The corrinoid B12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions and we previously demonstrated that experimental evolution of the non-requiring alga Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupted C1 metabolism, caused an accumulation of starch and triacylglycerides and a decrease in photosynthetic pigments, proteins and free amino acids. B12 deprivation also caused a substantial increase in reactive oxygen species (ROS), which preceded rapid cell death. Surprisingly, survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B12 limitation and an increase in B12 use-efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B12-producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly adapted to B12 deprivation, association with B12-producers can ensure long-term survival whilst also providing the environment to evolve mechanisms to better tolerate B12 limitation.

Physiological and molecular responses of a newly evolved auxotroph of Chlamydomonas to B12 deprivation