CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms.

TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases.

The rapid increase of the number of sequenced genomes asks for the functional annotation of the encoded enzymes. We used a combined computational-structural approach to determine the function of the TrpB2 subgroup of the tryptophan synthase ß chain/ß chain-like TrpB1-TrpB2 family (IPR023026). The results showed that TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases, whereas TrpB1 enzymes catalyze the l-serine dependent synthesis of tryptophan. We found a single residue being responsible for the different substrate specificities of TrpB1 and TrpB2 and confirmed this finding by mutagenesis studies and crystallographic analysis of a TrpB2 enzyme with bound O-phospho-l-serine.

Plants contain two distinct classes of functional tryptophan synthase beta proteins.

Tryptophan synthase beta-subunits (TSBs) catalyze the last step in tryptophan biosynthesis, i.e. the condensation of indole and serine yielding tryptophan. In microorganisms two subfamilies of TSBs (here designated as type 1 and type 2) are known, which are only distantly related. Surprisingly, in all genomes of multicellular plants analyzed genes encoding both types are present. While type 1 enzymes are well established as components of tryptophan synthase complexes, type 2 enzymes in plants have not yet been characterized. Tissue specific expression of the TSB genes from Arabidopsis thaliana was analyzed. While AtTSB1 is the predominantly expressed isoform in vegetative tissues, AtTSB1 and AtTSBtype2 reach similar transcript levels in seeds. AtTSBtype2 protein was expressed in Escherichia coli and purified. It converted indole and serine to tryptophan with a strikingly low K(m)-value for indole of ca. 74 nM. Attsbtype2 T-DNA insertion mutants showed no obvious deviation from the wild type phenotype, indicating that AtTSBtype2 function is not essential under standard growth conditions. As example for a monocot enzyme, maize TSBtype 2 was analyzed and found to be transcribed in various tissues. ZmTSBtype2 was also catalytically active and here a K(m)-value for indole of ca. 7 microM was determined. These data indicate that TSB type 2 enzymes generally are functionally expressed in plants. Their potential biological role is discussed.

Alpha-tryptophan synthase of Isatis tinctoria: gene cloning and expression.

Indole producing reaction is a crux in the regulation of metabolite flow through the pathways and the coordination of primary and secondary product biosynthesis in plants. Indole is yielded transiently from indole-3-glycerol phosphate and immediately condensed with serine to give tryptophan, by the enzyme tryptophan synthase (TS). There is evidence that plant TS, like the bacterial complex, functions as an alpha beta heteromer. In few species, e.g. maize, are known enzymes, related with the TS alpha-subunit (TSA), able to catalyse reaction producing indole, which is free to enter the secondary metabolite pathways. In this contest, we searched for TSA and TSA related genes in Isatis tinctoria, a species producing the natural blue dye indigo. The It-TSA cDNA and the full-length exons/introns genomic region were isolated. The phylogenetic analysis indicates that It-TSA is more closely related to Arabidopsis thaliana At-T14E10.210 TSA (95.7% identity at the amino acid level) with respect to A. thaliana At-T10P11.11 TSA1-like (63%), Zea mays indole-3-glycerol phosphate lyase (54%), Z. mays TSA (53%), and Z. mays indole synthase (50%). The It-TSA cDNA was also able to complement an Escherichia coli trpA mutant. To examine the involvement of It-TSA in the biosynthesis of secondary metabolism compounds, It-TSA expression was tested in seedling grown under different light conditions. Semi-quantitative RT-PCR showed an increase in the steady-state level of It-TSA mRNA, paralleled by an increase of indigo and its precursor isatan B. Our results appear to indicate an involvement for It-TSA in indigo precursor synthesis and/or tryptophan biosynthesis.