as much artemisinic acid as wormwood . But the yeast produced the drug much faster — four to five days compared to several months in wormwood . Two synthetic steps convert artemisinic acid to the active medicine . Amyris, a California company co-founded by Keasling, has licensed this technology to a pharmaceutical company and hopes microbially-produced artemisinin will be available commercially in 2013 . Other engineered microbes perform useful molecular Arteminisin modifications, like adding a fluorine atom to improve pharmaceutical efficacy . Researchers identified a rare enzyme, a fluorinase, that adds a fluorine atom to molecules . Then they used the fluorinase to replace a bacterial enzyme that adds a chlorine atom to an anti-cancer drug . The altered bacteria produced the fluorinated drug with low efficiency because the fluoride ion kills these cells .[35] The researchers planned to engineer fluoride ion resistance into a microbial host based on clues from another bacterial species . Biological Chemistry: Engineering New Functions For Natural Systems 15 fine ChemiCals Microbial fermentation is used to make lactic acid, propanediol, and citric acid on an industrial scale using sugars from corn or sugarcane . Other companies are working on making succinic acid and acrylic acid . Still other companies want to use cellulose, in wood and corn stalks, to make higher value chemicals beyond ethanol .[36] Through microbial engineering, sugars can replace petrochemicals currently used to make commodity chemicals for the rubber, plastics, and fragrance industries . These bio-based chemicals make process chemistry more environmentally friendly by using fewer solvents and reagents . A microbial factory also operates under mild conditions (ambient temperature and pressure, neutral pH) . A variety of engineered organisms can make precursors to most common plastics, though not on a commercial scale yet . Bacteria make styrene, yeast make a precursor to nylon from vegetable oil waste, and other microbes make lactic acid from corn sugar .[37] Biobased chemicals aren’t ready to replace petroleum-derived chemicals yet . Building large fermentation plants and scaling up the processes takes money — which young start-up companies lack and industry hesitates to provide to potentially risky business ventures . And once a manufacturing plant is built, there’s the question of economics . Petroleumderived chemicals are generally cheaper than these biobased chemicals . Technological improvements, declining feedstock costs, and rising oil prices are needed for these biobased chemicals to supplant their petrol competitors .[38] Bioplastics could find a big market in the trash by composting leaf litter and food waste .[39] And there are signs that the market could take off in other areas . Large companies like Coca-Cola, Colgate-Palmolive, and H .J . Heinz have shown interest in switching their packaging to bioplastics . But some think these companies’ interest in bioplastics is an excuse to advertise sustainability .[37] Another common material, rubber, could one day have microbial origins . Currently rubber comes from rubber trees or petrochemicals . But both these sources are shrinking due to worldwide demand . Engineered microbes can produce isoprene, butadiene, and isobutene, which are precursors to natural and synthetic rubber . Bio-derived rubber using isoprene could replace a fair chunk of the rubber consumed by tire company Goodyear, the company’s director for research and development told Chemical & Engineering News.[40] Again, others caution that petrochemical pathways are still cheaper than microbial creation . But functioning microbial pathways could still protect the rubber industry from price fluctuations due to limited feedstock supply . 16 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms Another group of molecules that suffer supply issues are fragrances and flavors . Like pharmaceuticals, many of these molecules have complicated structures that make total synthesis difficult . Others can only be isolated from plants or fruit peels . Natural disasters or political unrest in the habitat of a natural source can also limit the supply of these flavor molecules . Companies like Allylix and Isobionics are engineering microbes to produce two citrusy fragrance molecules: nootkatone and valencene . Amyris, a company that is also engineering microbes to make biofuels and cosmetic ingredients, is working on patchouli, a common aroma in incense . Nootkatone Valencene It’s too soon to tell how much of the flavor and fragrance market will move to biologically sourced chemicals . But these biotechnology companies are looking to sell more than one molecule . They hope to strengthen their ability to engineer microbes to produce any fragrance .[41] If they succeed, perhaps once rare aromas may appear in common consumer products . Engineered bacteria produce biodiesel from hemicellulose . biofuels Fuels derived from sugars synthesized by plants might one day replace the fossil fuels that power our cars . Engineered microbes can ferment those sugars into alcohol- and hydrocarbon-based fuels similar to what we use now . Chemical catalysts, too, can convert plant sugars, oils, and starches to fuel . Many major chemical firms and start-up companies alike are working to bring biofuels to the market .[42] But it’s anybody’s guess which approach — chemical or biological — will win . “Chemical technologies can be engineered to happen more quickly,” Jay Keasling, a synthetic biologist at University of California, Berkeley, Biological Chemistry: Engineering New Functions For Natural Systems 17 told Chemical & Engineering News.[42] “It does take a long time to engineer the biology . But the beauty of biology is that it can work under dirtier conditions, and you can get the specific molecule you want under a range of conditions .” Bacteria can produce ethanol, which is already blended with gasoline .[32] They can also be altered to create other fuels that work with our current infrastructure . In 2010, Jay Keasling and scientists from the company LS9 created bacteria to make fatty acid esters for biodiesel . [43] These engineered bacteria consume glucose or hemicellulose from partially digested plant cell walls . Cellulose must be part of United States biofuel feedstocks to meet the Renewable Fuel Standard established in the 2007 Energy Independence and Security Act . But breaking down cellulose is energy intensive, which limits its usefulness as a feedstock for efficient biofuel synthesis . Researchers are looking for genes that help microbes digest cellulose, so they can engineer its degradation in other organisms .[44] Last year, Keasling combined genes that break down cellulose from switchgrass with those that transform the sugars into fuel in the same microbe .[45] While not yet a commercial process, it’s a step towards addressing energyintensive cellulose digestion . But the business of biofuels is challenging . Many biofuel companies struggle to produce cellulose-based fuels cost-effectively, so they turn to other sugar sources, like corn .[46] Amyris slowed their production of microbially-derived biodiesel in February 2012 because they couldn’t make enough money to break even . But a partnership with oil company Total in August reinvigorated the company’s efforts to create biofuels from sugar cane . 18 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms V . Synthetic Biology: The Sum of the Parts The field of synthetic biology lacks a standard definition, even more than a decade after the term’s introduction .[47] In 2000, Eric Kool coined the term “synthetic biology” to describe research that probes the function of biological molecules using organic chemistry alterations .[48] Over time, “synthetic biology” grew into a field that encompasses genetic and metabolic engineering to create organisms with new functions . And now some definitions of the field include building a novel organism using standardized biological parts .[49] This section will focus on work that fits the last category . Synthetic biologists who dream of engineering biology from scratch want to pull genes from a bucket of standardized “parts,” often compared to transistors and resistors used in electrical engineering . The scientists would insert those assembled genetic instructions into a basic cell with minimal supplements and watch the cell come to life . That dream, however, is some ways off . Most work in this area focuses on assembling and testing the parts to create organisms with new functions .[50] Those parts are nucleic acid sequences with specific functions, like recruiting proteins for transcription, producing chemical signals in response to chemical inputs, and controlling gene expression by suppressing protein translation . Scientists link parts to create genes and connect genes to make pathways . And they’re not the only ones snapping biological Legos together . Since 2004, undergraduate and high school students from around the world contribute to a publicly available parts registry, called the Registry of Standard Biological Parts .[51] The students combine existing parts with new ones they’ve developed to create a microbe with a new function . In 2011, 160 student teams from 30 countries around the world participated in the iGEM (International Genetically Engineered Machine) competition . Some of the winning projects included a chemical sensor using a biofilm of fluorescent bacteria and bacteria that break down gluten, a wheat protein that interferes with digestion in some people . While scientists and students tinker with existing organisms bit by bit, scientists at the J . Craig Venter Institute have a different approach . They want to create new organisms with customized genomes built entirely from the building blocks of nucleic acids .[52] These researchers moved a step closer to their goal in 2010 when they “booted up” a synthetic genome from one bacterium inside a cell from another species .[53] The researchers built the genome from the bacteria Mycoplasma mycoides and transplanted it into a Mycoplasma capricolum cell . The altered cells resembled M . mycoides and replicated on their own . The Biological Chemistry: Engineering New Functions For Natural Systems 19 synthesized genome, while one of the simplest known, was more than one million base pairs long — an accomplishment for chemical DNA synthesis . The researchers hope this technology could help them build new genomes for algae that make biofuels or synthesize the genomes for a variety of influenza viruses so it’s easier for scientists to create new flu vaccines .[52] Some researchers think proteins could be part of synthetic biologist’s toolkit, too . Proteins naturally self-assemble into clusters that could link biomolecules into functional assemblies . [54] Andrew Thomson and Derek Woolfson, both at the University of Bristol, and colleagues recently designed coiled peptides that assemble into dimers, trimers, and tetramers . They entered those sequences and structures into a protein registry, similar to that for genes .[55] They hope proteins will be as “plug-and-play” as genes are today . Some of these synthetic biology parts, or “devices,” could help scientists build microbes that synthesize chemicals too .[56] To engineer a microbial chemical factory, a synthetic biologist’s toolkit needs genes, ways to control gene expression, and a minimal cell to host the genetic instructions . Breaking down the complicated networks of gene expression into controllable parts is quite a challenge . But researchers moved a step closer to this goal in 2012 when they systematically engineered RNA devices to control gene expression in bacteria .[57] Despite these advances, synthetic biology is still done largely by modifying small sets of genes through trial and error . Thus, the Defense Advanced Research Projects Agency (DARPA) launched its “Living Foundries” project in 2011 .[58] The agency recently awarded $17 .8 million to seven companies and universities to forward research that develops, tests, and models parts for synthetic biology . Though the field is young, many researchers from academic institutions and companies are systematically working to standardize biology . 20 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms VI: Regulating Synthetic Biology Research Policymakers discussing synthetic biology define the field as manipulating biological blocks to engineer new functions for microbes as well as create new organisms from the ground up . That may sound like an extension of genetic and metabolic engineering used in current biotechnology . But some advocacy groups feel synthetic biology is a radically new and unpredictable field lacking government oversight .[59] They worry an engineered organism will run amok, damaging the environment or human health . In 2009, researchers, policymakers, and industry executives from around the world met to discuss this emerging field . They tried to define the field and discussed safety, security, and regulatory oversight . They also acknowledged the importance of talking with the public to keep them informed about synthetic biology . After that meeting, researchers said that regulations governing the safe use of current biotechnology apply to synthetic biology .[59] The Presidential Commission for the Study of Bioethical Issues re-examined regulations for synthetic biology following Craig Venter’s 2010 announcement of creating a replicating cell with a synthetic genome .[60] Again, the regulators said the field has limited risk of misuse for now, but regulators should monitor new developments . So policymakers from China, the United States, and the United Kingdom are still holding meetings . In 2012 talks, attendees discussed the difficulty of creating regulations that cover the range of scientists in the synthetic biology community, which includes students and hobbyists tinkering in their garages . Amateurs have limited access to technology and resources compared to academic and industrial labs, so they may not engineer complicated organisms . Nevertheless, instilling ethical values among these scientists will be more important than the everyday regulations, Robert C . Wells, head of the biotechnology unit in the Directorate for Science, Technology & Industry at the Organisation for Economic Cooperation & Development, told Chemical & Engineering News.[61] The student competition iGEM requires competitors to study the safety risks of their engineered organisms . And the hobbyist community at DIYbio .org is working to develop a code of ethics for amateur scientists, too . Biological Chemistry: Engineering New Functions For Natural Systems 21 Synthetic biology carries dual-use implications, where the same research can be used for good and evil at the same time . No dual-use questions have been raised about synthetic biology yet . But a recent argument involving a mutant influenza virus could be a clue to the tenor of future dual-use debates . In early 2012, two research groups, one in the Netherlands and the other in the U .S ., independently engineered the H5N1 avian flu virus so it could be transmitted between mammals through the air, rather than the typical passage from birds to mammals . Such a virus would be more contagious and potentially more deadly than current versions of the flu . Such research helps scientists understand how the virus could evolve to be more dangerous . But a federal biosecurity advisory board argued that publishing the results and methods of this work could give those that would use the finding to do harm enough information to create viral bioweapons .[62] In a vigorous public debate, some scientists argued against the suggested redactions, saying that the government was trying to censor legitimate research . [63] Meanwhile, other scientists and members of the government advisory board argued that the redaction was more about protecting the general public than about censorship . The government released new policy guidelines for dual-use research with pathogens and toxins about two months after the original redaction recommendation .[64] The finalized policy requires funding agencies to review project proposals for potential dual-use issues before awarding any money .[65] The story of the flu papers ends with publication — one in Science, the other in Nature — in full . But the debate about dual-use research will likely continue, both in the U .S . and worldwide . One day, synthetic biology might be part of the discussion . 22 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms VII . Materials and Sensors Made From Biomolecules Another way to design new functions for biological molecules and organisms is to use them for entirely new purposes . Both DNA and peptides have natural structural properties that make them strong and reliable construction materials for defined molecular shapes . These biomaterials also have another advantage over traditional polymers: specific recognition . The nucleotide or amino acid sequence of the material can help glue the material together or recruit other biomolecules to the material . Cells, on the other hand, can behave like computers . With a few genetic tweaks, cells respond to a chemical input with a defined output like light . These engineered organisms can act as sentinels for environmental pollution . This section will highlight three ways biomolecules and microbes can be useful materials and sensors . building wiTh dna The defined helical structure of DNA, as well as its reliable construction using four nucleotides, makes the nucleic acid an ideal building material . For years, scientists had built cages, cubes, and octahedrons from DNA . In 2006, Paul Rothemund, at the California Institute of Technology, advanced the construction of complicated DNA structures when he published iconic images of 100-nm-wide smiley faces . [66] Rothemund created his original DNA origami using a single strand of bacteriophage DNA more than 7000 nucleotides long . Short strands of complementary DNA acted as “staples,” pulling and folding sections of the phage DNA so it formed stars, snowflakes and a map of the Americas . Scientists then expanded those flat structures into three dimensions by connecting flat panels to form 3D shapes, like a box with a lock and key .[67] DNA origami can create patterns as small as 5 nm long, which matches some of the smallest structures made with polymers to pattern computer chips . But practical applications for DNA origami have yet to appear . Scientists think folded DNA might pattern molecular tools like tweezers that tug apart a protein .[68] DNA origami could also be useful for futuristic devices like nucleic acid nanobots . In 2010, two teams of researchers created protein nanobots with DNA legs that walked across a spiky surface of DNA origami .[69] For one nanobot, one of its four legs bound tightly to the DNA posts on the surface . The other three legs were DNA Biological Chemistry: Engineering New Functions For Natural Systems 23 enzymes that cleaved and shortened some of the posts . Those three legs interact weakly with the shrunken spikes, so they wander around the surface until they find an unbreakable spike . Grabbing that new spike propels the nanobot forward . With three hands and four legs of single-stranded DNA, the other nanobot may resemble a child’s drawing more than a functional machine . Nevertheless, its hands can carry a gold nanoparticle as it moves across the spiky origami surface . The researchers attached their DNA bot to a spiky origami surface and forced it to walk by adding DNA strands complementary to the spikes on the surface . Those “fuel” strands displace the nanobot’s legs, which search for the next stable anchoring spot on the surface . In 2012, researchers tested a different DNA nanobot, this time to see if it could deliver drugs to a particular cell .[70] They built a hexagonal tube using slabs of DNA origami and sealed them together using a molecular glue targeted to specific cells . The glue is short strands of DNA called aptamers that bind to molecules on the surface of a cell . When the DNA nanobot reaches its target cell, the aptamers bind the cell surface molecules, opening the envelope and releasing the contents . In one experiment, the scientists delivered antibodies that shut down cultured cells . However, the structure of the nanobot would have to be optimized and likely redesigned if it was to kill cells, the researchers say .[70] Though DNA origami has not found practical applications yet, researchers are hopeful that it will . As materials go, DNA is hard to beat: It’s stable, durable, reliable, and chemically modifiable .[68] building wiTh pepTides The defined structure of peptides and proteins makes them attractive building blocks for new materials . Peptides easily assemble into twisted helices, ridged β-sheets, and aggregated amyloid fibers . Scientists control these shapes not only through specific sequences of amino acids, but also with temperature, pH, and the saltiness of a peptide’s environment . Peptides are commonly used to create hydrogel scaffolds for tissue engineering and controlled drug release .[71] Hydrogels are polymer networks that absorb water like a sponge and change from a solution to a gel . Natural peptides, like strands of elastin and collagen, structure cells and tissues in our bodies . These peptides, when purified, can build hydrogel networks . Synthetic versions of these peptides have several advantages over their natural counterparts . They are more readily available than the natural peptides . They can also be modified to alter the properties of the 24 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms hydrogel . And cell-recognition sequences can be built into the peptides structure to help cells colonize hydrogel . Amyloid proteins are another class of proteins with a usable, defined structure . These proteins clump into fibrils . Fibrils aggregate into long, tangled clusters that clog cells . These proteins are common in Alzheimer’s and Parkinson’s disease . The ready clumping of amyloid proteins makes their fibers easy to synthesize in the lab . Scientists can trigger fiber formation by changing the pH of a solution containing the proteins or by adding enzymes to connect precursor proteins that clump into fibrils . In 2008, mobile device company Nokia built a partially biodegradable cell phone containing amyloid fibrils .[72] The protein fibers in the body of the prototype device, called the Morph, degrade over time so that less plastic ends up in a landfill . However, amyloid fibrils might not be useful materials for implanted devices due to the toxicity of the fibrils themselves . miCRobial polluTion sensoRs Besides using biomolecules to build devices, researchers can also use organisms themselves as devices . Genetically engineered microbes can signal if concentrations of environmental pollutants are at safe levels . Scientists design pollution-sensitive microbes to produce fluorescent or luminescent proteins when the bugs encounter oily hydrocarbons or heavy metals like arsenic . Pollutants bind to a receptor inside the engineered bacteria, triggering the cells to produce the protein light source . Non-living biosensors or analytical laboratory instruments can detect these pollutants, too . But these instruments only detect concentrations of contaminants . Microbial sensors can tell scientists if the contaminants can enter cells and thus possibly affect living organisms .[73] Bacterial biosensors tend to have short lifetimes because the bacteria die or form insensitive films . In 2011, researchers built a bacterial array that measured contaminants in water for more than a week .[74] The scientists combined three different pollutant-sensitive bacteria, each detecting a different type of contaminant, in agar wells . Water flows across the wells and a light detector suspended above the wells picks up luminescence from the bacteria when they encounter specific pollutants . This experiment only detects groups of contaminants, not specific ones, thus the researchers stressed that the device was not ready for field trials . The next year, another research group took their light-producing bacteria into the field to measure arsenic in water in Bangladesh .[75] About 80% of wells in the country are not tested Biological Chemistry: Engineering New Functions For Natural Systems 25 for arsenic, even though the metal is known to be present at varying levels . The bacterial biosensor performed as well as two traditional chemical detection kits . And the researchers say that the new kit is easier to use and produces less waste than the others . They hope mass production will make the kit inexpensive, too . VIII . Conclusions With knowledge gained from trying to improve genes, proteins and microbes in the natural world, scientists now use that information to redesign nature and engineer natural systems to have unnatural functions . Scientists can alter genomes, design functional proteins from their constituent amino acids, and engineer microbes to be tiny chemical factories . Despite these accomplishments, their designed systems do not match the efficiency of natural systems — for now . Engineered proteins suffer from low activity compared to natural enzymes . Biofuel-producing microbes are picky eaters . They cannot yet digest the cheapest food source, cellulose, and so they consume corn sugars . Nevertheless, scientists engineering new functions for microbes, potentially even building them from the ground up, using a collection of standardized parts are hopeful they will succeed . They’re standardizing genetic and protein parts . They’re able to assemble or manipulate genes into synthetic genomes . And they’re studying how genes, proteins and small molecules fit together in metabolic networks that drive cells . With this new knowledge, scientists hope to exploit the exquisite selectivity and sensitivity of biological systems to build cheap, environmentally friendly catalysts, materials, sensors, and chemical factories . They are also chasing an intellectual challenge, too: Only by recreating nature do you test your knowledge of its principles . 26 Biological Chemistry: Engineering New Functions For Natural Systems Bio ms IX . Works Cited [1] Carr, P . A .; Church, G . M . Genome engineering . Nature Biotechnology 2009, 27, 1151–1162 . [2] Borman, S . DNA-Binding Proteins Turn Genes On And Off . Chemical & Engineering News, 2000, 78, 8: 34-35 . [3] Borman, S . New Way to Modify DNA . Chemical & Engineering News, 2012, 90, 22: 52-53 . [4] Borman, S . Bacterial Genome Made From Scratch . Chemical & Engineering News, 2008, 86, 4: 16 . [5] Wang, H . H .; Isaacs, F . J .; Carr, P . A .; Sun, Z . Z .; Xu, G .; Forest, C . R .; Church, G . M . Programming cells by multiplex genome engineering and accelerated evolution . Nature 2009, 460, 894–898 . [6] Singer, E . A Machine That Speeds Up Evolution . Technology Review 2009 . http://www . technologyreview .com/news/412573/a-machine-that-speeds-up-evolution/ (accessed August 25, 2012) . [7] Rouhi, M . From a Pore to a Cavity . Chemical & Engineering News, 2001, 79, 5: 10 . [8] Borman, S . Metalloprotein Made to Order . Chemical & Engineering News, 2009, 87, 49: 9 . [9] Borman, S . Bespoke Enzymes Push the Envelope . Chemical & Engineering News, 2012, 90, 19: 8 . [10] Jäckel, C .; Kast, P .; Hilvert, D . Protein Design by Directed Evolution . Annu . Rev . Biophys . 2008, 37, 153–173 . [11] Borman, S . Enzyme’s Activity Designed to Order . Chemical & Engineering News, 2000, 78, 8: 35-36 . [12] Wilson, E . K . Building Proteins Computationally . Chemical & Engineering News 2003, 81, 40: 35–36, 38–40 . [13] Pantazes, R . J .; Grisewood, M . J .; Maranas, C . D . Recent advances in computational protein design . Current Opinion in Structural Biology 2011, 21, 1–6 . [14] Borman, S . Proteins by Design . Chemical & Engineering News 1998, 76, 47: 9 . [15] Borman, S . New Protein Fold Made To Order . Chemical & Engineering News 2003, 81, 47: 11 . [16] Wilson, E . K . Virtually Created Enzymes . Chemical & Engineering News 2008, 86, 10: 13 . [17] Röthlisberger, D .; Khersonsky, O .; Wollacott, A . M .; Jiang, L .; DeChancie, J .; Betker, J .; Gallaher, J . L .; Althoff, E . A .; Zanghellini, A .; Dym, O .; Albeck, S .; Houk, K . N .; Tawfik, D . S .; Baker, D . Kemp elimination catalysts by computational enzyme design . Nature 2008, 453, 190–195 . [18] Halford, B . Build Your Own Enzyme . Chemical & Engineering News 2010, 88, 29: 5 . Biological Chemistry: Engineering New Functions For Natural Systems 27 28 [19] Kiss, G .; Röthlisberger, D .; Baker, D .; Houk, K . N . Evaluation and ranking of enzyme designs . Protein Science 2010, 19, 1760–1773 . [20] Baker, D . An exciting but challenging road ahead for computational enzyme design . Protein Science 2010, 19, 1817–1819 . [21] Liu, C . C .; Schultz, P . G . Adding New Chemistries to the Genetic Code . Annual Review of Biochemistry 2010, 79, 413–444 . [22] Dagani, R . Expanding Nature’s Protein Repertoire . Chemical & Engineering News 2003, 81, 25: 40–44 . [23] Drahl, C . Unnaturally Productive . Chemical & Engineering News 2011, 89, 34: 40–42 . [24] Brustad, E . M .; Arnold, F . H . Optimizing non-natural protein function with directed evolution . Current Opinion in Chemical Biology 2011, 15, 201–210 . [25] Liu, C . C .; Mack, A . V .; Brustad, E . M .; Mills, J . H .; Groff, D .; Smider, V . V .; Schultz, P . G . Evolution of Proteins with Genetically Encoded “Chemical Warheads .”J . Am . Chem . Soc . 2009, 131, 9616–9617 . [26] Khersonsky, O .; Kiss, G .; Röthlisberger, D .; Dym, O .; Albeck, S .; Houk, K . N .; Baker, D .; Tawfik, D . S . Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59 . Proc . Natl . Acad . Sci . 2012, 109, 40, 10358-10363 . [27] Eiben, C . B .; Siegel, J . B .; Bale, J . B .; Cooper, S .; Khatib, F .; Shen, B . W .; Foldit Players; Stoddard, B . L .; Popovic, Z .; Baker, D . Increased Diels-Alderase activity through backbone remodeling guided by Foldit players . Nature Biotechnology 2012, 30, 190–192 . [28] Thayer, A . M . Enzymes at Work . Chemical & Engineering News 2006, 84, 33: 15–25 . [29] Ritter, S . Greening Up Process Chemistry . Chemical & Engineering News 2010, 88, 43: 45-47 . [30] Bornscheuer, U . T .; Huisman, G . W .; Kazlauskas, R . J .; Lutz, S .; Moore, J . C .; Robins, K . Engineering the third wave of biocatalysis . Nature 2012, 485, 185–194 . [31] Thayer, A . M . Biocatalysis . Chemical & Engineering News 2012, 90, 22: 13–18 . [32] Wilson, E . K . Engineering Cell-Based Factories . Chemical & Engineering News 2005, 83, 12: 41–44 . [33] Kemsley, J . Boosting Taxol Production . Chemical & Engineering News 2010, 88, 40: 6 . [34] Ro, D .-K .; Paradise, E . M .; Ouellet, M .; Fisher, K . J .; Newman, K . L .; Ndungu, J . M .; Ho, K . A .; Eachus, R . A .; Ham, T . S .; Kirby, J .; Chang, M . C . Y .; Withers, S . T .; Shiba, Y .; Sarpong, R .; Keasling, J . D . Production of the antimalarial drug precursor artemisinic acid in engineered yeast . Nature 2006, 440, 940–943 . [35] Ritter, S . Fluorinase Success . Chemical & Engineering News 2010, 88, 5: 7 . [36] McCoy, M . Companies Advance Biobased Chemicals . Chemical & Engineering News 2011, 89, 17: 8 . [37] Reisch, M . S . Mainstreaming Biobased Plastics . Chemical & Engineering News 2012, 90, 17: 22–23 . [38] Thayer, A . M . Supplanting Oil . Chemical & Engineering News 2009, 87, 15: 22–26 . Biological Chemistry: Engineering New Functions For Natural Systems Bio ms [39] Tullo, A . H . Old Plastics, Fresh Dirt . Chemical & Engineering News 2012, 90, 12: 12–18 . [40] Bomgardner, M . M . Making Rubber From Renewables . Chemical & Engineering News 2011, 89, 50: 18–19 . [41] Bomgardner, M . M . The Sweet Smell of Microbes . Chemical & Engineering News 2012, 90, 29: 25-29 . [42] Ritter, S . Race To The Pump . Chemical & Engineering News 2011, 89, 7: 11-12, 14-17 . [43] Henry Arnaud, C . One-Pot Biodiesel . Chemical & Engineering News 2010, 88, 5: 11 . [44] Ritter, S . K . Genes to Gasoline . Chemical & Engineering News 2008, 86, 49: 10–17 . [45] Ritter, S . K . Doubly Engineered Microbes for Biofuels . Chemical & Engineering News 2011, 89, 49: 35 . [46] Bullis, K . To Survive, Some Biofuels Companies Give Up on Biofuels . Technology Review 2011 . http://www .technologyreview .com/news/426457/to-survive-some-biofuelscompanies-give-up-on/ (accessed August 23, 2012) . [47] What’s in a name? Nature Biotechnology 2009, 27, 1071–1073 . [48] Rawls, R . L . “Synthetic Biology” Makes its Debut . Chemical & Engineering News 2000, 78, 17: 49 . [49] Borman, S . Designed Pathways and Microbes . Chemical & Engineering News 2008, 86, 46: 62-66 . [50] Khalil, A . S .; Collins, J . J . Synthetic biology: applications come of age . Nature Reviews Genetics 2010, 11, 367–379 . [51] Borman, S . A Jamboree With Reusable Parts . Chemical & Engineering News 2008, 86, 46: 64-65 . [52] Pittman, D . Custom-Made Cells . Chemical & Engineering News 2010, 88, 29: 34-36 . [53] Pittman, D . Synthetic Biology Takes a Step Forward . Chemical & Engineering News 2010, 88, 21: 10 . [54] Bromley, E . H . C .; Channon, K .; Moutevelis, E .; Woolfson, D . N . Peptide and Protein Building Blocks for Synthetic Biology: From Programming Biomolecules to Self-Organized Biomolecular Systems . ACS Chem . Biol . 2008, 3, 38–50 . [55] Fletcher, J . M .; Boyle, A . L .; Bruning, M .; Bartlett, G . J .; Vincent, T . L .; Zaccai, N . R .; Armstrong, C . T .; Bromley, E . H . C .; Booth, P . J .; Brady, R . L .; Thomson, A . R .; Woolfson, D . N . A Basis Set of de Novo Coiled-Coil Peptide Oligomers for Rational Protein Design and Synthetic Biology . ACS Synth . Biol . 2012, 1, 240–250 . [56] Keasling, J . D . Synthetic Biology for Synthetic Chemistry . ACS Chem . Biol . 2008, 3, 64–76 . [57] Borman, S . Factory-Style RNA Engineering . Chemical & Engineering News 2012, 90, 1: 24 . [58] Mukhopadhyay, R . DARPA To Launch Synthetic Biology Program . Chemical & Engineering News 2011, 89, 28: 26 . [59] Erickson, B . E . Synthetic Biology . Chemical & Engineering News 2009, 87, 31: 23–25 . [60] Erickson, B . E . Synthetic Biology Has Limited Risks . Chemical & Engineering News 2010, 88, 51:31 . Biological Chemistry: Engineering New Functions For Natural Systems 29 30 [61] Erickson, B . E . Advancing Synthetic Biology . Chemical & Engineering News 2012, 90, 27: 20–21 . [62] Schulz, W . Censoring Research Results . Chemical & Engineering News 2012, 90, 6: 6 . [63] Schulz, W . A Dual-Use Debate . Chemical & Engineering News 2012, 90, 8: 34-37 . [64] Schulz, W . Government Acts on Biosecurity . Chemical & Engineering News 2012, 90, 15: 7 . [65] Holtcamp, W . One Study, Two Paths: The Challenge of Dual-Use Research . Environmental Health Perspectives 2012, 120, a238–a242 . [66] Halford, B . DNA Origami . Chemical & Engineering News 2006, 84, 12: 10 . [67] Halford, B . Building in 3-D With DNA Origami . Chemical & Engineering News 2009, 87, 19: 30-31 . [68] Halford, B . More than Just a Smiley Face . Chemical & Engineering News 2012, 90, 28: 30-31 . [69] Halford, B . Rise of the DNA Robots . Chemical & Engineering News 2010, 88, 20: 8 . [70] Halford, B . Delivery Via DNA Nanobots . Chemical & Engineering News 2012, 90, 8: 8 . [71] Jonker, A . M .; Löwik, D . W . P . M .; van Hest, J . C . M . Peptide- and Protein-Based Hydrogels . Chem . Mater . 2012, 24, 759–773 . [72] Arnold, C . From Diseases to Devices . Chemical & Engineering News 2008, 86, 29: 48–50 . [73] Tecon, R .; Van der Meer, J . R . Bacterial Biosensors for Measuring Availability of Environmental Pollutants . Sensors 2008, 8, 4062–4080 . [74] Sanderson, K . Device Tests Toxic Waters . Chemical & Engineering News 2011 . http://cen . acs .org/articles/89/web/2011/09/Device-Tests-Toxic-Waters .html [75] Sanderson, K . New Portable Kit Detects Arsenic In Wells . Chemical & Engineering News 2012 . http://cen .acs .org/articles/90/web/2012/02/New-Portable-Kit-Detects-Arsenic .html Biological Chemistry: Engineering New Functions For Natural Systems Bio ms NOTES Biological Chemistry: Engineering New Functions For Natural Systems 31 NOTES 32 Biological Chemistry: Engineering New Functions For Natural Systems