Highly Efficient Synthesis of Rare Sugars from Glycerol in Endotoxin-Free ClearColi by Fermentation.

Rare sugars possess potential applications as low-calorie sweeteners, especially for anti-obesity and anti-diabetes. In this study, a fermentation biosystem based on the "DHAP-dependent aldolases strategy" was established for D-allulose and D-sorbose production from glycerol in endotoxin-free ClearColi BL21 (DE3). Several engineering strategies were adopted to enhance rare sugar production. Firstly, the combination of different plasmids for aldO, rhaD, and yqaB expression was optimized. Then, the artificially constructed ribosomal binding site (RBS) libraries of aldO, rhaD, and yqaB genes were assembled individually and combinatorially. In addition, a peroxidase was overexpressed to eliminate the damage or toxicity from hydrogen peroxide generated by alditol oxidase (AldO). Finally, stepwise improvements in rare sugar synthesis were elevated to 15.01 g/L with a high yield of 0.75 g/g glycerol in a 3 L fermenter. This research enables the effective production of rare sugars from raw glycerol in high yields.

Highly efficient expression and secretion of human lysozyme using multiple strategies in Pichia pastoris.

BACKGROUND: Human lysozyme (hLYZ), an emerging antibacterial agent, has extensive application in the food and pharmaceutical industries. However, the source of hLYZ is particularly limited. RESULTS: To achieve highly efficient expression and secretion of hLYZ in Pichia pastoris, multiple strategies including G418 sulfate screening, signal sequence optimization, vacuolar sorting receptor VPS10 disruption, and chaperones/transcription factors co-expression were applied. The maximal enzyme activity of extracellular hLYZ in a shaking flask was 81,600 ± 5230 U mL-1 , which was about five times of original strain. To further reduce the cost, the optimal medium RDMY was developed and the highest hLYZ activity reached 352,000 ± 16,696.5 U mL-1 in a 5 L fermenter. CONCLUSION: This research provides a very useful and cost-effective approach for the hLYZ production in P. pastoris and can also be applied to the production of other recombinant proteins.

Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins.

Herein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins, including synaptosomal-associated protein 25 (SNAP25). BoNT-LCs are metalloproteases, and each BoNT-LC recognizes and cleaves conserved domains in SNAREs termed the SNARE domain. In the budding yeast Saccharomyces cerevisiae, the SNAP25 ortholog Spo20 is required for production of the spore plasma membrane; thus, defects in Spo20 cause sporulation deficiencies. We found that chimeric SNAREs in which SNARE domains in Spo20 are replaced with those of SNAP25 are functional in yeast cells. The Spo20/SNAP25 chimeras, but not Spo20, are sensitive to digestion by BoNT-LCs. We demonstrate that spo20∆ yeasts harboring the chimeras exhibit sporulation defects when various SNAP25-targeting BoNT-LCs are expressed. Thus, the activities of BoNT-LCs can be assessed by colorimetric measurement of sporulation efficiencies. Although BoNTs are notorious toxins, they are also used as therapeutic and cosmetic agents. Our assay system will be useful for analyzing novel BoNTs and BoNT-like genes, as well as their manipulation.

Studies on the Proteinaceous Structure Present on the Surface of the Saccharomyces cerevisiae Spore Wall.

The surface of the Saccharomyces cerevisiae spore wall exhibits a ridged appearance. The outermost layer of the spore wall is believed to be a dityrosine layer, which is primarily composed of a crosslinked dipeptide bisformyl dityrosine. The dityrosine layer is impervious to protease digestion; indeed, most of bisformyl dityrosine molecules remain in the spore after protease treatment. However, we find that the ridged structure is removed by protease treatment. Thus, a ridged structure is distinct from the dityrosine layer. By proteomic analysis of the spore wall-bound proteins, we found that hydrophilin proteins, including Sip18, its paralog Gre1, and Hsp12, are present in the spore wall. Mutant spores with defective hydrophilin genes exhibit functional and morphological defects in their spore wall, indicating that hydrophilin proteins are required for the proper organization of the ridged and proteinaceous structure. Previously, we found that RNA fragments were attached to the spore wall in a manner dependent on spore wall-bound proteins. Thus, the ridged structure also accommodates RNA fragments. Spore wall-bound RNA molecules function to protect spores from environmental stresses.

Rare sugar L-sorbose exerts antitumor activity by impairing glucose metabolism.

Rare sugars are monosaccharides with low natural abundance. They are structural isomers of dietary sugars, but hardly be metabolized. Here, we report that rare sugar L-sorbose induces apoptosis in various cancer cells. As a C-3 epimer of D-fructose, L-sorbose is internalized via the transporter GLUT5 and phosphorylated by ketohexokinase (KHK) to produce L-sorbose-1-phosphate (S-1-P). Cellular S-1-P inactivates the glycolytic enzyme hexokinase resulting in attenuated glycolysis. Consequently, mitochondrial function is impaired and reactive oxygen species are produced. Moreover, L-sorbose downregulates the transcription of KHK-A, a splicing variant of KHK. Since KHK-A is a positive inducer of antioxidation genes, the antioxidant defense mechanism in cancer cells can be attenuated by L-sorbose-treatment. Thus, L-sorbose performs multiple anticancer activities to induce cell apoptosis. In mouse xenograft models, L-sorbose enhances the effect of tumor chemotherapy in combination with other anticancer drugs. These results demonstrate L-sorbose as an attractive therapeutic reagent for cancer treatment.

Receptor for advanced glycation end-products (RAGE) mediates phagocytosis in nonprofessional phagocytes.

In mammals, both professional phagocytes and nonprofessional phagocytes (NPPs) can perform phagocytosis. However, limited targets are phagocytosed by NPPs, and thus, the mechanism remains unclear. We find that spores of the yeast Saccharomyces cerevisiae are internalized efficiently by NPPs. Analyses of this phenomenon reveals that RNA fragments derived from cytosolic RNA species are attached to the spore wall, and these fragments serve as ligands to induce spore internalization. Furthermore, we show that a multiligand receptor, RAGE (receptor for advanced glycation end-products), mediates phagocytosis in NPPs. RAGE-mediated phagocytosis is not uniquely induced by spores but is an intrinsic mechanism by which NPPs internalize macromolecules containing RAGE ligands. In fact, artificial particles labeled with polynucleotides, HMGB1, or histone (but not bovine serum albumin) are internalized in NPPs. Our findings provide insight into the molecular basis of phagocytosis by NPPs, a process by which a variety of macromolecules are targeted for internalization.

Triosephosphate Isomerase and Its Product Glyceraldehyde-3-Phosphate Are Involved in the Regulatory Mechanism That Suppresses Exit from the Quiescent State in Yeast Cells.

Cells of the budding yeast Saccharomyces cerevisiae form spores or stationary cells upon nutrient starvation. These quiescent cells are known to resume mitotic growth in response to nutrient signals, but the mechanism remains elusive. Here, we report that quiescent yeast cells are equipped with a negative regulatory mechanism which suppresses the commencement of mitotic growth. The regulatory process involves a glycolytic enzyme, triosephosphate isomerase (Tpi1), and its product, glyceraldehyde-3-phosphate (GAP). GAP serves as an inhibitory signaling molecule; indeed, the return to growth of spores or stationary cells is suppressed by the addition of GAP even in nutrient-rich growth media, though mitotic cells are not affected. Reciprocally, dormancy is abolished by heat treatment because of the heat sensitivity of Tpi1. For example, spores commence germination merely upon heat treatment, which indicates that the negative regulatory mechanism is actively required for spores to prevent premature germination. Stationary cells of Candida glabrata are also manipulated by heat and GAP, suggesting that the regulatory process is conserved in the pathogenic yeast. IMPORTANCE Our results suggest that, in quiescent cells, nutrient signals do not merely provoke a positive regulatory process to commence mitotic growth. Exit from the quiescent state in yeast cells is regulated by balancing between the positive and negative signaling pathways. Identifying the negative regulatory pathway would provide new insight into the regulation of the transition from the quiescent to the mitotic state. Clinically, quiescent cells are problematic because they are resistant to environmental stresses and antibiotics. Given that the quiescent state is modulated by manipulation of the negative regulatory mechanism, understanding this process is important not only for its biological interest but also as a potential target for antifungal treatment.

Establishment of a Novel Cell Surface Display Platform Based on Natural "Chitosan Beads" of Yeast Spores.

Cell surface display technology, which expresses and anchors proteins on the surface of microbial cells, has broad application prospects in many fields, such as protein library screening, biocatalysis, and biosensor development. However, traditional cell surface display systems have disadvantages: the molecular weight of phage display proteins cannot be too large; bacterial display lacks the post-translational modification process for eukaryotic proteins; yeast display is prone to excessive protein glycosylation and misfolding of multisubunit proteins; and the compatibility of Bacillus subtilis spore display needs to be further improved. Therefore, it is extremely valuable to develop an efficient surface display platform with strong universality and stress resistance properties. Although yeast surface display systems have been extensively investigated, the establishment of a surface display platform using yeast spores has rarely been reported. In this study, a novel cell surface display platform based on natural "chitosan beads" of yeast spores was developed. The target protein in fusion with the chitosan affinity protein (CAP) exhibited strong binding capability with "chitosan beads" of yeast spores in vitro and in vivo. Moreover, this protein display system showed highly preferable enzymatic properties and stability. As an example, the displayed LXYL-P1-2-CAP demonstrated high thermostability and reusability (60% of the initial activity after seven cycles of reuse), high storage stability (75% of original activity after 8 weeks), and excellent tolerance to a concentration up to 75% (v/v) organic reagents. To prove the practicability of this surface display system, the semisynthesis of paclitaxel intermediate was demonstrated and its highest conversion rate was 92% using 0.25 mM substrate. This study provides a novel and useful platform for the surface display of proteins, especially for multimeric macromolecular proteins of eukaryotic origin.

Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension.

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71-Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.

Identification of novel O-GlcNAc transferase substrates using yeast cells expressing OGT.

O-GlcNAc modification mediated by O-GlcNAc transferase (OGT) is a reversible protein modification in which O-GlcNAc moieties are attached to target proteins in the cytosol, nucleus, and mitochondria. O-GlcNAc moieties attached to proteins can be removed by O-GlcNAcase (OGA). The addition of an O-GlcNAc moiety can influence several aspects of protein function, and aberrant O-GlcNAc modification is linked to a number of diseases. While OGT and OGA are conserved across eukaryotic cells, yeasts lack these enzymes. Previously, we reported that protein O-GlcNAc modification occurred in the budding yeast Saccharomyces cerevisiae when OGT was ectopically expressed. Because yeast cells lack OGA, O-GlcNAc moieties are stably attached to target proteins. Thus, the yeast system may be useful for finding novel OST substrates. By proteomic analysis, we identified 468 O-GlcNAcylated proteins in yeast cells expressing human OGT. Among these proteins, 13 have human orthologues that show more than 30% identity to their corresponding yeast orthologue, and possible glycosylation residues are conserved in these human orthologues. In addition, the orthologues have not been reported as substrates of OGT. We verified that some of these human orthologues are O-GlcNAcylated in cultured human cells. These proteins include an ubiquitin-conjugating enzyme, UBE2D1, and an eRF3-similar protein, HBS1L. Thus, the yeast system would be useful to find previously unknown O-GlcNAcylated proteins and regulatory mechanisms.

PGAP6, a GPI-specific phospholipase A2, has narrow substrate specificity against GPI-anchored proteins.

PGAP6, also known as TMEM8A, is a phospholipase A2 with specificity to glycosylphosphatidylinositol (GPI) and expressed on the surface of various cells. CRIPTO, a GPI-anchored co-receptor for a morphogenic factor Nodal, is a sensitive substrate of PGAP6. PGAP6-mediated shedding of CRIPTO plays a critical role in an early stage of embryogenesis. In contrast, CRYPTIC, a close family member of CRIPTO, is resistant to PGAP6. In this report, chimeras between CRIPTO and CRYPTIC and truncate mutants of PGAP6 were used to demonstrate that the Cripto-1/FRL1/Cryptic domain of CRIPTO is recognized by an N-terminal domain of PGAP6 for processing. We also report that among 56 human GPI-anchored proteins tested, only glypican 3, prostasin, SPACA4, and contactin-1, in addition to CRIPTO, are sensitive to PGAP6, indicating that PGAP6 has a narrow specificity toward various GPI-anchored proteins.

Engineered disulfide bonds improve thermostability and activity of L-isoleucine hydroxylase for efficient 4-HIL production in Bacillus subtilis 168.

4-Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l-Isoleucine hydroxylase specifically converts l-Ile to 4-hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4-hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l-isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half-life of variant T181C was 4.03 h at 50°C, 10.27-fold the half-life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56-fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole-cell biotransformation, Bacillus subtilis 168/pMA5-ido T181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4-hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4-hydroxyisoleucine. Rational design of disulfide bond improved l-isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.

Functional characteristics of Svl3 and Pam1 that are required for proper cell wall formation in yeast cells.

In the budding yeast Saccharomyces cerevisiae, Svl3 and Pam1 proteins work as functional homologues. Loss of their function causes increased levels of chitin deposition in the cell wall and temperature sensitivity, suggesting their involvement in cell wall formation. We found that the N- and C-termini of these proteins have distinctive and critical functions. They contain an N-terminal part that has a probable 2-dehydropantoate 2-reductase domain. In Svl3, this part can be replaced with the yeast 2-dehydropantoate 2-reductase, Pan5, suggesting that Svl3 and its homologues may be able to mediate 2-dehydropantoate 2-reductase function. On the other hand, Svl3 is recruited to the bud tip and bud neck via multiple localization signals in the C-terminal part. One of such signals is the lysine-rich region located in the C-terminal end. The function and localization of Svl3 are significantly disrupted by the loss of this lysine-rich region; however, its localization is not completely abolished by the mutation because another localization signal enables appropriate transport. Svl3 and Pam1 orthologues are found in cells across fungal species. The Svl3 orthologues of Candida glabrata can complement the loss of Svl3 and Pam1 in S. cerevisiae. C. glabrata cells lacking the SVL3 and PAM1 orthologue genes exhibit phenotypes similar to those observed in svl3∆pam1∆ S. cerevisiae cells. Thus, Svl3 homologues may be generally required for the assembly of the cell wall in fungal cells.

Encapsulation of Mannose-6-phosphate Isomerase in Yeast Spores and Its Application in l-Ribose Production.

A mannose-6-phosphate isomerase (MPI) from Geobacillus thermodenitrificans was expressed and successfully encapsulated into the Saccharomyces cerevisiae spores. Our results demonstrated that compared to the free enzyme, the MPI triple mutant encapsulated in osw2Δ spores exhibited much preferred enzymatic properties, such as enhanced catalytic activity, excellent reusability, thermostability, and tolerance to various harsh conditions. In combination with an l-arabinose isomerase (AI) also from G. thermodenitrificans, this technique of spore encapsulation was applied for producing a high-value rare sugar l-ribose from biomass-derived l-arabinose. Using a 10 mL reaction system, 350 mg of l-ribose was produced from 1 g of l-arabinose with a conversion yield of 35% by repeatedly reacting with 200 mg of AI-encapsulated spores and 300 mg of MPI-encapsulated spores. This study provides a very useful and concise approach for the synthesis of rare sugars and other useful compounds.

Characteristics of SNARE proteins are defined by distinctive properties of SNARE motifs.

BACKGROUND: Syntaxin-1A and Sso1 are syntaxin family SNARE proteins engaged in synaptic vesicle fusion and yeast exocytosis. The syntaxin-1A SNARE motif can form a fusogenic SNARE complex with Sso1 partners. However, a chimera in which the SNARE motif in syntaxin-1A is introduced into Sso1 was not functional in yeast because the chimera is retained in the ER. Through the analysis of the transport defect of Sso1/syntaxin-1A chimeric SNAREs, we found that their SNARE motifs have distinctive properties. METHODS: Sso1, syntaxin-1A, and Sso1/syntaxin-1A chimeric SNAREs were expressed in yeast cells and their localization and interaction with other SNAREs are analyzed. RESULTS: SNARE proteins containing the syntaxin-1A SNARE motif exhibit a transport defect because they form a cis-SNARE complex in the ER. Ectopic SNARE complex formation can be prevented in syntaxin-1A by binding to a Sec1/Munc-18-like (SM) protein. In contrast, the SNARE motif of Sso1 does not form an ectopic SNARE complex. Additionally, we found that the SNARE motif in syntaxin-1A, but not that in Sso1, self-interacts, even when it is in the inactive form and bound to the SM protein. CONCLUSIONS: The SNARE motif in syntaxin-1A, but not in Sso1, likely forms ectopic SNARE complex. Because of this property, the SM protein is necessary for syntaxin-1A to prevent its promiscuous assembly and to promote its export from the ER. GENERAL SIGNIFICANCE: Properties of SNARE motifs affect characteristics of SNARE proteins. The regulatory mechanisms of SNARE proteins are, in part, designed to handle such properties.

Studies on the Properties of the Sporulation Specific Protein Dit1 and its Product Formyl Tyrosine.

The dityrosine layer is a unique structure present in the spore wall of the budding yeast Saccharomyces cerevisiae. The primary constituent of this layer is bisformyl dityrosine. A sporulation-specific protein, Dit1 is localized in the spore cytosol and produces a precursor of bisformyl dityrosine. Although Dit1 is similar to isocyanide synthases, the loss of Dit1 is not rescued by heterologous expression of the Pseudomonas aeruginosa isocyanide synthase, PvcA, indicating that Dit1 does not mediate isocyanidation. The product of Dit1 is most likely formyl tyrosine. Dit1 can produce its product when it is expressed in vegetative cells; however, formyl tyrosine was not detected in the crude cell lysate. We reasoned that formyl tyrosine is unstable and reacts with some molecule to form formyl tyrosine-containing molecules in the cell lysate. In support of this hypothesis, formyl tyrosine was detected when the lysate was hydrolyzed with a mild acid. The same property was also found for bisformyl dityrosine. Bisformyl dityrosine molecules assemble to form the dityrosine layer by an unknown mechanism. Given that bisformyl dityrosine can be released from the spore wall by mild hydrolysis, the process of formyl tyrosine-containing molecule formation may resemble the assembly of the dityrosine layer.

Construction of functional chimeras of syntaxin-1A and its yeast orthologue, and their application to the yeast cell-based assay for botulinum neurotoxin serotype C.

BACKGROUND: Botulinum neurotoxins (BoNTs) prevent synaptic transmission because they hydrolyze synaptic N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). BoNT serotype C (BoNT/C) targets syntaxin-1A and SNAP-25, and is expected to be applied to cosmetic and therapeutic uses. SNAREs are evolutionally conserved proteins and in yeast a syntaxin-1A orthologue Sso1 is involved in exocytosis. The substrate specificity of BoNT/C is strict and it cannot cleave Sso1. METHODS: Domain swapping and mutational screenings were performed to generate functional chimeras SNAREs of syntaxin-1A and Sso1. Such chimeras are expressed in yeast cells and assessed whether they are susceptible to BoNT/C digestion. RESULTS: The Sso1 and syntaxin-1A chimera (Sso1/STX1A), in which the SNARE domain in Sso1 was replaced with that of syntaxin-1A, was not functional in yeast. The functional incompatibility of Sso1/STX1A was attributable to its accumulation in the ER. We found several mutations that could release Sso1/STX1A from the ER to make the chimera functional in yeast. Yeast cells harboring the mutant chimeras grew similarly to wild-type cells. However, unlike wild-type, yeast harboring the mutant chimeras exhibited a severe growth defect upon expression of BoNT/C. Results of further domain swapping analyses suggest that Sso1 is not digested by BoNT/C because it lacks a binding region to BoNT/C (α-exosite-binding region). CONCLUSIONS: We obtained functional Sso1/STX1A chimeras, which can be applied to a yeast cell-based BoNT/C assay. BoNT/C can recognize these chimeras in a similar manner to syntaxin-1A. GENERAL SIGNIFICANCE: The yeast cell-based BoNT/C assay would be useful to characterize and engineer BoNT/C.

Efficient chiral synthesis by Saccharomyces cerevisiae spore encapsulation of Candida parapsilosis Glu228Ser/(S)-carbonyl reductase II and Bacillus sp. YX-1 glucose dehydrogenase in organic solvents.

BACKGROUND: Saccharomyces cerevisiae AN120 osw2∆ spores were used as a host with good resistance to unfavorable environment. This work was undertaken to develop a new yeast spore-encapsulation of Candida parapsilosis Glu228Ser/(S)-carbonyl reductase II and Bacillus sp. YX-1 glucose dehydrogenase for efficient chiral synthesis in organic solvents. RESULTS: The spore microencapsulation of E228S/SCR II and GDH in S. cerevisiae AN120 osw2∆ catalyzed (R)-phenylethanol in a good yield with an excellent enantioselectivity (up to 99%) within 4 h. It presented good resistance and catalytic functions under extreme temperature and pH conditions. The encapsulation produced several chiral products with over 70% yield and over 99% enantioselectivity in ethyl acetate after being recycled for 4-6 times. It increased substrate concentration over threefold and reduced the reaction time two to threefolds compared to the recombinant Escherichia coli containing E228S and glucose dehydrogenase. CONCLUSIONS: This work first described sustainable enantioselective synthesis without exogenous cofactors in organic solvents using yeast spore-microencapsulation of coupled alcohol dehydrogenases.

Production of l-Ribulose Using an Encapsulated l-Arabinose Isomerase in Yeast Spores.

The rare sugar l-ribulose is produced from the abundant sugar l-arabinose by enzymatic conversion. An l-arabinose isomerase (AI) from Geobacillus thermodenitrificans was efficiently expressed and encapsulated in Saccharomyces cerevisiae spores. Deletion of the yeast OSW2 gene, which causes a mild defect in the integrity of the spore wall, substantially improved the activity of encapsulated AI, without damaging its superior enzymatic properties of thermostability, pH tolerance,and resistance toward SDS and proteinase treatments. In a 10 mL reaction, 100 mg of dry AI encapsulated in spores produced 250 mg of l-ribulose from 1 g of l-arabinose, indicating a 25% conversion rate. Notably, the product of l-ribulose was directly purified from the reaction solution with an approximately 91% recovery using a Ca2+ ion exchange column. Our results describe not only a facile approach for the production of l-ribulose but also a useful strategy for the enzymatic conversion of rare sugars in "Izumoring".

Yeast Dop1 is required for glycosyltransferase retrieval from the trans-Golgi network.

BACKGROUND: Glycosyltransferases are type II membrane proteins that are responsible for glycan modification of proteins and lipids, and localize to distinct cisternae in the Golgi apparatus. During cisternal maturation, retrograde trafficking helps maintain the steady-state localization of these enzymes in the sub-compartments of the Golgi. METHODS: To understand how glycosyltransferases are recycled in the late Golgi complex, we searched for genes that are essential for budding yeast cell growth and that encode proteins localized in endosomes and in the Golgi. We specifically analyzed the roles of Dop1 and its binding partner Neo1 in retaining Golgi-resident glycosyltransferases, in the late Golgi complex. RESULTS: Dop1 primarily localized to younger compartments of the trans-Golgi network (TGN) and seemed to cycle within the TGN. In contrast, Neo1, a P4-ATPase that interacts with Dop1, localized to the TGN. Abolition of DOP1 expression led to defects in the FM4-64 endocytic pathway. Dop1 and Neo1 were required for correct glycosylation of invertase, a secretory protein, at the Golgi. In DOP1-shutdown cells, Och1, a mannosyltransferase that is typically located in the cis-Golgi, mislocalized to the TGN. In addition, the function of multiple glycosyltransferases required for N- and O-glycosylation were impaired in DOP1-shutdown cells. CONCLUSIONS: Our results indicate that Dop1 is involved in vesicular transport at the TGN, and is critical for retrieving glycosyltransferases from the TGN to the Golgi in yeast. GENERAL SIGNIFICANCE: Golgi-resident glycosyltransferases recycling from the TGN to the Golgi is dependent on Dop1 and the P4-ATPase Neo1.