Ashbya gossypii is a splendid riboflavin biofactory that suppresses nearly half of the worldwide
demand for vitamin B2. The availability of its genome sequence allowed a better understanding
of this fungus metabolism and the development of targeted genome editing tools. Such
progresses allowed increasing the vitamin B2 natural production levels, and to expand the
potential of this fungus towards the production of nucleosides, folic acid, single cell oil and
fragrances [1,2].
Now, with a broadening range of potential genetic targets, modular and flexible pathway
assembly tools are more convenient than ever. There is also an urgent need for a larger set of
well characterized promoters for metabolic flux optimization towards improved production of
target products. As such, this work aimed at developing a modular, flexible and fast pathway
assembly platform for targeted chromosomal integration in A. gossypii that offers a wide range
of expression strengths (promoters).
The developed pathway assembly platform is in an early-stage, designed to easily allow fast
cloning and exchange of selection markers, promoters, genes and terminators. As endogenous
promoters provide the required regulatory elements for gene expression, several
transcriptomic datasets were scanned for medium to high strength promoters. A set of uni- and
bi-directional promoters was selected, and each promoter cloned into promoter-probes for
characterization in terms of expression strength and regulation, a task that is under way using
multiple reporter systems. Preliminary results revealed that the bi-directional promoter
AgCCW12/HOG1p drives moderate gene expression through the HOG1p side and strong
expression through the CCW12p side. The datasets were also used for transcription factor
binding motif mining, in order to find important regulation elements that confer strong gene
expression values. Five transcription factor binding motifs were identified as strong targets for
promoter engineering. This information can hereafter enable the rational design of
synthetic/semi-synthetic minimal promoters.
With the current genetic engineering turnaround time to genetically modify A. gossypii being a
limiting factor to establish this fungus as a state-of-the-art microbial factory, this pathway assembly platform has great relevance. With a growing number of promoters characterized, it
will become increasingly easier to genetically channel the A. gossypii metabolism towards the production of value-added chemicals.Study supported by FCT, Compete2020 and Portugal2020 through the strategic funding of UIDB/04469/2020 and
Project EcoBioInks4SmartTextiles (PTDC/CTM-TEX/30298/2017–POCI-01-0145-FEDER-030298)info:eu-repo/semantics/publishedVersio
