biological chemistry: engineering new functions · 2 biological chemistry: engineering new...

Biological Chemistry: Engineering New Functions For Natural Systems 1

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 .

2 Biological Chemistry: Engineering New Functions For Natural Systems Biological Chemistry: Engineering New Functions For Natural Systems

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 .

Biological Chemistry: Engineering New Functions For Natural Systems Biological Chemistry: Engineering New Functions For Natural Systems 3

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,


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]


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 .


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 copper-

containing 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 .


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]


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-butadiene-

1-carbamate and the dienophile N,N-dimethylacrylamide


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]


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 .


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


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]


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


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


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 anti-

malaria 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

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

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 .

Arteminisin

Taxadine is a key intermediate

in the biosynthesis of Taxol


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 . Petroleum-

derived 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 .


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 .

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 .

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,

Nootkatone Valencene

Engineered bacteria produce biodiesel from hemicellulose .


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 energy-

intensive 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 .


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


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 .


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 .


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 .


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


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


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


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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32 Biological Chemistry: Engineering New Functions For Natural Systems

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biological chemistry: engineering new functions · 2 biological chemistry: engineering new...

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