Download Biological Chemistry: Engineering New Functions for Natural Systems

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 . . . . . . . . . . . . . . . . . . . . .
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V. Synthetic Biology: The Sum of the Parts
. . . . . . . . . . . . . . . . . .
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VI. Regulating Synthetic Biology Research . . . . . . . . . . . . . . . . . .
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VII. Materials and Sensors Made From Biomolecules . . . . . . . . . . . . . .
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VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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IX. Works Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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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 .
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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 .
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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,
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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]
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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 .
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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 .
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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]
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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
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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]
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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 .
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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
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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]
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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
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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
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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 .
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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 .
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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,
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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 .
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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
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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 .
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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 .
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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 .
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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
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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
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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
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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 .
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