Insertional mutations exhibiting high cell-culture density HCD phenotypes are enriched through continuous subcultures in Chlamydomonas reinhardtii

Low efficiency in microalgal biomass production was largely attributed to the low density of algal cell cultures. Though mutations that reduced the level of chlorophyll or pigment content increased efficiency of photon usage and thus the cell-culture density under high-illumination growth conditions (e.g., >$500{\mu}mol\;photon\;m^{-2}\;s^{-1}$), it was unclear whether algae could increase cell-culture density under low-illumination conditions (e.g., ${\sim}50{\mu}mol\;photon\;m^{-2}\;s^{-1}$). To address this question, we performed forward genetic screening in Chlamydomonas reinhardtii. A pool of >1,000 insertional mutants was constructed and subjected to continuous subcultures in shaking flasks under low-illumination conditions. Complexity of restriction fragment length polymorphism (RFLP) pattern in cultures indicated the degree of heterogeneity of mutant populations. We showed that the levels of RFLP complexity decreased when cycles of subculture increased, suggesting that cultures were gradually populated by high cell-culture density (HCD) strains. Analysis of the 3 isolated HCD mutants after 30 cycles of subcultures confirmed that their maximal biomass production was 50-100% higher than that of wild type under low-illumination. Furthermore, levels of chlorophyll content in HCD mutant strains were similar to that of wild type. Inverse polymerase chain reaction analysis identified the locus of insertion in two of three HCD strains. Molecular and transcriptomic analyses suggested that two HCD mutants were a result of the gain-of-function phenotype, both linking to the abnormality of mitochondrial functions. Taken together, our results demonstrate that HCD strains can be obtained through continuous subcultures under low illumination conditions.