SCRaMbLE-ing Synthetic Yeast

Today’s guest post is written by Jack Ho, a synthetic biology PhD student and one of the authors on the paper “Rapid host strain improvement by in vivo rearrangement of a synthetic yeast chromosome” about the SCRaMbLE system for modular rearrangement of chromosomes in yeast. 

 

If you like beer or bread, then you know why yeast is important to us.

Saccharomyces cerevisiae, or brewer’s yeast, has been in close relationship with us throughout human history. The technology developed from using these culinary microbes has been vital in the progression of modern biotechnology. It was the first microbe observed under a microscope, the first fully sequenced eukaryote, and was used for the production of the first genetically modified vaccine. It’s quite easy to imagine that our lives would be quite different without them.

A collaborative effort between some of the largest research institutes around the world, the Synthetic Yeast Genome project (Sc2.0 or Yeast 2.0) aims to redesign and synthesise the 16 yeast chromosomes from scratch. A particularly interesting system designed into the synthetic yeast genome is the SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution) system – a DNA sequence called LoxPsym is able to carry out homologous recombination with other LoxPsym sites when an enzyme called cre-EBD is induced by a chemical (b-estradiol). These LoxPsym sites were placed downstream of every non-essential gene, which allows the large-scale shuffling of the genome. This system creates one or more of the following mutations:

I was involved in the recent work published from the Tom Ellis Lab – where useful metabolic pathways on plasmids (without loxPsym sites) were put into yeast containing a synthetic version of chromosome V (synV). The pathways were xylose utilisation and violacein biosynthesis. I induced SCRaMbLE on the synthetic yeast and screened for an improved activity of the respective pathways. Then I selected a winner strain from each category and called them XD4 (xylose utilisation) and VB2 (violacein biosynthesis).

XD4 was able grow quicker on xylose as a sugar source by almost 6-fold. And VB2 was able to produce violacein with a 2-fold increase in yield. We also tested VB2 on the penicillin pathway – it also showed a 2-fold increase in yield!

To find out how these two strains have managed to improve these pathways with only chromosome V being SCRaMbLEd, we used Nanopore sequencing to look for the rearrangements that had occurred on the chromosome. Both strains only had two rearrangements: a deletion and an inversion, which deleted or altered the transcription of 2-3 genes. What’s so interesting is that even such small changes in a single chromosome could lead to such a drastic improvement in the activity of foreign metabolic pathways – imagine if all 16 chromosomes could be rearranged!

 

 

 

 

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