Hi Daniel,

I think it is a very important issue. You state that the life-time of cell-free enzymatic systems is an issue. Hence, what is the life-time of a cell free system? And which variables determine the life-time of a functional cell-free enzymatic system? Important questions, especially when we start thinking about the early evolution of (biochemistry) and life on Earth. Did the fundamental processes of living organisms (DNA replication and photosynthesis, among others) take place within a cell or outside of it. For me it is difficult to grasp that for example processes like DNA replication or photosynthesis have evolved outside the confines of a cell.

Research like you are performing might give clues to which boundary conditions must be met for an extracellular system to stay functional for log periods of time. In some phase of biomolecular evolution a phase must have occured where DNA replication and/or photosynthesis may have taken place outside (even simple) cells, fro example on membranes floating in the early seas of the Earth. Do you use membranes for example to stabilize your extra-cellular systems? Interesting to know or test both for DNA replication as well as photosynthesis.

So if you ask me about the potential of  functional cell free systems, then my answer is, I do not know about the potential, I guess rather big, but the importance to understand early biochemical evolution on Earth is maybe orders of magnitude more important. And tying to answer this question is maybe the road to developing important applications as well in biotechnology. You never know! Often science has made progress rather unexpectedly.

Cheers,

Frank

Cell-free systems are sometimes used to produce proteins, and there are commercial products for lab-scale work. It's an expensive way to do it compared with overexpression in living cells, but sometimes it's the most effective way. I don't know whether any biotech products are made this way.

Here is some information about cell-free translation methodology:

https://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/cellfree_expression_systems/

http://149.250.246.19/wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA_3_01_p27-28.pdf

Technically, cell-free systems are more feasible to produce biofuels such as alcohols, fatty acids, H2 and electricity, because the biochemical pathways are less complicated and also well studied, compared to the biosynthesis of drugs that are usually complex secondary metabolites. In addition, the enzymes and coenzymes responsible for those "simpler" processes are well characterized and are more available.  

Economically,biofuels are usually considered as low-value added. Therefore,  it is very critical evaluate the cost and the potential benefits. However, this is not such critical for biosynthesis of drugs by cell-free systems because drugs are usually high-value added and will compensate the production cost.  

OK Daniel,

But how long do these systems you describe stay operational? And if they destabilize after some time, how do you try stabilize them for a longer time? Do you use a supporting system like a cell membrane? I keep coming back to this, because you don't really mention this in your answer, and I am curious about how oyu rpoceed with stabilization. Or are you using simple chemical reactors?

What about the more complex synthesis of proteins or DNA (replication) or photosynthesis. Have you tried one of these?In a chemical reactor or on a supporting system like a membrane.

Apparently you can produce H2 and electricity! Why biofuels when you can  produce chemical energy with H2. Do you use light in this process? Or do you isolate  (bacterial) H2 producing enzymaytic systems to use them in an extracellular mode?

Why produce biofuels when you can produce energy stored in H2 or electrical energy? Isn't it more efficient to produce H2 or electricity directly?  Maybe because these processes are much more complicated, they are much less stable?! And with electricity, do you mean electron transport? Electron transport is an important process in many energy producing systems in eukaryotic as well as prokaryotic cells. How do you store the energy produced with the electromotive force (emf) of the electron transport system? In chemical energy? In an osmotic potential created with a closed membrane system (cfr. chemi-osmotic theory!).

Or are you only trying to produce phamaceuticals extra-cellularly?

Cheers,

Frank

@Frank, Thank you very much for your thoughts and many interesting and fundamental questions. I am looking at the cell-free systems from a view of their potential applications.  I am not saying to construct an artificial cell system but to build an artificial biochemical pathway by combining relevant enzymes together into a buffer to produce biofuels and other chemicals from simple sugars or plant biomass or other cheap substrates. The stability of the mixed enzyme systems has to be at least several days to weeks under the sterile condition. 

Below are some examples of applications of cell-free systems.

http://www.nature.com/ncomms/2014/140121/ncomms4026/full/ncomms4026.html?WT.ec_id=NCOMMS-20140122

http://www.sciencedirect.com/science/article/pii/S073497501400158X

OK Thanks Daniel,

Got the point now,

Success,

Frank