Marie Vandermies

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

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.

Identification and characterization of EYK1, a key gene for erythritol catabolism in Yarrowia lipolytica

Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipolytica, in response to osmotic stress. This metabolite has application as food additive due to its sweetening properties. Although Y. lipolytica can produce erythritol at a high level from glycerol, it is also able to consume it as carbon source. This ability negatively affects erythritol productivity and represents a serious drawback for the development of an efficient erythritol production process. In this study, we have isolated by insertion mutagenesis a Y. lipolytica mutant unable to grow on erythritol. Genomic characterization of the latter highlighted that the mutant phenotype is directly related to the disruption of the YALI0F01606g gene. Several experimental evidences suggested that the identified gene, renamed EYK1, encodes an erythrulose kinase. The mutant strain showed an enhanced capacity to produce erythritol as compared to the wild-type strain. Moreover, in specific experimental conditions, it is also able to convert erythritol to erythrulose, another compound of biotechnological interest.

Identification and characterization of EYD1, encoding an erythritol dehydrogenase in Yarrowia lipolytica and its application to bioconvert erythritol into erythrulose

In this study, gene YALI0F01650g has been isolated and characterized. Several experimental evidences suggest that the identified gene, renamed EYD1, encodes an erythritol dehydrogenase. An efficient bioreactor process for the bioconversion of erythritol into erythrulose was also developed. Using constitutive expression of EYD1 in a Y. lipolytica mutant containing a disrupted EYK1 gene, which encodes erythrulose kinase, erythrulose could be synthesized from erythritol at a rate of 0.116g/gDCW.h and with a bioconversion yield of 0.64g/g.

Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering

Erythritol (1,2,3,4-butanetetrol) is a four-carbon sugar alcohol with sweetening properties that is used by the agrofood industry as a food additive. In this study, we demonstrated that metabolic engineering can be used to improve the production of erythritol from glycerol in the yeast Yarrowia lipolytica. The best results were obtained using a mutant that overexpressed GUT1 and TKL1, which encode a glycerol kinase and a transketolase, respectively, and in which EYK1, which encodes erythrulose kinase, was disrupted; the latter enzyme is involved in an early step of erythritol catabolism. In this strain, erythritol productivity was 75% higher than in the wild type; furthermore, the culturing time needed to achieve maximum concentration was reduced by 40%. An additional advantage is that the strain was unable to consume the erythritol it had created, further increasing the processs efficiency. The erythritol productivity values we obtained here are among the highest reported thus far.

EYK1 encoding erythrulose kinase as a catabolic selectable marker for genome editing in the non-conventional yeast Yarrowia lipolytica

We report here on EYK1, encoding erythrulose kinase, as an efficient catabolic selectable marker for genome editing in Y. lipolytica. Compared to auxotrophic markers, EYK1 increases the growth rate of transformants and allows improved efficiency of transformation. The utility of the marker EYK1 in a replicative vector was also demonstrated.

Yarrowia lipolytica morphological mutant enables lasting in situ immobilization in bioreactor

AbstractIn the present study, we have isolated and characterized a Yarrowia lipolytica morphological mutant growing exclusively in the pseudohyphal morphology. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the metallic structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture. The system of medium aspersion on the packing ensured oxygen availability in the absence of agitation and minimized the potential release of cells in the culture medium. Additionally, the metallic packing proved its facility of cleaning and sterilization after fermentation. This combined use of morphological mutation and bioreactor design is a promising strategy to develop continuous processes for the production of recombinant protein and metabolites using Y. lipolytica. Graphical Abstract

Erratum to: Identification and characterization of EYK1, a key gene for erythritol catabolism in Yarrowia lipolytica (Applied Microbiology and Biotechnology, (2017), 10.1007/s00253-017-8361-y)

Multiple alignments of YALY0F01606g genes with the DAK1, DAK2 and DAK3 in the lipolytica clade. Homologs of EYK1 in Yarrowia species were identified by Blast on the private site of GRYC using EYK1 gene as template and retrieved using the download functionality. Color code: red, similar in all gene; green, similar in two of the three genes; yellow highlighted, different in DAK3 group compared to DAK1 and DAK2 gene. Sequences are from strains YALI: Yarrowia lipolytica CLIB122; YAGA: Yarrowia galli CBS 9722 (96.77%); YAYA: Yarrowia yakushimensis CBS 10253 (91.62%); YAAL: Yarrowia alimentaria CBS 10151 (87.22%) and YAPH: Yarrowia phangngensis CBS 10407 (85.01%). Maximal identities with Yarrowia lipolytica EYK1 (YALI0F01606g) are indicated in bracket.