Science1 publisher2 min readPublished
Looping bacterial mRNA into a ring raised reporter protein 5.95-fold in E. coli
A Seoul National University group rearranged a self-splicing intron so bacterial transcripts join their own ends inside the cell. Most of the extra protein came from mRNA that lasts longer, and the design worked in three hosts.
The Scientist · Science desk
What happened
- A team led by Sang Woo Seo at Seoul National University built CRESEnT, a system that converts linear messenger RNA into a ring inside living microbial cells using a rearranged self-splicing intron.
- The ring forms without added enzymes or test-tube processing: the intron's front and back portions were reassembled in reverse order so the transcript joins its own ends in the cell.
- Two design rules set efficiency, the first being untranslated region length, where too long exposes more RNA to degrading enzymes and too short keeps the intron from folding properly.
Compiled by The ScientistSomething wrong?How this is made
Why it matters
- capability Because the loop closes itself from a DNA-encoded construct, a strain team can try this by changing the expression cassette, without synthesising RNA in vitro or expressing an extra ligating enzyme.
- constraint The gain reaches product titer only where enzyme abundance is the limiting step; in a pathway held back by carbon supply or by an enzyme's turnover number, more enzyme protein buys little.
- decision Anyone choosing between promoter and ribosome-binding-site engineering and mRNA stabilisation now has a decomposition to reason with: 3.95-fold of the effect was transcript abundance and 1.51-fold was translation per transcript.
- precedent With UTR length and the zipper sequences published as rules, circularization efficiency becomes a figure other groups can report, which makes a failed construct diagnosable instead of anecdotal.
The three fold-changes are linked. Divide the protein gain by the mRNA gain: 5.95 / 3.95 = 1.51, and 1.51-fold is exactly the per-transcript figure the team reports [2][3][15]. So most of the roughly sixfold protein increase is more mRNA present in the cell at any moment, and a factor of about 1.5 is each surviving transcript being translated more productively [15].
Bacterial mRNA has no cap and no poly(A) tail, so RNases work inward from the ends and a transcript lasts only a few minutes [7]. Join the ends and there are no ends to attack [9]. The part that makes this usable in a strain is that the loop closes itself: the researchers split a self-splicing intron and reassembled its front and back portions in reverse order, so the transcript excises the intron and ligates its own ends inside living E. coli, with no added enzyme and no in vitro step [10].
Two parameters decided how well that worked. Untranslated region length has an optimum at each end, because a long UTR gives RNases more to chew and a short one keeps the intron from folding, and the team measured where those optima sit [11]. They also added short sequences designed to interlock like zipper teeth, which pair the two intron fragments more precisely and raise circularization efficiency [12].
The 5.95-fold number is a fluorescent protein, measured against a construct in which circularization did not occur [2]. That is the comparison that isolates the shape of the transcript, and it is the right control here. A reporter also draws no pathway precursors and does not feed back on growth. The phys.org account says CRESEnT was applied to flaviolin, itaconic acid, lycopene and violacein, and does not report titers for those four compounds [5][16]. Whether a longer-lived transcript moves a titer depends on the team's own premise, that cell factory productivity is set by whether enough of the needed enzyme can be made [14].
The same design worked in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum [4]. Earlier work on raising expression in bacteria concentrated on promoters and ribosome-binding sites [8], which sit upstream of the problem CRESEnT addresses, so a strain with a tuned promoter should in principle be able to carry the circularizing cassette as well. Amino acids for seasonings and animal feed are already fermented at a scale of millions of tons a year [13]. The work is published in Nucleic Acids Research [6].
What to watch
- Titers for flaviolin, itaconic acid, lycopene and violacein from the Nucleic Acids Research paper, measured against the same non-circularizing control.
- Whether the gain survives hours of fed-batch culture, where transcript stability competes with growth rate and plasmid burden.
- Whether the reversed-intron cassette closes multi-gene transcripts, since pathway engineering usually expresses several enzymes at once.