Structure-function analysis of chloroplast proteins via random mutagenesis using error-prone PCR

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone PCR of a gene of interest with chloroplast complementation of the knock-out Chlamydomonas reinhardtii mutant. As a proof-of-concept, we targeted a 300-bp sequence of the petD gene, which encodes subunit IV of the thylakoid membrane-bound cytochrome b6f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multi-subunit and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or probe the mutational robustness of chloroplast-encoded proteins, and propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast.