Functional overexpression of genes involved in erythritol synthesis in the yeast Yarrowia lipolytica

Abstract

BackgroundErythritol, a four-carbon polyol synthesized by microorganisms as an osmoprotectant, is a natural sweetener produced on an industrial scale for decades. Despite the fact that the yeast Yarrowia lipolytica has been reported since the 1970s as an erythritol producer, the metabolic pathway of this polyol has never been characterized. It was shown that erythritol synthesis in yeast occurs via the pentose phosphate pathway (PPP). The oleaginous yeast Y. lipolytica is a good host for converting inexpensive glycerol into a value-added product such as erythritol. Glycerol is a renewable feedstock which is produced on a large scale as a waste product by many branches of industry.ResultsIn this study, we functionally overexpressed four genes involved in the pentose phosphate pathway (PPP): gene YALI0E06479g encoding transketolase (TKL1), gene YALI0F15587g encoding transaldolase (TAL1), gene YALI0E22649g encoding glucose-6-phosphate dehydrogenase (ZWF1), and gene YALI0B15598g encoding 6-phosphogluconate dehydrogenase (GND1). Here, we show that the crucial gene for erythritol synthesis in Y. lipolytica is transketolase. Overexpression of this gene results in a twofold improvement in erythritol synthesis during a shake-flask experiment (58 g/L). Moreover, overexpression of TKL1 allows for efficient production of erythritol independently from the supplied dissolved oxygen. Fermentation conducted in a 5-L bioreactor at low agitation results in almost 70% higher titer of erythritol over the control strain.ConclusionThis work presents the importance of the PPP in erythritol synthesis and the feasibility for economic production of erythritol from glycerol by the yeast Y. lipolytica.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0772-6) contains supplementary material, which is available to authorized users.

Homology‐independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica

“…Yarrowia lipolytica , an unconventional and oleaginous yeast, has attracted an increasing attention in recent years (Darvishi, Ariana, Marella, & Borodina, ; Hussain et al, ; Markham and Alper, ; Xie, ) and has been exploited to produce a variety of products, such as fatty acid derivatives (Tai & Stephanopoulos, ; Xue et al, ), succinic acid (Cui et al, ; C. Gao et al, ), erythritol (Carly et al, ; Mirończuk, Biegalska, & Dobrowolski, ), and lipids (Qiao, Wasylenko, Zhou, Xu, & Stephanopoulos, ). Y. lipolytica is also an excellent host for the production of secondary metabolites such as α‐farnesene (Yang, Nambou, Wei, & Hua, ), lycopene (Matthäus, Ketelhot, Gatter, & Barth, ), and β‐carotene (S. Gao et al, ; Larroude et al, ), because it contains many precursors and cofactors.…”

Erythritol production by yeasts: a snapshot of current knowledge

“…It is then dephosphorylated by an erythrose‐4‐P phosphatase, yielding erythrose. This latter is then reduced by an erythrose reductase (ER) into erythritol (Ishizuka, Tokuoka, Sasaki, & Taniguchi, ; Mirończuk, Biegalska, & Dobrowolski, ). In addition to this anabolic pathway, some yeasts are also able to use erythritol as a carbon source.…”

“…Similarly to Y. lipolytica , in M. megachiliensis expression of transketolase is increased when grown under high osmotic pressure (Iwata et al, ). In Y. lipolytica , glucose‐6‐P dehydrogenase, the first enzyme from the oxidative pentose phosphate pathway, is also overexpressed during erythritol synthesis (Mirończuk et al, ).…”

“…Moreover, this strain showed higher glycerol uptake (Mirończuk, Rzechonek, Biegalska, Rakicka, & Dobrowolski, ). Strains overexpressing both transketolase and ER also showed important benefits as compared with wild‐type strains, highlighting the central gene of these genes in erythritol synthesis (Janek et al, ; Mirończuk et al, ). Gene deletion has also been used for strain improvement.…”

Effects of osmotic pressure and pH on citric acid and erythritol production from waste cooking oil by Yarrowia lipolytica

“…Yield of products from carbon resource is usually caused by the overflow metabolites resulting from changes in key enzymes involved in the metabolic process [31]. According to hypothetical pathway of erythritol synthesis in Y. lipolytica described by Mirończuk et al [32], Pyruvate kinase (PK) and transketolase (TK) created bottlenecks in carbon flow during conversion of glyceraldehydes-3-P into citric acid or erythritol respectively, and the last steps of citric acid and erythritol synthesis are respectively catalyzed by citrate synthase (CS) and erythrose reductase (ER) (Fig. 2).…”