Team:SCUT/Project/Analysis and Discussion

Analysis and Discussion

We are the first team to bring up the conception of PAN-compartmentalization, which considers both the inner side and the outer side of the compartment as the target. In this project, we chose mitochondria as the target compartment, to connect its inner and outer sides.

The n-butanol production pathway we chose is the n-butanol biosynthetic pathway from Clostridium beijerinckii in which isozymes from a number of different organisms. Although Clostridia are the traditional organisms employed in biobutanol production, a significant and growing amount of research is centered on the engineering of more robust strains capable of elevated production impeded by a lack of characterization and genetic tools. Considering a vast availability of genetic tools for its engineering, E. coli is always the first choice. However, the susceptibility of E. coli to high butanol concentrations complicates its development as a butanol production strain.

We chose Saccharomyces cerevisiae as our host cell for n-butanol production because it is not only a genetically tractable, well-characterized organism; but also the current industrial strain alcohol (ethanol) producer, which means it is able to tolerate high concentrations of n-butanol by the same mechanisms it tolerates ethanol.

Since the previous researches have already testified the poor production in S. cerevisiae cytoplasm, we decided to locate the whole pathway into mitochondrial matrix.
Abundant of NADH and acetyl-CoA and thus having a lower oxygen concentration, a higher pH and a more reducing redox potential than the cytoplasm, mitochondrial matrix is a perfect place for the n-butanol biosynthetic reactions, which lead to a higher production.

By analysis, the result of the above-mentioned re-experiment is probably because when the secondly culture started, there was still oxygen in the fermentation flasks, which allowed yeast to partially continue aerobic growing. As a result, CA expressing cells would reduce CO₂ around and force aerobic respiration going on and hence reduce ethanol output, a representative product in anaerobic fermentation. These did not happen in those cells that did not express CA and gave rise to the contrary results among the groups. For further research, there is, of course, a lot of work to be done here.

Our experiment data show that making use of high ATP and CO₂ concentration around mitochondria brings an apparent increase of ethanol yield. In addition, we have successfully constructed other six leading peptides to different subcellular compartments, which is a good start for further study in PAN-compartmentalization. This may be going to play a significant role in solving the problem of greenhouse effect? Plus there are still relatively isolated organelles to be modified, we confidently believe that utilizing cellular compartmentalization would be a hot research trend. Frankly speaking, though, what we`ve done are very limited and, there are much more knowledge about comfortable zones and metabolic engineering on them for us to explore. Nevertheless, then again we would say, once you make up your mind to do something special and spare no efforts holding on to it, big thing happens.