Engineering Yarrowia lipolytica for the Synthesis of Glutathione from Organic By-Products

Abstract

Tripeptide glutathione, which plays important roles in many cellular mechanisms, is also a biotechnology-oriented molecule with applications in medicine, food and cosmetic. Here, the engineering of the yeast Yarrowia lipolytica for the production of this metabolite at high titer values from various agro-industrial by-products is reported. The constitutive overexpression of the glutathione biosynthetic genes GSH1 and GSH2 encoding respectively γ-glutamylcysteine synthetase and glutathione synthetase, together with the INU1 gene from Kluyveromyces marxianus encoding inulinase yielded a glutathione titer value and a productivity of 644 nmol/mg protein and 510 µmol/gDCW, respectively. These values were obtained during bioreactor batch cultures in a medium exclusively comprising an extract of Jerusalem artichoke tuber, used as a source of inulin, and ammonium sulfate, used as a nitrogen source.

Biosynthesis of the antioxidant γ‐glutamyl‐cysteine with engineered Yarrowia lipolytica

“…In Y. lipolytica , genes YALI0E30129g ( GSH1 ) and YALI0C17831g ( GSH2 ) encode GCL and GS, respectively. [ 27 ] With the goal of constructing a γ‐GC producer strain, gene GSH2 was disrupted in strain RIY176, resulting in the generation of strain RIY441 (Table 1). Subsequently, in the disrupted strain, gene GSH1 was overexpressed under the control of the strong constitutive promoter pTEF, both in one and two copies, leading to the strains RIY455 and RIY445, respectively.…”

“…These γ‐GC titers fall within the range of the GSH titer obtained with strain RIY231, which overexpresses GSH1 and GSH2 (340 nmol GSH mg −1 protein). [ 27 ]…”

“…[ 30,31 ] qPCRs were performed following procedures outlined elsewhere, with the specific primers listed in Table S2. [ 27 ]…”

“…[30,31] qPCRs were performed following procedures outlined elsewhere, with the specific primers listed in Table S2. [27] For the GSH2 gene disruption, a 1 kb fragment (Pro_GSH2) upstream of the start codon and a 1.2 kb fragment (Ter_GSH2) downstream of the stop codon of gene YALI0C17831g were each PCR-amplified using the primer pairs GSH2pro_for/GSH2pro_rev and GSH2ter_for/GSH2ter_rev, respectively, and with Y. lipolytica RIY129 genomic DNA serving as the template. The URA3 selection marker was amplified using the primer pair Auxo_fo/Auxo_rev, utilizing plasmid RIP110 as the template.…”

“…[24] While the reaction achieved a conversion yield of 0.8, it requires a pure GSH solution, as well as the production, purification, and immobilization of recombinant NsPCS, which can be cumbersome and time-consuming. We previously reported on the engineering of the yeast Yarrowia lipolytica for efficient GSH production, [27,28] achieving GSH titers and productivity rates among the highest reported in the literature. Therefore, we employed this yeast as a chassis strain for γ-GC production.…”