Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica

Abstract

BackgroundIn recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for protein production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting recombinant protein production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study’s aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources.ResultspPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic acid. Indeed, no distinct subpopulations that specialized on glucose versus oleic acid were observed; such an outcome would have led to producer and non-producer phenotypes. In medium containing both glucose and oleic acid, cells tended to directly metabolize oleic acid instead of storing it in lipid bodies.ConclusionsThis study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for recombinant protein production by Y. lipolytica used as cell factory.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0558-8) contains supplementary material, which is available to authorized users.

Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering

“…Shake-flask cultures were grown in EPF medium for 24 h. Cells were then collected at an OD 600 of 0.5 and stored at −80°C. RNA extraction and cDNA synthesis were performed as previously described (Sassi et al, 2016). Primers for RT-qPCR are listed in Supplementary table 2.…”

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

“…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 . Instead, it is strongly induced in the presence of erythritol, which can be used both as a carbon source and as an inducer, and it is repressed in the presence of glucose or glycerol.…”

Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris

“…DNA sequencing was performed by GATC Biotech (https://www.gatc-biotech.com), and the primers were synthetized by Eurogentec (https://secure.eurogentec.com/). Transcriptional analyses were performed as described in Sassi et al () with primer pair listed in Table . The reference actin gene (ACT) was amplified using primers Act‐F and Act‐R.…”