ABSTRACT
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.
Subject(s)
Oxidoreductases/metabolism , Yarrowia , Bioreactors , Erythritol/metabolism , TetrosesABSTRACT
In 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.
Subject(s)
Bioreactors/microbiology , Industrial Microbiology/methods , Yarrowia/genetics , MutationABSTRACT
Erythritol is a four-carbon sugar alcohol produced by microorganisms as an osmoprotectant. It could be used as a natural sweetener in the pharmaceutical and food industries. Here, a snapshot of current knowledge on erythritol metabolism and synthesis, optimization of its production and more precise process and producer strain improvement is presented.
Subject(s)
Erythritol/biosynthesis , Yeasts/metabolism , Carbon/chemistry , Carbon/classification , Conservation of Natural Resources , Culture Media/chemistry , Erythritol/genetics , Erythritol/metabolism , Fermentation , Genetic Engineering , Mutation , Osmotic Pressure/physiology , Proteomics , Yeasts/classification , Yeasts/enzymology , Yeasts/geneticsABSTRACT
BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: Novel 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.
Subject(s)
Fungal Proteins/genetics , Yarrowia/metabolism , Base Sequence , Gene Expression/drug effects , Plasmids/genetics , Plasmids/metabolism , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Sequence Alignment , Tandem Repeat Sequences/genetics , Tetroses/pharmacology , Yarrowia/growth & developmentABSTRACT
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 process's efficiency. The erythritol productivity values we obtained here are among the highest reported thus far.
