How do you turn an orange into a grapefruit? It’s an important question, according to Alize Pennec, biotransformation development manager at Oxford Biotrans, speaking in an interview for Oxford University portal Oxford Sparks.
Nootkatone from grapefruits is one of the world’s most expensive flavour ingredients, costing £3000-£4000 per kg. ‘It takes 400,000kg of grapefruits to make just 1kg of this oily molecule,’ Pennec said. Grapefruits are not always easily available, so ‘supply is struggling to keep up with growing demand’.
Luckily, nootkatone can be produced from a similar molecule – valencene – which is sourced more easily from plentiful oranges. The chemical route to make nootkatone involves heavy metal catalysts and peroxides, but the resulting compound is no longer so desirable as it’s not natural, Pennec explained. Instead, Oxford Biotrans has devised an environmentally friendlier pathway – by engineering the bacterium E. Coli to produce the enzyme cytochrome P450 for catalysing the efficient bio-conversion of valencene to nootkatone.
Tremendous progress
The business of reprogramming biology in this way is currently attracting a lot of interest – and not just from grapefruit lovers. According to data by synthetic biology hub SynBioBeta, Oxford Biotrans is one of over 50 synthetic biology start-ups and more established synbio companies now operating in the UK – a number that’s been increasing by around 20%/year between 2009 and 2015. The UK government, meanwhile, has earmarked synthetic biology as one of ‘Eight Great Technologies’ set to trigger a fourth industrial revolution: one where it believes the UK could be a world leader.
‘There has been tremendous progress,’ enthused Richard Kitney, a co-director of the UK’s national synbio centre, SynbiCITE, based at Imperial College London, speaking at the first IET/SynbiCITE meeting on engineering biology in London in December 2016. ‘But there’s still a long way to go before the discipline realises its full commercial potential.’
Kitney was one of the main authors of a synthetic biology roadmap back in 2012, widely credited with creating much of the impetus for the field’s rapid evolution. The roadmap set out a series of actions to accelerate progress, including investing in a network of multidisciplinary centres, establishing a UK national industrial centre and forming a national Synthetic Biology Leadership Council (SBLC).
At a global level, too, SynBioBeta’s annual industry investment update released in March 2017 reveals that synthetic biology is gathering momentum. Worldwide, there are 410 synbio companies currently in operation, SynBioBeta reports, while the falling costs of reading and writing DNA are making biology ‘faster, better and more cost effective’ to engineer.
‘The global synthetic biology industry raised over US$1bn in 2016, and the industry continues to show very strong signs of growth,’ says SynBioBeta founder John Cumbers. A study by Allied Market Research in 2014 predicts a global market for synthetic biology of around $38bn by 2020, growing at a CAGR of 44% between 2013 and 2020.
Back in the UK, spurred on by the success of the first roadmap, Kitney and his SBLC coauthors, published a second version in early 2016. Subtitled Biodesign for the bioeconomy, this updated roadmap redefines synthetic biology as a ‘facilitating toolbox’ that allows the field’s practitioners to transform possibilities into practical results. What’s needed now, says its authors, is agreement on exactly what that toolbox should comprise, including a set of reliable genetic parts (Nature, 2016, doi: 10.1038/531401a).
‘To boost productivity will require the use of standardised components and processes, automation and robotics – underpinned by computation,’ write Kitney and his coauthors in a recent review paper (Synthetic and Systems Biotechnology, 2016, 1, 243). Users’ time is then freed up to focus on biodesign, they point out - in other words ‘what can we use synthetic biology to do?’ rather than the ‘underlying mechanics’ of how to get there.
A common language
Critically, the plan depends on the sharing of resources and information by different research groups, pointed out Anil Wipat of Newcastle University, another speaker at the IET/SynbiCITE meeting. And, equally importantly, on ensuring that everyone adheres to the same standards, he added - before quoting the US computer scientist Andy Tanenbaum who once famously remarked that: ‘the nice thing about standards is there are so many to choose from’.
Synthetic Biology Open Language (SBOL) is a computational language that aims to provide a standardised format for sharing genetic data - which is critical in order to reduce errors and ensure reproducibility, explained Wipat, who is also current chair of the SBOL developers group.
Current synthetic biology operations can be broken down into three main steps: selecting an appropriate ‘DNA part’ of interest – often enzymes or stretches of DNA coding for some desired characteristic or trait; synthesising the DNA at scale; and inserting it into cells.
One major use of SBOL, Wipat said, is for designing and synthesising DNA parts. These parts can either be designed online or obtained from biological repositories, likened to DNA libraries, which store both physical samples and sequence information on interesting pieces of DNA previously isolated and studied by other labs. The US National Institutes of Health (NIH) public biological repository GenBank, for example, contains nucleotide sequences for hundreds of thousands of ‘formally described species’ (Nucleic Acids Research, 2013, 41(D1), D36-42).
However, Wipat pointed out the information available from SBOL is far more information rich than that available from GenBank, and also encodes other useful data along with the genetic sequence information - about proteins and small molecules and the interactions between them - which allows researchers to design truly novel DNA sequences. SBOL was selected as the ‘language of choice’ by ACS Synthetic Biology journal in 2016. A new SBOL Visual tool also standardises depictions of DNA regions such as promoters and coding sequences - by representing them as glyphs or symbols.
A difficult subject
Despite some advances, however, biology remains notoriously difficult to work with. ‘Right now, working with biology is hard,’ said Sean Ward, Chief Technology Officer of London biotech Synthace: ‘It takes too long, costs too much and fails too often.’
Reproducibility, in particular, is a huge problem. A recent review of 441 random scientific publications, for example, found that only one was theoretically reproducible in having enough detailed information to be accurately repeatable, Ward pointed out, adding that the annual economic impact of irreproducible research in the life sciences is estimated at $28bn.
Ward blames many of the problems on the sheer complexity of the biological systems studied, and also on the vagueness of journal paper descriptions such as ‘shake vigorously’, ‘leave overnight’ or ‘at room temperature’. A problem that another speaker at the meeting also suggested may be addressed if future journal papers were written in computer code rather than words.
Synthace’s Antha operating system is a step in that direction, Ward explained. It takes common laboratory processes and procedures and translates them into a ‘high level’ computer code, so they can be shared and more accurately repeated by others.
‘Antha takes these workflows and compiles them into computer code required to directly run automation and analytical equipment for each experiment,’ he said. ‘It tracks the flow of information and physical samples through the workflows, so every piece of data generated is linked to full provenance of the experimental procedure.’
Importantly, he added, it’s also interoperable between different makes and models of lab hardware.
Finding a common language is one mechanism to expedite progress. Another approach is to bring different groups together to share facilities and resources. At UK national centre SynbiCITE, for example, Kitney explained that the aim is to promote commercialisation of synthetic biology by encouraging collaboration between business, academe and supporting organisations and regional government and local authorities. One of seven Innovation and Knowledge Centres (IKCs) set up to nucleate new industries, SynbiCITE was established in 2013, backed by a five-year £28m commitment from Engineering and Physical Sciences Research Council (EPSRC), Biotechnology and Biological Sciences Research Council (BBSRC), Innovate UK and its industrial and academic partners.
The idea is to pool not just expensive resources, facilities and equipment, but also people and ideas. A £2m London DNA Foundry opened at SynbiCITE in April 2016, for example, hosts suites of equipment for automated DNA assembly and delivery of DNA to living cells, allowing researchers to design and scale up the volumes of DNA produced to more easily test their new function. The UK now has five of these DNA foundries, including the Edinburgh Genome Foundry at the university's School of Biological Sciences in Scotland.
Progress update
Technological developments, too, have played a big role in expediting progress. Thanks to companies like California-headquartered Illumina, for example, the costs of DNA sequencing have plummeted in recent years. DNA synthesis can now often be performed in the lab or outsourced relatively inexpensively to any one of a number of specialist biotech firms who will synthesise it for you. Gene editing technologies such as CRISPR and TALENs, meanwhile, have greatly improved the accuracy of procedures for inserting foreign genetic material into cells – though not yet, admittedly, to the levels of precision desired for certain medical and gene therapy applications.
Not too surprisingly, synthetic biology success stories are becoming easier to find. The commercial production of a ‘semi-synthetic version’ of antimalarial drug artemisinin from reprogrammed E. coli bacteria by San Francisco biotech Amyris is one of the most notable, retold at the meeting by Jay Kiesling, one of the company’s cofounders. Shown to be functionally equivalent to the plant-derived drug, semi-synthetic artemisinin was approved in 2013 by the World Health Organization. Other examples include the rapid development of new bird flu vaccine by another CA biotech, Synthetic Genomics, in collaboration with Novartis, following the outbreak of a novel infection outbreak H7N9 in 2013. And the production of low-cost energy fuels and high value chemicals such as 2,3-butanediol and acetic acid from waste gas resources, including industrial flue gas, via engineered microbes created by New Zealand firm LanzaTech.
Given the right tools, it seems that synthetic biologists can do just about anything - within legal and ethical boundaries. The era of bio design is almost upon us – and turning oranges into grapefruits is just the beginning.
Twist on DNA archiving
DNA has been storing information in the form of genetic code for millennia, according to Ross Kettleborough, field applications specialist at California biotech Twist Bioscience, speaking at the IET/SynbiCITE meeting. Now, researchers are also beginning to look at its potential for digital data archiving, he pointed out, referring to the announcement in April 2016 that Microsoft Corp. has agreed to buy 10m of Twist’s ‘long oligonucleotides’ for the purpose of encoding digital data.
A single gram of DNA can store almost a zettabyte of digital data, Kettleborough said. That’s more than a third of the total 2.7 zettabytes or 2.7 x 1021 bytes of information the entire world is estimated to have stored back in 2012 (http://dx.doi.org/10.1155/2016/8072463). Unlike traditional data storage media, DNA storage also lasts thousands of years without deterioration.
DNA archiving is now feasible for two reasons, Kettleborough said: the development of affordable DNA sequencing, and new synthesis technology for making DNA at scale more cheaply. The classical way to synthesise DNA is on a 96 well plate, he explained. However, what’s different about Twist’s DNA synthesis technology is that it automates the whole process on silicon.
‘Making 100, 000 genes in a fortnight was not possible until Twist came along,’ Kettleborough said. ‘The company’s silicon based synthesis platform has the same footprint as the standard 96 well plate but it makes 9600 genes instead of one.’
‘Twist’s goal is to make highest-quality DNA cheaper and faster than anyone else,’ he added.
Twist technology uses standard phosphoramidite chemistry. Whereas the clusters of a standard 96-well plate contain around 100 microlitres of reagent, Twist’s silicon wafer clusters each hold hundreds of 100nL. A silicon chip the size of a 96-well plate therefore has 9600 clusters, each containing all of the chemistry needed to make and to stitch together the various oligos and bases together in the same well or reaction pot.
Twist’s typical turnaround time for a synthesised DNA part is currently around 15-20 days, Kettleborough said, and costs around 9 cents per base. By the end of 2017, the aim is to reduce delivery times to just 10 days.
In November 2015, Twist signed a deal with scents and flavours firm Ginkgo Bioworks to make 100m base pairs of synthetic DNA by the end of 2016 – a quantity then equal to roughly 10% of the market. A further 300m base pairs is now on order for delivery by the end of 2017.
Big scale DNA synthesis
Companies and researchers wanting large amounts of high quality DNA have traditionally hit a roadblock. Until now, all of the available methods of making it – either by lab synthesis or by fermentation in bacterial cells – have proved both difficult and expensive to scale.
However, London based Touchlight Genetics has hit on a way around the problem, with a two-enzyme process to make multi-gram quantities of DNA in just two weeks. The company’s ‘Doggybone DNA’ technology is ‘at least an order of magnitude cheaper’ than plasmid fermentation with difficult constructs, said Sarah Milsom, Touchlight’s group business development manager, speaking at the IET/synbiCITE meeting. And, because it is made in vitro in the lab on a benchtop, it doesn’t require large fermentation reactors or microbes, so avoiding the problems of handling bacterial materials and genetic sequences that often plague other synthesis methods.
Doggybone DNA (dbDNA) is so-called because of its shape, Milsom explained. Apart from two very short enzyme binding sites flanking the desired DNA construct, there are virtually no extraneous DNA sequences in this linear construct.
To create dbDNA, the process starts with a double stranded circular DNA template, such as a plasmid. The two DNA strands are then separated or denatured, allowing the first, polymerase, enzyme to copy or amplify the strand containing the sequence of interest by modified polymerase chain reaction (PCR). Next, the desired sequence is then removed by a second ‘protelomerase’ enzyme, which cuts the DNA at the two flanking enzyme binding sites.
The resulting dbDNA contains very few sequence errors – the reported error rate is less than 1 in 107, Milsom pointed out. And because it doesn’t involve bacteria, no bacterial genes remain floating around to contaminate the process or confer future problems of antibiotic resistance.
DNA vaccines and gene therapies are just a couple of the medical applications that could benefit, Milsom pointed out, with multiple grams of DNA needed every year for gene therapy applications. Outside of therapeutic areas, the group is currently working with researchers at Newcastle University to make DNA templated nanowires for sensing air pollutants such as VOCs. Another group at the University of Utah is exploiting the technology to develop DNA hydrogel bio-batteries that use lactase enzyme in tears to power devices. Materials applications such as these would require 100s of grams or kilograms of DNA to commercialise.
Synthetic yeast genome
The genome of common baker’s yeast, Saccharomyces cerevisiae, contains around 12m base pairs of DNA and has 16 chromosomes – compared with the 23 pairs of chromosomes found in humans. Yet despite this complexity, researchers – including hundreds of undergraduate students – are well on track to create a synthetic version of S. cerevisiae before 2020, reported Leslie Mitchell of New York University, speaking at the IET/SynbiCITE meeting.
When complete, this genome would be the first reported designer synthetic ‘eukaryotic’ genome, Mitchell pointed out, since yeast also stores its DNA in a nucleus within cells, like plants and animals.
Mitchell is one of a team of researchers in the NYU laboratory of Jef Boeke, who had the original idea to synthesise the baker’s yeast genome back in 2010 (Nature, doi:10.1038/nature.2014.14941). Boeke’s plan was not to recreate the whole genome, but to streamline the system by stripping out some non-essential parts and adding new features to open up new research possibilities, Mitchell explained. To reduce costs and save time, he also recruited undergraduate students – many of them enrolled on the ‘Build a Genome’ course he originally started at Johns Hopkins University – to make different genome stretches by stitching together very short lengths of DNA made by a DNA synthesis machine.
Boeke and his team of around 70 undergraduate students achieved the first milestone in 2014 with the seven-year synthesis of the first synthetic yeast chromosome, synIII, comprising 273,871 base pairs of DNA, reported in the journal Science (doi:http://dx.doi.org/10.1126/science.1249252). To construct synIII, the team made more than 500 alterations, amounting to a sequence alteration approximately every 500 base pairs and a 13.8% overall reduction in size, Mitchell explained. Despite these extensive changes, amazingly synIII could be swapped back into the native yeast genome with little effect on yeast growth and replication.
Three years later, Mitchell reported that the Synthetic Yeast Genome Project (Sc2.0; www.syntheticyeast.org) - involving researchers from the US, China, Australia, Singapore and the UK - is now well on the way towards completing Sc2.0, with the design of the synthetic genome complete, and an additional five synthetic chromosomes synthesised and shown to direct growth of yeast identically to their wild-type counterparts. Construction of the remaining synthetic chromosomes is under way in labs around the world.
Synthetic ‘start’ and ‘stop’ signals
‘Nature knows best,’ so the saying goes. But researchers at Synpromics might not always agree. The Edinburgh-based biotech is in the business of making artificial ‘promoters’ – small sequences of DNA that function as the start and stop signals for switching genes on or off.
Natural promoters have evolved over many generations for different biological functions in living organisms, said Synpromics’ founder and Chief Scientific Officer, Michael Roberts, speaking at the IET/SynbiCITE meeting. Every gene sits beside a promoter that tells it when to stop and start transcription, the first stage whereby genes are expressed as proteins.
Most current biotech applications employ natural viral or gene-specific promoters. But Synpromics’ synthetic promoters have many advantages as they are specially tailored for the end application of interest, Roberts said. They can also be made with higher specificity and fewer side or off-target effects, which could improve safety – particularly valuable for gene therapies.
In January 2017, Synpromics announced a research collaboration with GE Healthcare to develop synthetic promoters for use in Chinese Hamster Ovary (CHO) cells - living factories for the production of protein ‘biologic’ drugs or biopharmaceuticals. Initial testing of synthetic CHO promoters have shown they boost protein expression or production levels five to ten times compared with the natural promoters, Roberts reported. Based on these results, Synpromics will now produce a library of promoters for screening in GE’s CHO expression system, and the resulting promoter toolbox is expected to increase the yields of a range of protein biologic drugs and biopharmaceutical drugs, including difficult to manufacture proteins.
‘We believe our synthetic promoters will provide a much more efficient production system and we are aiming to help GE Healthcare boost efficiency for its biopharma customers,’ said Synpromics’ CEO David Venables.
The company’s proprietary Promoter Precision Technology (PROMPT) evaluates what are the optimal factors for gene transcription, Roberts explained. It employs algorithms to unravel the different combinations of transcription factors that control a gene’s expression profile - which ultimately dictates whether a cell becomes a lung, liver or brain cell. This expression pattern varies if cells become diseased or infected, or are treated with chemicals, he said, so it is possible to design synthetic promoters that are active only under certain conditions, say in response to infection or only in one particular body tissue.
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