New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica

Abstract

BackgroundThe oleaginous yeast Yarrowia lipolytica is increasingly used as alternative cell factory for the production of recombinant proteins. At present, several promoters with different strengths have been developed based either on the constitutive pTEF promoter or on oleic acid inducible promoters such as pPOX2 and pLIP2. Although these promoters are highly efficient, there is still a lack of versatile inducible promoters for gene expression in Y. lipolytica. ResultsWe have isolated and characterized the promoter of the EYK1 gene coding for an erythrulose kinase. pEYK1 induction was found to be impaired in media supplemented with glucose and glycerol, while the presence of erythritol and erythrulose strongly increased the promoter induction level. Promoter characterization and mutagenesis allowed the identification of the upstream activating sequence UAS1EYK1. New hybrid promoters containing tandem repeats of either UAS1XPR2 or UAS1EYK1 were developed showing higher expression levels than the native pEYK1 promoter. Furthermore, promoter strength was improved in a strain carrying a deletion in the EYK1 gene, allowing thus the utilization of erythritol and erythrulose as free inducer.ConclusionsNovel tunable and regulated promoters with applications in the field of heterologous protein production, metabolic engineering, and synthetic biology have been developed, thus filling the gap of the absence of versatile inducible promoter in the yeast Y. lipolytica.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0755-0) contains supplementary material, which is available to authorized users.

Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B

“…CalB sequence analysis and cloning. The DNA fragment containing the coding sequence of lipase CalB and its pro-region was codon optimized and synthesized during previous work 11 (for pro-CalB coding sequence, see Supplementary Fig. 1).…”

“…Strains and plasmids used in this study are listed in Supplementary Table 2. Construction of Y. lipolytica strain RIY368 is described in Park et al 11 . Briefly, the DNA fragment containing the CalB sequence codon optimized for Y. lipolytica by Biocatalysts LTD (Cardiff, UK) and synthesized by Geneart (Regensburg, Germany) was cloned as a BamH1/AvrII fragment in vector JME4266 at the corresponding restriction sites.…”

“…When combined with efficient bioreactor process strategies, these tools have been successfully used for the production of a large number of rProt 8,9 . We have recently developed a novel set of expression vectors based on the promoter of EYK1 gene encoding erythrulose kinase 10,11 . Compared to previously available inducible systems like pPOX2 and pLIP2, this promoter does not depend on the utilization of hydrophobic inducers such as oleic acid 12 .…”

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

“…In addition, we included three hybrid promoters that are inducible by erythritol. They are based on the recently developed p EYK1 promoter and incorporate three to five repeated UAS sequences (Trassaert et al ., ; Park et al ., ). Consequently, in total, there are nine promoters available for the three TUs.…”

Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose

“…Targeted genetic disruptions and markerless gene integrations are possible with an endonuclease active CRISPR‐Cas9 system, while transcriptional knockdowns have been demonstrated with CRISPR interference (CRISPRi) . The synthetic biology tools available for engineering Y. lipolytica also include well‐characterized hybrid and native promoters with constitutive and inducible expression …”