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Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae.

ABSTRACT: Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp). In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2(-) and ura3(-) for YIplac128 and YEp352, respectively. The heterologous expression of phytase was used to determine the expression capability of the host strain. Expression in the new host strain (2805-a7) was as high as that in the parental strain (2805), which expresses high levels of several foreign genes. Following transformation, Northern and Western blot analyses demonstrated the expression of H- and L-chain genes. The recombinant yeast was more iron tolerant, in that transformed cells formed colonies on plates containing more than 25 mM ferric citrate, whereas none of the recipient strain cells did. Prussian blue staining indicated that the expressed isoferritins were assembled in vivo into a complex that bound iron. The expressed subunits showed a clear preference for the formation of heteropolymers over homopolymers. The molar ratio of H to L chains was estimated to be 1:6.8. The gel-purified heteropolymer took up iron faster than the L homopolymer, and it took up more iron than the H homopolymer did. The iron concentrations in transformants expressing the heteropolymer, L homopolymer, and H homopolymer were 1,004, 760, and 500 micro g per g (dry weight) of recombinant yeast cells, respectively. The results indicate that heterologously expressed H and L subunits coassemble into a heteropolymer in vivo and that the iron-carrying capacity of yeast is further enhanced by the expression of heteropolymeric isoferritin.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC154789 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae.

Kim Hye-Jin HJ Kim Hyang-Mi HM Kim Ji-Hye JH Ryu Kyeong-Seon KS Park Seung-Moon SM Jahng Kwang-Yeup KY Yang Moon-Sik MS Kim Dae-Hyuk DH

Applied and environmental microbiology 20030401 4

Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp). In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2(-) and ura3(-) for YIplac128 and YEp352, respectively. The heterologous expression of phytase was used to determine the expression capability of the host strain. Expression in the ne ...[more]

PMID: 12676675

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Project description:Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.

Project description:BACKGROUND:Cocoa butter (CB) extracted from cocoa beans (Theobroma cacao) is the main raw material for chocolate production, but CB supply is insufficient due to the increased chocolate demand and limited CB production. CB is mainly composed of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0-C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0). In general, Saccharomyces cerevisiae produces TAGs as storage lipids, which consist of C16 and C18 fatty acids. However, cocoa butter-like lipids (CBL, which are composed of POP, POS and SOS) are not among the major TAG forms in yeast. TAG biosynthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT), and it is essential to modulate the yeast TAG biosynthetic pathway for higher CBL production. RESULTS:We cloned seven GPAT genes and three LPAT genes from cocoa cDNA, in order to screen for CBL biosynthetic gene candidates. By expressing these cloned cocoa genes and two synthesized cocoa DGAT genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some of the strains. In the best producer, the potential CBL content was eightfold higher than the control strain, suggesting the cocoa genes expressed in this strain were functional and might be responsible for CBL biosynthesis. Moreover, the potential CBL content increased 134-fold over the control Y29-TcD1 (IMX581 sct1? ale1? lro1? dga1? with TcDGAT1 expression) in strain Y29-441 (IMX581 sct1? ale1? lro1? dga1? with TcGPAT4, TcLPAT4 and TcDGAT1 expression) further suggesting cocoa GPAT and LPAT genes functioned in yeast. CONCLUSIONS:We demonstrated that cocoa TAG biosynthetic genes functioned in S. cerevisiae and identified cocoa genes that may be involved in CBL production. Moreover, we found that expression of some cocoa CBL biosynthetic genes improved potential CBL production in S. cerevisiae, showing that metabolic engineering of yeast for cocoa butter production can be realized by manipulating the key enzymes GPAT, LPAT and DGAT in the TAG biosynthetic pathway.

Dataset Information

Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae.

Publications

Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae.

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