Engineering metabolic pathways, whether for pharmaceuticals or fuels, involves many trade-offs. Flux imbalances (for example where flux through the first step exceeds flux through the second) can lead to the accumulation of toxic intermediates. Moreover, expressing heterologous proteins beyond that which is necessary can place a burden upon the cell for protein, rather than product, synthesis. Sometimes lowering expression levels of heterologously expressed proteins improves product titers (e.g. here and here). Continue reading
There is a PhD position available with Paul Race at Bristol that I will be co-supervising (subject to securing the funding). If successful this will be funded through the South West Doctoral Training Partnership. The PhD will be a fully-funded, four-year programme designed to provide training in cutting edge world-class bioscience and food security research skills.
The project details are below (and also here), and the deadline for application is Friday 10th January 2014. Coincidentally the last day of the SEB Synthetic Biology workshop (shameless plug): deadline for abstracts and registration looming (Dec 6th)!
Main supervisor: Dr Paul Race (School of Biochemistry, University of Bristol)
Second supervisor: Dr Thomas Howard (I really should get my web-page updated) (Department of Biosciences, University of Exeter)
With: Dr Ross Anderson (School of Biochemistry, University of Bristol) & Prof Chris Willis (School of Chemistry, University of Bristol)
Nature has evolved many elegant strategies for the assembly of complex bioactive natural products. The most sophisticated of these involves the action of giant assembly line like megasynthases, that fuse and tailor simple carboxylic acid monomers into a vast array of elaborate carbon scaffolds. This biosynthetic logic is found extensively in the polyketide pathway, and has provided us with many of our most important clinically used antibiotics, e.g. the tetracyclines. There is considerable interest in the rational reengineering of these enzymatic machines, en route to the production of ‘non-natural’ natural products with enhanced bioactivities. To date reengineering strategies have focused on the deletion, insertion, or transplantation of defined synthase components within or between pathways. These modifications have been predominantly directed by bioinformatic analysis of nucleotide sequences. Using molecular level structural information of megasynthase components to guide synthase reengineering is an inherently more powerful approach for manipulation, as module/domain boundaries, enzyme active sites, protein-protein interaction interfaces and inter-module/domain linkers can be accurately defined and interrogated. Further, by using these data as a starting point one is well placed to begin constructing megasynthases de novo, introducing specified biosynthetic functionality to yield desired chemical and stereochemical output in the final pathway product.
This project will marry the complementary expertise of Race (structural enzymology natural product biosynthesis), Howard (pathway redesign and genome engineering), Anderson (enzyme design) and Willis (analytical and synthetic chemistry), in attempting to construct, from first principals, minimal assembly line like megasynthases for the production of new to science polyketide antibiotics. Our approach will be to use the considerable volume of detailed structural and functional information garnered in the applicants’ laboratories (crystal/NMR structures, enzymatic analyses, PPI studies, chemical information, etc.) to construct minimal synthase mosaics with defined biosynthetic activities. We will explore and define the rules of synthase construction and introduce our new systems into microbial hosts en route to the scaleable production of new chemical entities. All novel compounds isolated from this study will be tested for antimicrobial activity and where appropriate taken forward for clinical evaluation.
During the first year of the project the student will undertake 2 rotation projects. The first of these will be based in the Race lab, University of Bristol and will focus on the design and synthesis of the first iteration ofde novo minimal synthases. During this rotation training will be provided in protein structure analysis and enzymological methods. The second rotation project will be performed in the Howard lab, University of Exeter. During this placement training will be provided in genome engineering, molecular biology and associated analytical methods. Please note, as the second rotation project will be located in Exeter, depending on your preference you will be either required to travel to Exeter on a daily basis or accommodation will be provided at Exeter for yourself.
As promised here are a few notes and links on the key points of today’s lecture: Synthetic Biology and its Application (28th November 2013). Follow the links for further information.
Synthetic Biology – it’s not what you do, it’s the way that you do it.
Three key aims of synthetic biology were highlighted: the first was ‘making biology easier to engineer’. From this flow the two other aims, ‘learning by building’ and ‘commercialisation of product’ (being an equally applied and fundamental science).
Making Biology Easier to Engineer
For the last 30-40 years, genetic engineering has been based on PCR and recombinant DNA technologies for writing, and automated sequencing for reading DNA. Automated DNA synthesis and new assembly methods (e.g. Biobrick, Gibson, Golden Gate etc.) and an industry (e.g. DNA2.0, IDT etc.) that can supply these products cheaply and quickly mean that the emphasis is now on what you write, not how you write it.
This synthesis and assembly capacity allows well characterised parts, engineered to behave in a predictable, standard way. This is recognised in recent high profile papers such as Mutalik et al. 2013a, Mutalik et al. 2013b and Kosuri et al 2013 in which these methods have been used to build and assess different combinations of regulatory units (primarily promoters and 5’UTRs). The Nat. Methods papers are behind paywall and not open access (which kind of defeats the object of communicating science) so you can get an overview here and here. The strategies were different but the outcome the same: defined, quantifiable functions relating to strength and quality of the part. Also, see the Synthetic Biology Open Language standard (SBOL) and part characterisation performed for example, during iGEM.
Parts like this can be assembled into more complex devices that perform predictable functions. In turn these can be represented in an abstract manner (abstraction) allowing the designer to focus on a few key design parameters at a time. You don’t need to go to the DNA level if you don’t want to. Computer-aided design (e.g. Clotho, GeneDesigner) is becoming more and more common. Also, as we create more synthetic, engineered sequences and learn the rules of DNA-coding, we are likely to move further and further away from ‘natural’ to truly ‘synthetic’ genes.
As an example the two papers on a genetically-encoded oscillator and toggle switch were briefly introduced. Taken together this encourages people to think of biosciences as the place to be for driving economic growth and the next industrial revolution.
Learning By Building
Firstly, taking parts from nature and finding out they don’t do what we thought they did is a pretty quick way of testing the literature!
Secondly, building synthetic diversity (based on rational, statistical choices) is preferable to natural diversity for understanding biology. In the artificial situation we can deliberately maximise our search of the design space, but in natural systems we are restricted to the places evolution has taken biology. See here and here. This point was returned to later.
Commercialisation of product
Production of artemisinin via metabolic engineering of S. cerevisiae used 14 upregulated/introduced genes and 2 repressed/omitted. This approach highlights the importance of taking a holistic approach – in this instance it is better to produce a chemical precursor (artemisinic acid) rather than the final product – chemical and biological engineering working in tandem – and learning how to ferment these strains has the biggest impact on improving yield.
Biofuels are a different prospect. The recent elucidation of the genetic basis for alkane biosynthesis in cyanobacteria and the higher plant Arabidopsis thaliana has resulted in a series of reports in which alkane biosynthesis has been tailored towards the production of desirable retail fuel molecules. These include the biosynthesis of n-alkanes and -alkenes of different chain lengths (here here and here) and of the structural complexity (iso-alkanes, here) required to supply the ability to blend fuel.
However, if you work on therapeutics, drugs or cosmetics sugar is cheap. If you work on biofuels, sugar is expensive.
Strategies to exploit cheaper (and preferably non-food) feed stocks (esp. lignocellulosic material) are therefore underway. These follow similar strategies to the artemisinin and biofuels work above. A bigger challenge is maximising conversion efficiency (I didn’t get to the trade-off between this % conversion and concentration (mg/L) and yield (mg/h) – but it is a key processing problem – which do you want more of? It is unlikely one solution will give you everything). So, how do we engineer a system whose complexity so far escapes us? Taking a leaf out of computer science we can look to couple machine learning approaches to our new automated DNA writing technologies and the creation of synthetic diversity described previously. Biology as a computer science problem. But that’s not for now.
The final question was: that sounds all well and good, but,
How easy is it to engineer biology?
How do you even test this? The answer is, give it to a bunch of amateurs….
The results (of course) can be amazing. Here are a couple of posts I wrote some time ago about iGEM projects, (Grand Prizes and Materials Science), but the best thing to do is to go and browse around yourself: iGEM main page, and Exeter 2012 and Exeter 2013.
Here are some other links you may find interesting:
Video: Synthetic Biology Explained
Any questions, feel free to ask. The best thing to do if any of this interests you is to get involved.
For German coverage of our biofuels paper, look no further than 3sat. The article starts at 22mins and features interviews and cameos from many in the mezz. lab. Keep your eyes pealed for a stealth technician around the 23.40min mark.
Further good news is a nomination for a University of Exeter Impact Awards in the ‘Outstanding impact in technology’ category – programming bacteria to produce retail-grade diesel. Also nominated in this category, fellow Bioscience’s researcher Chris Thornton – good luck Chris….really ;-p
Cas9 – could it change Biotech forever? Well, you’ll have to follow that link to find out. In the meantime, here’s a very brief introduction.
CRISPR – clustered, regularly interspersed, short palindromic repeats – are used by bacteria and archaea to provide protection against foreign nucleic acid sequences. Continue reading
Just a very quick note to say that Claes Gustafsson, co-founder of synbio company DNA2.0 (far right), will be giving a seminar on gene design and their protein/pathway engineering platform on Wednesday 20th March at 3pm in GP328a. Continue reading