The evolution of the GALactose utilization pathway in budding yeasts.

The Leloir galactose utilization or GAL pathway of budding yeasts, including that of the baker's yeast Saccharomyces cerevisiae and the opportunistic human pathogen Candida albicans, breaks down the sugar galactose for energy and biomass production. The GAL pathway has long served as a model system for understanding how eukaryotic metabolic pathways, including their modes of regulation, evolve. More recently, the physical linkage of the structural genes GAL1, GAL7, and GAL10 in diverse budding yeast genomes has been used as a model for understanding the evolution of gene clustering. In this review, we summarize exciting recent work on three different aspects of this iconic pathway's evolution: gene cluster organization, GAL gene regulation, and the population genetics of the GAL pathway.

Towards universal synthetic heterotrophy using a metabolic coordinator.

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pSyn4.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

Divergence of alternative sugar preferences through modulation of the expression and activity of the Gal3 sensor in yeast.

Optimized nutrient utilization is crucial for the progression of microorganisms in competing communities. Here we investigate how different budding yeast species and ecological isolates have established divergent preferences for two alternative sugar substrates: Glucose, which is fermented preferentially by yeast, and galactose, which is alternatively used upon induction of the relevant GAL metabolic genes. We quantified the dose-dependent induction of the GAL1 gene encoding the central galactokinase enzyme and found that a very large diversification exists between different yeast ecotypes and species. The sensitivity of GAL1 induction correlates with the growth performance of the respective yeasts with the alternative sugar. We further define some of the mechanisms, which have established different glucose/galactose consumption strategies in representative yeast strains by modulating the activity of the Gal3 inducer. (1) Optimal galactose consumers, such as Saccharomyces uvarum, contain a hyperactive GAL3 promoter, sustaining highly sensitive GAL1 expression, which is not further improved upon repetitive galactose encounters. (2) Desensitized galactose consumers, such as S. cerevisiae Y12, contain a less sensitive Gal3 sensor, causing a shift of the galactose response towards higher sugar concentrations even in galactose experienced cells. (3) Galactose insensitive sugar consumers, such as S. cerevisiae DBVPG6044, contain an interrupted GAL3 gene, causing extremely reluctant galactose consumption, which is, however, improved upon repeated galactose availability. In summary, different yeast strains and natural isolates have evolved galactose utilization strategies, which cover the whole range of possible sensitivities by modulating the expression and/or activity of the inducible galactose sensor Gal3.

Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells.

Glycosylation of recombinant therapeutics like monoclonal antibodies (mAbs) is a critical quality attribute. N-glycans in mAbs are known to affect various effector functions, and thereby therapeutic use of such glycoproteins can depend on a particular glycoform profile to achieve desired efficacy. However, there are currently limited options for modulating the glycoform profile, which depend mainly on over-expression or knock-out of glycosyltransferase enzymes that can introduce or eliminate specific glycans but do not allow predictable glycoform modulation over a range of values. In this study, we demonstrate the ability to predictably modulate the glycoform profile of recombinant IgG. Using CRISPR/Cas9, we have engineered nucleotide sugar synthesis pathways in CHO cells expressing recombinant IgG for combinatorial modulation of galactosylation and fucosylation. Knocking out the enzymes UDP-galactose 4'-epimerase (Gale) and GDP-L-fucose synthase (Fx) resulted in ablation of de novo synthesis of UDP-Gal and GDP-Fuc. With Gale knock-out, the array of N-glycans on recombinantly expressed IgG is narrowed to agalactosylated glycans, mainly A2F glycan (89%). In the Gale and Fx double knock-out cell line, agalactosylated and afucosylated A2 glycan is predominant (88%). In the double knock-out cell line, galactosylation and fucosylation was entirely dependent on the salvage pathway, which allowed for modulation of UDP-Gal and GDP-Fuc synthesis and intracellular nucleotide sugar availability by controlling the availability of extracellular galactose and fucose. We demonstrate that the glycoform profile of recombinant IgG can be modulated from containing predominantly agalactosylated and afucosylated glycans to up to 42% and 96% galactosylation and fucosylation, respectively, by extracellular feeding of sugars in a dose-dependent manner. By simply varying the availability of extracellular galactose and/or fucose, galactosylation and fucosylation levels can be simultaneously and independently modulated. In addition to achieving the production of tailored glycoforms, this engineered CHO host platform can cater to the rapid synthesis of variably glycoengineered proteins for evaluation of biological activity.

The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Site-Specific Mutations of GalR Affect Galactose Metabolism in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen that is capable of asymptomatically colonizing the nasopharynx. Progression from colonization to invasive disease involves adaptation to distinct host niches, which vary markedly in the availability of key nutrients such as sugars. We previously reported that cell-cell signaling via the autoinducer 2 (AI-2)/LuxS quorum-sensing system boosts the capacity of S. pneumoniae to utilize galactose as a carbon source by upregulation of the Leloir pathway. This resulted in increased capsular polysaccharide production and a hypervirulent phenotype. We hypothesized that this effect was mediated by phosphorylation of GalR, the transcriptional activator of the Leloir pathway. GalR is known to possess three putative phosphorylation sites, S317, T319, and T323. In the present study, derivatives of S. pneumoniae D39 with putative phosphorylation-blocking alanine substitution mutations at each of these GalR sites (singly or in combination) were constructed. Growth assays and transcriptional analyses revealed complex phenotypes for these GalR mutants, with impacts on the regulation of both the Leloir and tagatose 6-phosphate pathways. The alanine substitution mutations significantly reduced the capacity of pneumococci to colonize the nasopharynx, middle ear, and lungs in a murine intranasal challenge model.IMPORTANCE Pneumococcal survival in the host and capacity to transition from a commensal to a pathogenic lifestyle are closely linked to the organism's ability to utilize specific nutrients in distinct niches. Galactose is a major carbon source for pneumococci in the upper respiratory tract. We have shown that both the Leloir and tagatose 6-phosphate pathways are necessary for pneumococcal growth in galactose and demonstrated GalR-mediated interplay between the two pathways. Moreover, the three putative phosphorylation sites in the transcriptional regulator GalR play a critical role in galactose metabolism and are important for pneumococcal colonization of the nasopharynx, middle ear, and lungs.

The past determines the future: sugar source history and transcriptional memory.

Transcriptional reinduction memory is a phenomenon whereby cells "remember" their transcriptional response to a previous stimulus such that subsequent encounters with the same stimulus can result in altered gene expression kinetics. Chromatin structure is thought to play a role in certain transcriptional memory mechanisms, leading to questions as to whether and how memory can be actively maintained and inherited to progeny through cell division. Here we summarize efforts towards dissecting chromatin-based transcriptional memory inheritance of GAL genes in Saccharomyces cerevisiae. We focus on methods and analyses of GAL (as well as MAL and INO) memory in single cells and discuss the challenges in unraveling the underlying mechanisms in yeast and higher eukaryotes.

N-glycosylation of the human ß1,4-galactosyltransferase 4 is crucial for its activity and Golgi localization.

ß1,4-galactosyltransferase 4 (B4GalT4) is one of seven B4GalTs that belong to CAZy glycosyltransferase family 7 and transfer galactose to growing sugar moieties of proteins, glycolipids, glycosaminoglycans as well as single sugar for lactose synthesis. Herein, we identify two asparagine-linked glycosylation sites in B4GalT4. We found that mutation of one site (Asn220) had greater impact on enzymatic activity while another (Asn335) on Golgi localization and presence of N-glycans at both sites is required for production of stable and enzymatically active protein and its secretion. Additionally, we confirm B4GalT4 involvement in synthesis of keratan sulfate (KS) by generating A375 B4GalT4 knock-out cell lines that show drastic decrease in the amount of KS proteoglycans and no significant structural changes in N- and O-glycans. We show that KS decrease in A375 cells deficient in B4GalT4 activity can be rescued by overproduction of either partially or fully glycosylated B4GalT4 but not with N-glycan-depleted B4GalT4 version.

A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG.

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson's r correlation coefficient of 0.8208 between the two methods.

Microhomology-mediated end joining induces hypermutagenesis at breakpoint junctions.

Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated end joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden.

Galactofuranose-Related Enzymes: Challenges and Hopes.

Galactofuranose is a rare form of the well-known galactose sugar, and its occurrence in numerous pathogenic micro-organisms makes the enzymes responsible for its biosynthesis interesting targets. Herein, we review the role of these carbohydrate-related proteins with a special emphasis on the galactofuranosidases we recently characterized as an efficient recombinant biocatalyst.

Exploiting Single-Cell Quantitative Data to Map Genetic Variants Having Probabilistic Effects.

Despite the recent progress in sequencing technologies, genome-wide association studies (GWAS) remain limited by a statistical-power issue: many polymorphisms contribute little to common trait variation and therefore escape detection. The small contribution sometimes corresponds to incomplete penetrance, which may result from probabilistic effects on molecular regulations. In such cases, genetic mapping may benefit from the wealth of data produced by single-cell technologies. We present here the development of a novel genetic mapping method that allows to scan genomes for single-cell Probabilistic Trait Loci that modify the statistical properties of cellular-level quantitative traits. Phenotypic values are acquired on thousands of individual cells, and genetic association is obtained from a multivariate analysis of a matrix of Kantorovich distances. No prior assumption is required on the mode of action of the genetic loci involved and, by exploiting all single-cell values, the method can reveal non-deterministic effects. Using both simulations and yeast experimental datasets, we show that it can detect linkages that are missed by classical genetic mapping. A probabilistic effect of a single SNP on cell shape was detected and validated. The method also detected a novel locus associated with elevated gene expression noise of the yeast galactose regulon. Our results illustrate how single-cell technologies can be exploited to improve the genetic dissection of certain common traits. The method is available as an open source R package called ptlmapper.

Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae.

The concept of "protein-based inheritance" defines prions as epigenetic determinants that cause several heritable traits in eukaryotic microorganisms, such as Saccharomyces cerevisiae and Podospora anserina. Previously, we discovered a non-chromosomal factor, [NSI+], which possesses the main features of yeast prions, including cytoplasmic infectivity, reversible curability, dominance, and non-Mendelian inheritance in meiosis. This factor causes omnipotent suppression of nonsense mutations in strains of S. cerevisiae bearing a deleted or modified Sup35 N-terminal domain. In this work, we identified protein determinants of [NSI+] using an original method of proteomic screening for prions. The suppression of nonsense mutations in [NSI+] strains is determined by the interaction between [SWI+] and [PIN+] prions. Using genetic and biochemical methods, we showed that [SWI+] is the key determinant of this nonsense suppression, whereas [PIN+] does not cause nonsense suppression by itself but strongly enhances the effect of [SWI+]. We demonstrated that interaction of [SWI+] and [PIN+] causes inactivation of SUP45 gene that leads to nonsense suppression. Our data show that prion interactions may cause heritable traits in Saccharomyces cerevisiae.

Insights into the Pathophysiology of Infertility in Females with Classical Galactosaemia.

Classical galactosaemia (CG) (OMIM 230400) is a rare inborn error of galactose metabolism caused by the deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT, EC 2.7.7.12). Primary ovarian insufficiency (POI) is the most common long-term complication experienced by females with CG, presenting with hypergonadotrophic hypoestrogenic infertility affecting at least 80% of females despite new-born screening and lifelong galactose dietary restriction. In this review, we describe the hypothesized pathophysiology of POI from CG, implications of timing of the ovarian dysfunction, and the new horizons and future prospects for treatments and fertility preservation.

Galactosylation of the Secondary Cell Wall Polysaccharide of Bacillus anthracis and Its Contribution to Anthrax Pathogenesis.

Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats with the structure, [→4)-ß-ManNAc-(1→4)-ß-GlcNAc(O3-α-Gal)-(1→6)-α-GlcNAc(O3-α-Gal, O4-ß-Gal)-(1→]6-12 The genes whose products promote the galactosylation of B. anthracis SCWP are not yet known. We show here that the expression of galE1, encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required for B. anthracis SCWP galactosylation. The galE1 mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-type B. anthracis, but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion of galE1 diminishes the capsulation of B. anthracis with poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of the B. anthracis cell wall envelope and for the pathogenesis of anthrax disease.IMPORTANCE Unlike virulent Bacillus anthracis isolates, B. anthracis strain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growth in vitro in broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants of B. anthracis strains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genes galE1 and galE2 We identified galE1 as necessary for SCWP galactosylation. Deletion of galE1 decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form of B. anthracis and increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae.

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate.

Inflammation-induced Gro1 triggers senescence in neuronal progenitors: effects of estradiol.

BACKGROUND: Inflammation has been proposed to contribute to the decline in adult hippocampal neurogenesis. Proinflammatory cytokines activate transcription of chemokine growth-regulated oncogene α (Gro1) in human and murine hippocampal neuronal progenitor cells (NPC). The goal of this study was to investigate the effects of Gro1 on hippocampal neurogenesis in the presence of inflammation. METHODS: Human hippocampal NPC were transfected with lentivirus expressing Gro1, and murine NPC and hippocampal neuronal HT-22 cells were treated with Gro1 protein. A plasmid expressing mGro1 was electroporated in the hippocampus of newborn mice that were sacrificed 10 days later. Adult male and female mice were injected with lipopolysaccharide (LPS; 1 mg/kg, i.p in five daily injections) or normal saline. Adult male mice were implanted with pellets releasing 17-ß estradiol (E2; 2.5 mg/pellet, 41.666 µg/day release) or placebo for 6 weeks and challenged with LPS or normal saline as above. In both experiments, mice were sacrificed 3 h after the last injection. Hippocampal markers of neurogenesis were assessed in vitro and in vivo by Western blot, real-time PCR, and immunohisto/cytochemistry. RESULTS: Gro1 induced premature senescence in NPC and HT-22 cells, activating senescence-associated ß-galactosidase and the cell cycle inhibitor p16 and suppressing neuroblast proliferation and expression of doublecortin (DCX) and neuron-specific class III beta-tubulin (Tuj-1), both neuroblast markers, while promoting proliferation of neural glial antigen 2 (Ng2)-positive oligodendrocytes. Gro1 overexpression in the hippocampus of newborn mice resulted in decreased neuroblast development, as evidenced by decreased DCX expression and increased expression of platelet-derived growth factor α receptor (PDGFαR), a marker of oligodendrocyte precursors. In adult mice, Gro1 was induced in response to LPS treatment in male but not in female hippocampus, with a subsequent decrease in neurogenesis and activation of oligodendrocyte progenitors. No changes in neurogenesis were observed in females. Treatment with E2 blunted LPS-induced Gro1 in the male hippocampus. CONCLUSIONS: Inflammation-induced Gro1 triggers neuroblast senescence, thus suppressing new neuron development in the hippocampus. Sex-dependent differences in Gro1 response are attributed to estradiol, which blunts these changes, protecting the female hippocampus from the deleterious effects of inflammation-induced Gro1 on neurogenesis.

Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNAc transporter.

Cryptococcus neoformans, an opportunistic fungal pathogen, produces a glycan capsule to evade the immune system during infection. This definitive virulence factor is composed mainly of complex polysaccharides, which are made in the secretory pathway by reactions that utilize activated nucleotide sugar precursors. Although the pathways that synthesize these precursors are known, the identity and the regulation of the nucleotide sugar transporters (NSTs) responsible for importing them into luminal organelles remain elusive. The UDP-galactose transporter, Ugt1, was initially identified by homology to known UGTs and glycan composition analysis of ugt1Δ mutants. However, sequence is an unreliable predictor of NST substrate specificity, cells may express multiple NSTs with overlapping specificities, and NSTs may transport multiple substrates. Determining NST activity thus requires biochemical demonstration of function. We showed that Ugt1 transports both UDP-galactose and UDP-N-acetylgalactosamine in vitro. Deletion of UGT1 resulted in growth and mating defects along with altered capsule and cellular morphology. The mutant was also phagocytosed more readily by macrophages than wild-type cells and cleared more quickly in vivo and in vitro, suggesting a mechanism for the lack of virulence observed in mouse models of infection.

Characterization of the dTDP-Fuc3N and dTDP-Qui3N biosynthetic pathways in Campylobacter jejuni 81116.

The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.

Improvement of betulinic acid biosynthesis in yeast employing multiple strategies.

BACKGROUND: Betulinic acid (BA) is a lupane-type triterpene which has been considered as a promising agent to cure melanoma with no side effects. Considering that BA is naturally produced in small quantities in plants, we previously reported the success in engineering its production in yeast. In the present study, we attempted to improve BA biosynthesis in yeast by the use of different strategies. RESULTS: We first isolated a gene encoding a lupeol C-28 oxidase (LO) from Betula platyphylla (designated as BPLO). BPLO showed a higher activity in BA biosynthesis compared to the previously reported LOs. In addition, two yeast platforms were compared for engineering the production of BA, which demonstrated that the WAT11 strain was better to host BA pathway than the CEN.PK strain. Based on the WAT11-chassiss, the Gal80p mutant was further constructed. The mutant produced 0.16 mg/L/OD600 of BA, which was 2.2 fold of that produced by the wild type strain (0.07 mg/L/OD600). CONCLUSIONS: This study reported our efforts to improve BA production in yeast employing multiple strategies, which included the identification of a novel LO enzyme with a higher activity in BA biosynthesis, the evaluation of two yeast strains for hosting the BA pathway, and the up-regulation of the expression of the BA pathway genes by managing yeast GAL gene regulon circuit.