When developing bioprocessing conditions for a new product, you may be faced with a choice between using whole cells in a fermentation
system, or production using cell-free enzymes
. Before enzyme optimization
can be achieved for any process, you have to know the basic conditions under which the enzymes will need to function. Here are a few things to consider when making that choice:
1. Number of Steps in the Biosynthesis
Is the process of making your new bioproduct simple? Does it entail a single step, or is it a multi-step process requiring several enzymes? In the latter case, the process is more suited to whole cells where all required enzymes, and appropriate ligands
, can be present at once, and in close proximity.
2. Competing Side ReactionsSince whole cells contain a complete metabolic compliment of enzymes, there is potential for your substrate to undergo more than one biochemical reaction. If that is the case, substrate sources may be exhausted by the presence of competing side reactions, resulting in reduced yields.
Using whole cells may complicate protein purification
and contribute to higher concentrations of impurities, resulting from the formation of metabolic by-products, or cell components themselves. When chosing culture conditions, consider how easily a purified product can be produced, without destroying it in the process.
4. Cell Culture Viability
Is it realistic to propose growing a scaled-up version of your cell culture? It may not be if the only strain you can come up with is one that is anaerobic, or is very sensitive to changes in its environment. Alternatively, if you are working with an extremophile
, tolerance to harsh growth conditions might make it more forgiving during the scale-up process. Growth rate is another factor to consider, since some transgenic
strains may grow quite slowly.
5. Sterility IssuesThe larger the bioprocessing scale, the harder it is to keep conditions absolutely sterile. Biological contamination is likely to interfere with production yields, especially if growth of the desired culture is slow.
6. Enzyme Stability
Not all enzymes like to be exposed outside of the cell that made them, while some enzymes are intended to be excreted. Before you begin, consider the stability of the enzyme(s) you are working with, or whether it can be produced for extracellular processes at all. For example, the tertiary structure, and activity
, of some membrane proteins is highly dependent on their being held in place within the bipolar lipid membrane.
7. Permeability IssuesThe semi-permeable nature of cell membranes can complicate substrate entry and/or product release from a whole cell system. For this reason, whole cell biocatalysis reactions tend to be 1-2 orders of magnitude slower than enzymes alone. In addition, slow export of products can result in accumulation within the cell, which can cause feedback inhibition. Chemical and physical means of improving permeability are not always feasible for larger-scale processes.
The importance of developing a cost-effective process goes without saying, but at the early steps of discovery, economics are not always foremost on everyone's minds. Whole cell biocatalytic processes that are inefficient, because of complications like carbon dioxide buildup, restrictions on permeability, and product purification, are often not cost-effective. Finding an appropriate CSO
, with suitable production equipment for your choice of either cell-free, or whole cell processes, is another consideration when looking to save money on production.