SCRaMbLE: A Study of Its Robustness and Challenges through Enhancement of Hygromycin B Resistance in a Semi-Synthetic Yeast

Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed “essential raft”, where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast.

Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas

Background Chlamydomonas reinhardtii is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including C. reinhardtii. Thus, we investigated whether NAT was useful and effective for selection of transgenic strains in C. reinhardtii. The successful use of NAT would provide alterative choice for selectable markers in this organism and likely in other microalgae. Results C. reinhardtii was sensitive to NTC at concentrations as low as 5 µg/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized NAT from Streptomyces noursei was synthesized and assembled into different expression vectors followed by transformation into Chlamydomonas. Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 µg/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that NAT could be used as a selectable marker for ectopic expression of IFT54-HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for IFT54-HA positive clones was greatly increased by fusing IFT54-HA to NAT and processing with the FMDV 2A peptide. Conclusions This work represents the first demonstration of stable expression of NAT in the nuclear genome of C. reinhardtii and provides evidence that NAT can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in C. reinhardtii. NAT is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.

The Transformation in Trichoderma viride Protoplasts by Restriction Enzyme Mediated Integration (REMI)

The transformation conditions of the protoplasts from Trichoderma viride mediated by restriction enzyme were studied in this paper. The optimum generation conditions of protoplasts were as followed: 8 mg/ml glucanex was added into the phosphate buffer (pH 6.98), the mycelial that cultured for 24 hr was hydrolyzed for 4 hr at 30°C under 40 r/min shaking speed. The protoplast yield was 4.7×107 cfu/mg. The regeneneration rate of protoplast was 14.5% on CM medium contained 0.3 mol/L KCl and 0.3 mol/L Inositol. Transformants were obtained by transfering hygromycin B resistance gene into T. viride by restriction enzyme mediated integration (REMI), The preliminary identification of the transformants indicated that the exogenous gene had been integrated into T. viride genome.