Grey is the new green? Heterologous reconstruction of plant metabolism.

Plants are large and thirsty. They require a lot of space and if you want to extract something from them you’ll have to find a way to efficiently extract it from the smorgasbord of chemicals in each and every plant cell. And for that matter plant cells are not all equal, and you may find plenty of product in one cell type but precious little in others.

With all that in mind it seems a pretty great idea to take desirable metabolic pathways out of plants and get them working in friendly microbes such as E. coli and budding yeast. And that is precisely what many synthetic biology/ metabolic engineering groups do as their bread and butter. If this is news to you just check out some of the articles below:

Pinene in E. coli

Mycerne production in E. coli

Producing plant monoterpene Indole alkaloids in yeast 

and of course there was the flagship synbio project, taking the enzymes for the biosynthesis of anti-malarial drug Artemisinin from Artemisia annua into yeast. That work spawned a company, Amyris, in California. Whilst over on the East Coast of the USA Ginko Bioworks follow a similar strategy.

So if microbes are so great, why use plants at all?

Well, perhaps to extract the power for metabolism from photosynthesis?

Still no dice. There are plenty of photosyntheic mircoorganisms (Cyanobacteria and unicellular eukaryotic algae such as Chlamydomonas).Birger Lindberg Møller’s group at the University of Copenhagen are turning the chloroplasts of microalgae and mosses into biorefineries.

 

So what are the advantages of leafy plants? 

Actually there are several. But here are the three most currently relevant:

You can eat them. Think Golden Rice and Purple Tomatoes.

They are cheap to grow. Sunlight and rainwater are free. So is soil, and if you nurture it properly and grow nitrogen fixers in rotation expenditure on fertilizers can be kept down. By contrast, micro-organisms need constantly aerated (or artificially anoxic) environments, that need to be supplied with the right nutrients whilst being protected from contamination. All that adds up to running costs, which have been a major stumbling block in the way of algal biofuel production, for example. So if you want to produce vast amounts of a relatively low-value product plants are likely the best way to go.

Tobacco is an efficient soluble protein producing machine. This has spawned a huge amount of research focus into harnessing tobacco for the production of bio-pharmaceuticals and ‘plantibodies’.

Taking plant pathways into microbes is a neat trick, and one that definitely makes sense. But there is still plenty of reason to hold on to our leafy friends.

 

Advertisements

The X prize in Plant Synbio

A sudden breeze, warm and not unwelcome, whips at the hem of your dress as you step aboard the vessel. You mentally scan back through the training sessions you did last month and you take your seat. Helmet on, you sit back in your chair, humming with excitement, ready for your first trip into Space. 5…4….3…2…1

If you think that’s pure science fiction, visit the website of Virgin Galactic. Upwards of 300 people have proffered $250,000 each for a ticket on a Virgin Galactic space craft. Whilst the date of the Maiden voyage has been pushed back considerably this is no flight of collective fantasy, it is a realistic proposition built on the vision and technology of Mojave Aerospace Ventures, which in 2004, launched SpaceShipOne into the first manned private space flight.

That 2004 flight was born out of the first Xprize, which offers a hefty financial reward to any team able to address a daunting engineering challenge with an innovative solution. Visit http://www.Xprize.org and you’ll be inundated with such cliches and buzzwords. But at its core the idea is simple and powerful: goal + incentive + competition = innovation.

Anybody who has ever been involved with iGem knows this. Unfortunately iGem projects rarely reach full fruition. What we need is an Xprize for synthetic biology. And of course my bias is for an Xprize in Plant synthetic biology.

There are plenty of existing projects that have been a very long time coming and could lend themselves perfectly to a little boost from the Xprize model:

And how about some more:

  • A toolkit to specifically induce any gene in response to any plant hormone.
  • Grow a genetically modified plant into a cube.
  • Produce a fool-proof kill-switch to prevent GM crop escape.

The model I propose would be to offer up a prize with some money attached and each year the team closest to the goals gets the prize, and with it the glory. The prize could continue for as many years as is felt necessary to really tackle the challenge.

It keeps people focused, it keeps people driven.

Synthetic biology is often trapped by the inefficiencies of basic research. Not only does curiosity have a tendency to trump engineering requirement, leading to ‘wasted’ efforts. But also the process is inefficient: PhD students and postdocs making individual, piecemeal contributions to challenges that are ill-defined and constantly shifting.

Of course prize-money needs donors. As distasteful as some may find them, this is a role of the big Agrochemical companies. Whether its through a prize or not, they have their eyes on the plant synthetic biology community and they WILL be looking to turn our discoveries into profits either way. Why not work together on goals that we all share. And after all, don’t we want to be creating marketable products i.e. things that people would actually want and benefit their lives in some way?

We all know there are glittering prizes on the horizons of plant bionengineering right now. It’s time to pluck them out of the sky, inject them with cash, and let the wonders of competition and incentive accelerate discovery.

Temperature sensitive degron for plants

Engineering needs tools. Tools such as restriction enzymes and CRISPR-Cas9 are vital to synthetic biology. Tools can also be methods such as PCR and transformation techniques.

Plant synthetic biology suffers from a lack of tools to manipulate molecular processes inside plant cells in vivo.

Enter stage right: A modular temperature sensitive degron that can be used inside living plants.

The authors, from across a number of German universities/institutes, demonstrated their tool in Arabidopsis, Nicotiana benthamiana and even fruit flies.

So  that’s one more tool for the tool box 🙂

 

ncomms12202-f1

 

 

Synbio Leapfrogging GM in Africa?

Get them lab supplies, funding, and higher education infrastructure. That’s my naive response to anyone asking me how to boost agricultural biotechnology in Africa. And I would suggest that implementing existing technologies is more important than developing new ones: in particular access to clean seed and high-yield crop varieties as well as classical breeding programs for African crops. I wouldn’t think ‘SynBio’. So imagine my surprise to read the recently published: “Review of Synthetic Biology and the African Biotechnology Revolution” by two researchers in Kenya.

Two things stood out to me from that review. Firstly the regional differences :the biotechnology activities in South Africa dwarf those of all other countries on the list. With that said Kenya also stands out, perhaps from hosting the Central and East African agricultural biosciences hub BECA.

The second thing that really stood out though is that this review implicitly suggests leapfrogging conventional biotech approaches in favor of synthetic biology. The agrobiotech approaches taken for granted elsewhere i.e. marker assisted-breeding, gene knock-outs and gene-knock ins, are not employed widely by researchers in Africa. This review, however, promotes a very ambitious synthetic biology approach. In fact it promoters the J. Craig Venter approach of total genome synthesis to create wholly novel organisms.

I don’t think synthesizing and transplanting novel crop genomes is a realistic goal right-now anywhere in the world. But I am intrigued by the suggestion, or exhortation, to leapfrog older methods and implement a synthetic biology approach.

There could be something to that idea. Synthetic biologists spend a lot of time straining to convince their colleagues on the usefulness of the approach. Challenging old methods. Maybe that fight simply wont take place in many African biotech centers because there won’t be ingrained approaches to replace. Maybe Africa will host some of the most innovative agricultural synthetic biology for the coming decades.

How to grow a brain: just add sunlight

There was a sensation last week over research that showed pea plants exhibit evidence of risk sensitivity. Apparently the first demonstration in non-humans let alone non-animals*. No molecular mechanisms are given in that paper but the last decades of plant molecular biology have unearthed a veritable goldmine of plant decision making, showing that our leafy cousins are a little sharper than we might have imagined:

Whether you call this intelligence or wish to reserve that term for organisms with nervous systems is not really that important. Clearly plants are equipped with molecular machinery to do pretty complex computation.

Synthetic biology has made serious headway with computation based on promoters and transcription factors, receiving inputs from small molecule inducers or light (optogenetics).  But plants have a veritable army of computational mechanisms that function post-transcriptionally or post-translationally. miRNAs and other small RNAs seem to play key roles in developmental switches in plants, and we know how to engineer small RNAs, indeed RNA synthetic biology is already a healthy field.

Ubiquitination-based control is another mechanism that has been domesticated, reasonably well. Ubiquitin tags are a standard way to mark out a protein for degradation within the cell. What’s more there are a suite of ubiquitin ligase adapters within the cell that bridge target proteins to the ligase. Fuse the adapter target domain to a protein of interest and you have a simple way to control the degradation of an arbitrary protein within a cell. This has been used by the group I now work in to create CRISPR-Cas9 based transcription factors that are degraded in the presence of plant hormone auxin.

There are two other covalent post-translational modifications that are standard in form, but not in effect, making them a little harder to work with: phosphorylation and sumoylation.  The interplay of these protein tags to control function is beautifully demonstrated in the control of salicylic acid receptor NPR1. Similarly, calcium is an  ionic small molecule protein modifier, that triggers conformational changes in target proteins bearing calcium binding domains. For example calcium binding domains allow CCamK, a key organogenesis inducer in symbiosis, to sense oscillations of nuclear calcium. The problem with these ‘tags’ is that the effect they have on protein structure and therefore function is totally specific to the protein in question. Poly-ubiquitination (pretty much) always targets a protein for degredation. The effects of phosphorylation, sumoylation and calcium binding are harder to predict.

The best way to make use of this ‘protein code’ is to define standard domains which receive these inputs in a standard way. A domain that when phosphorylated always does X to the protein its attached to. For example phosphodegron motifs that recruit ubiquitin ligases when phosphorylated. But it would be interesting to look at protein domains that fold in defined ways upon phosphorylation to unite or separate two functional domains.

If we can tame the regulatory bestiary of the plant cell, not just transcription factors and genetic circuits, but a toolbox of phosphorylation, ubiquitination, sumoylation and miRNAs, each responding to different inputs the possibilities for cellular computation are, well, damn exciting. So no, I don’t think we’ll ever grow brains like veiny, wet tomatoes but to have the capability to programme plants, to make photosynthetic computers, well that’s a fruit worth climbing for.

 

*Personally I found it very hard to follow the connection between the weak growth effects in the data and the exciting conclusions when I think there are multiple models that explain the data without assuming a mechanism for plants to assess risk (I think they’ve discovered a good way to grow out roots by pulsing fertiliser at low concentration, though). Anywho

Plant transformation in the next generation

I last wrote about the vision of plant synthetic biology, generating genetic circuits encoding defined functions in plant cells.  This process involves the design, assembly and testing before integration into a plant of interest. Of these processes assembly is by far the most developed.  Current understanding of plant molecular biology is also already good enough to design systems with certain qualitative behaviors, even if the quantitative rigor necessary to allow computer assisted design is lacking. And transient systems (protoplast transfection, Nicotiana benthamiana transient transformation, leaf-tissue biolistics) or A. thaliana floral dip allow for designs to be tested in a plant cell.

But what about the next step?

You have a great design, and it actually seems to work in Arabidopsis or whatever test chassis you used. If, like me, your end goal is crop improvement you actually need to get your circuit into a crop plant’s genome. And you also don’t want just one variety transformed, if the crop is to be successful you’ll need to integrate your circuit into a number of commercially viable varieties. You could move it between varieties with plant breeding, but that could be tricky if your circuit involves 10 genes (though if they are all sitting in a row in the genome it might be fine, anyone have any insight there?). Getting 10 genes even into a test chassis (A. thaliana for example) could be really difficult for that matter.

Plant transformation is a major bottleneck for any successful plant synthetic biology project. A recent review in Plant Cell explores exactly that problem, offering a range of possible future solutions. These include the use of novel bacterial vectors: alternatives to Agrobacterium tumefaciens as well as modified versions of that tried and trusted workhorse. Another suggestion is to modify the host you intend to transformation later, turning on or off particular genes that are known to affect transformation efficiency. Essentially using plant synbio to enable plant synbio, which seems pretty neat. I think that such efforts would be well worth while to employ for a range of model plants that are commonly used in basic research (Medicago, tomato, Brassica, maize, rice) all spring to mind. Essentially making plant transformation as simple as floral dip for as many species as possible.

This still doesn’t help with the issue of transforming large numbers of crop varieties, though. And I think that will be one of the biggest hurdles in the next decade for the employment of plant synthetic biology in the agricultural sector.

At least until better methods are developed we’ll have to rely and brute force using labour-intensive tissue-culturing methods. If so then I think that regional centres for large-scale plant transformation operating as a service for academic institutions will be invaluable. An example of such a centre is BRACT in the Norwich research park (UK). Hopefully if early plant synbio work proves its worth in generating financially viable plant products funding will become available to expand or multiply such centres.

I’ll be honest I’m totally sold on the vision of assembly/cloning – design/testing – stable transformation all happening separately with the first and last carried out as fee-based services, leaving design and testing as the preserve of the research lab. Yes, I have been influenced by working in a lab operating a cloning automation service .

I will leave off my dreaming here, time to get back to the floral dips.

 

 

Towards programmable plant genetic circuits

I have stolen the title of this blog post directly from the publication I want to write about. It’s a review just published in The Plant Journal. The review is co-authored by June Medford at Colorado Stat. Her lab published one of the milestone studies, developing humble Arabidopsis thaliana into a TnT biosensor, and the Medford lab remains the best representative and pioneer of a certain vision of plant synthetic biology. That vision is laid out very neatly in the introduction so I’ll quote from it:

“Rather than endlessly searching fields and forests for precursors of the foods, materials, and pharmaceuticals that nature provides, synthetic biology provides a means to develop plants, and hence sustainable systems, to directly address human and environmental needs. For example, if there is individualized medicine, why not design plants to provide individualized nutrients? If we can build a house from wooden boards, why not design a plant’s vascular cambium and waxy cuticle layer to, in effect, “grow a house”?”

This vision is allied in this review with what is arguably the canonical synthetic biology methodology: modular assembly of characterized parts to create genetic circuits that can be described mathematical and ideally function at a higher level using Boolean logic operators. If that all sounds like gibberish then read the review, the authors do a very good job of describing these methods in an approachable way.

What I took from this review was a re-affirmation not only of the promise of plant synthetic biology but of how little has so far been achieved in plants. Even relatively simple circuits achieved long ago in microbes have yet to be attempted in plants.

Yet, a word of caution, as I’ve written about previously this same lab just published a characterization of synthetic genetic parts for plants, and newsflash: plant cells are complicated and noisy!

Still, there has been comparatively little effort but into genetic circuit design in plants compared to other systems, including mammalian systems (check out this recent paper on engineered immune cells). Plant synthetic biology is in its infancy, and maybe we’ll never be able to grow a house, but we won’t know if we don’t try.