Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter

The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-side-out Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi. In vitro assays using a Golgi-enriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1- MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed in S. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.     

Cytosolic invertases contribute to cellulose biosynthesis and influence carbon partitioning in seedlings of Arabidopsis thaliana

In plants, UDP‐glucose is the direct precursor for cellulose biosynthesis, and can be converted into other NDP‐sugars required for the biosynthesis of wall matrix polysaccharides. UDP‐glucose is generated from sucrose by two distinct metabolic pathways. The first pathway is the direct conversion of sucrose to UDP‐glucose and fructose by sucrose synthase. The second pathway involves sucrose hydrolysis by cytosolic invertase (CINV), conversion of glucose to glucose‐6‐phosphate and glucose‐1‐phosphate, and UDP‐glucose generation by UDP‐glucose pyrophosphorylase (UGP). Previously, Barratt et al. (Proc. Natl Acad. Sci. USA, 106, 2009 and 13124) have found that an Arabidopsis double mutant lacking CINV1 and CINV2 displayed drastically reduced growth. Whether this reduced growth is due to deficient cell wall production caused by limited UDP‐glucose supply, pleiotropic effects, or both, remained unresolved. Here, we present results indicating that the CINV/UGP pathway contributes to anisotropic growth and cellulose biosynthesis in Arabidopsis. Biochemical and imaging data demonstrate that cinv1 cinv2 seedlings are deficient in UDP‐glucose production, exhibit abnormal cellulose biosynthesis and microtubule properties, and have altered cellulose organization without substantial changes to matrix polysaccharide composition, suggesting that the CINV/UGP pathway is a key metabolic route to UDP‐glucose synthesis in Arabidopsis. Furthermore, differential responses of cinv1 cinv2 seedlings to exogenous sugar supplementation support a function of CINVs in influencing carbon partitioning in Arabidopsis. From these data and those of previous studies, we conclude that CINVs serve central roles in cellulose biosynthesis and carbon allocation in Arabidopsis.     

Genetic alteration of UDP‐rhamnose metabolism in Botrytis cinerea leads to the accumulation of UDP‐KDG that adversely affects development and pathogenicity

Botrytis cinerea is a model plant‐pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)‐glucose, the major fungal wall nucleotide sugar precursor, to UDP‐rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose‐containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP‐rhamnose pathway intermediate UDP‐4‐keto‐6‐deoxy‐glucose (UDP‐KDG) in hyphae of the Δbcer strain. UDP‐KDG could not be detected in hyphae of the wild‐type strain, indicating fast conversion to UDP‐rhamnose by the BcEr enzyme. The correlation between high UDP‐KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP‐KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP‐KDG may lead to the development of new antifungal drugs.     

Two nucleotide sugar transporters are important for cell wall integrity and full virulence of Magnaporthe oryzae

Cell wall polysaccharides play key roles in fungal development, virulence, and resistance to the plant immune system, and are synthesized from many nucleotide sugars in the endoplasmic reticulum (ER)-Golgi secretory system. Nucleotide sugar transporters (NSTs) are responsible for transporting cytosolic-derived nucleotide sugars to the ER lumen for processing, but their roles in plant-pathogenic fungi remain to be revealed. Here, we identified two important NSTs, NST1 and NST2, in the rice blast fungus Magnaporthe oryzae. Both NSTs were localized in the ER, which was consistent with a function in transporting nucleotide sugar for processing in the ER. Sugar transport property analysis suggested that NST1 is involved in transportation of mannose and glucose, while NST2 is only responsible for mannose transportation. Accordingly, deletion of NSTs resulted in a significant decrease in corresponding soluble saccharides abundance and defect in sugar utilization. Moreover, both NSTs played important roles in cell wall integrity, were involved in asexual development, and were required for full virulence. The NST mutants exhibited decreasing external glycoproteins and exposure of inner chitin, which resulted in activation of the host defence response. Altogether, our results revealed that two sugar transporters are required for fungal cell wall polysaccharides accumulation and full virulence of M. oryzae.     

Identification of a residue responsible for UDP‐sugar donor selectivity of a dihydroxybenzoic acid glycosyltransferase from Arabidopsis natural accessions

UDP‐glycosyltransferase (UGT) plays a major role in the diversity and reactivity of plant specialized metabolites by catalyzing the transfer of the sugar moiety from activated UDP‐sugars to various acceptors. Arabidopsis UGT89A2 was previously identified from a genome‐wide association study as a key factor that affects the differential accumulation of dihydroxybenzoic acid (DHBA) glycosides in distinct Arabidopsis natural accessions, including Col‐0 and C24. The in vitro enzyme assays indicate that these distinct metabolic phenotypes reflect the divergence of UGT89A2 enzyme properties in the Col‐0 and C24 accessions. UGT89A2 from Col‐0 is highly selective toward UDP‐xylose as the sugar donor, and the isoform from C24 can utilize both UDP‐glucose and UDP‐xylose but with a higher affinity to the glucose donor. The sequences of the two isozymes only differ at six amino acid residues. Examination of these amino acid residues in more natural accessions revealed a strong correlation between the amino acid polymorphism at position 153 and the DHBA glycoside accumulation pattern. Site‐directed mutagenesis that swapped residue 153 between UGT89A2 from Col‐0 and C24 reversed the UDP‐sugar preferences, indicating that residue 153 plays an important role in determining sugar donor specificity of UGT89A2. This study provides insight into the key amino acid changes that confer sugar donor selectivity on UGTs, and demonstrates the usefulness of natural variation in understanding the structure–function relationship of enzymes involved in specialized metabolism.     

Genome-wide annotation and functional identification of aphid GLUT-like sugar transporters

Background

Phloem feeding insects, such as aphids, feed almost continuously on plant phloem sap, a liquid diet that contains high concentrations of sucrose (a disaccharide comprising of glucose and fructose). To access the available carbon, aphids hydrolyze sucrose in the gut lumen and transport its constituent monosaccharides, glucose and fructose. Although sugar transport plays a critical role in aphid nutrition, the molecular basis of sugar transport in aphids, and more generally across all insects, remains poorly characterized. Here, using the latest release of the pea aphid, Acyrthosiphon pisum, genome we provide an updated gene annotation and expression profile of putative sugar transporters. Finally, gut expressed sugar transporters are functionally expressed in yeast and screened for glucose and fructose transport activity.

Results

In this study, using a de novo approach, we identified 19 sugar porter (SP) family transporters in the A. pisum genome. Gene expression analysis, based on 214, 834 A. pisum expressed sequence tags, supports 17 sugar porter family transporters being actively expressed in adult female aphids. Further analysis, using quantitative PCR identifies 4 transporters, A. pisum sugar transporter 1, 3, 4 and 9 (ApST1, ApST3, ApST4 and ApST9) as highly expressed and/or enriched in gut tissue. When expressed in a Saccharomyces cerevisiae hexose transporter deletion mutant (strain EBY.VW4000), only ApST3 (previously characterized) and ApST4 (reported here) transport glucose and fructose resulting in functional rescue of the yeast mutant. Here we characterize ApST4, a 491 amino acid protein, with 12 predicted transmembrane regions, as a facilitative glucose/fructose transporter. Finally, phylogenetic reconstruction reveals that ApST4, and related, as yet uncharacterized insect transporters are phylogenetically closely related to human GLUT (SLC2A) class I facilitative glucose/fructose transporters.

Conclusions

The gut enhanced expression of ApST4, and the transport specificity of its product is consistent with ApST4 functioning as a gut glucose/fructose transporter. Here, we hypothesize that both ApST3 (reported previously) and ApST4 (reported here) function at the gut interface to import glucose and fructose from the gut lumen.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-647) contains supplementary material, which is available to authorized users.     

Segregation of the Qb‐SNAREs GS27 and GS28 into Golgi Vesicles Regulates Intra‐Golgi Transport

The Golgi apparatus is the main glycosylation and sorting station along the secretory pathway. Its structure includes the Golgi vesicles, which are depleted of anterograde cargo, and also of at least some Golgi‐resident proteins. The role of Golgi vesicles remains unclear. Here, we show that Golgi vesicles are enriched in the Qb‐SNAREs GS27 (membrin) and GS28 (GOS‐28), and depleted of nucleotide sugar transporters. A block of intra‐Golgi transport leads to accumulation of Golgi vesicles and partitioning of GS27 and GS28 into these vesicles. Conversely, active intra‐Golgi transport induces fusion of these vesicles with the Golgi cisternae, delivering GS27 and GS28 to these cisternae. In an in vitro assay based on a donor compartment that lacks UDP‐galactose translocase (a sugar transporter), the segregation of Golgi vesicles from isolated Golgi membranes inhibits intra‐Golgi transport; re‐addition of isolated Golgi vesicles devoid of UDP‐galactose translocase obtained from normal cells restores intra‐Golgi transport. We conclude that this activity is due to the presence of GS27 and GS28 in the Golgi vesicles, rather than the sugar transporter. Furthermore, there is an inverse correlation between the number of Golgi vesicles and the number of inter‐cisternal connections under different experimental conditions. Finally, a rapid block of the formation of vesicles via COPI through degradation of ϵCOP accelerates the cis‐to‐trans delivery of VSVG. These data suggest that Golgi vesicles, presumably with COPI, serve to inhibit intra‐Golgi transport by the extraction of GS27 and GS28 from the Golgi cisternae, which blocks the formation of inter‐cisternal connections .     

Completion of the canonical pathway for assembly of anticancer drugs vincristine/vinblastine in Catharanthus roseus

Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP‐β‐l ‐rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDP‐β‐l ‐rhamnose. The mutant H357Q exhibited activity with both UDP‐β‐l ‐rhamnose and UDP‐glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor‐His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism.     

UDP‐sugar pyrophosphorylase is essential for arabinose and xylose recycling,and is required during vegetative and reproductive growth in Arabidopsis

Numerous nucleotide sugars are needed in plants to synthesize cell wall polymers and glycoproteins. The de novo synthesis of nucleotide sugars is of major importance. During growth, however, some polymers are broken down to monosaccharides. Reactivation of these sugars into nucleotide sugars occurs in two steps: first, by a substrate‐specific sugar‐1‐kinase and, second, by UDP‐sugar‐pyrophosphorylase (USP), which has broad substrate specificity. A knock‐out of the USP gene results in non‐fertile pollen. By using various genetic complementation approaches we obtained a strong (>95%) knock‐down line in USP that allowed us to investigate the physiological role of the enzyme during the life cycle. Mutant plants show an arabinose reduction in the cell wall, and accumulate mainly two sugars, arabinose and xylose, in the cytoplasm. The arabinogalactanproteins in usp mutants show no significant reduction in size. USP is also part of the myo‐inositol oxygenation pathway to UDP‐glucuronic acid; however, free glucuronic acid does not accumulate in cells, suggesting alternative conversion pathways of this monosaccharide. The knock‐down plants are mostly sterile because of the improper formation of anthers and pollen sacks.     

A flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase responsible for terminal modification of pollen‐specific flavonols in Arabidopsis thaliana

Flavonol 3‐O‐diglucosides with a 1→2 inter‐glycosidic linkage are representative pollen‐specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild‐type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3‐O‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild‐type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP‐glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen‐specific flavonol structure. Kaempferol and quercetin 3‐O‐glucosyl‐(1→2)‐glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild‐type plants. Recombinant UGT79B6 protein converted kaempferol 3‐O‐glucoside to kaempferol 3‐O‐glucosyl‐(1→2)‐glucoside. UGT79B6 recognized 3‐O‐glucosylated/galactosylated anthocyanins/flavonols but not 3,5‐ or 3,7‐diglycosylated flavonoids, and prefers UDP‐glucose, indicating that UGT79B6 encodes flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase. A UGT79B6‐GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.     

<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1

Transport of nucleotide-sugars across the Golgi membrane is required for the lumenal synthesis of a variety of essential cell surface components, and is mediated by nucleotide sugar transporters (NSTs) which are members of the large drug/metabolite superfamily of transporters. Despite the importance of these proteins in plants, so far only two have been described, GONST1 and AtUTr1 from Arabidopsis thaliana. In this work, our aim was to identify further Golgi nucleotide-sugar transporters from Arabidopsis. On the basis of their sequence similarity to GONST1, we found four additional proteins, which we named GONST2, 3, 4 and 5. These putative NSTs were grouped into three clades: GONST2 with GONST1; GONST3 with GONST4; and GONST5 with six further uncharacterized proteins. Transient expression in tobacco cells of a member of each clade, fused to the Green Fluorescent Protein (GFP), suggested that all these putative NSTs are localised in the Golgi. To obtain evidence for nucleotide sugar transport activity, we expressed these proteins, together with the previously characterised GONST1, in a GDP-mannose transport-defective yeast mutant ( vrg4-2). We tested the transformants for rescue of two phenotypes associated with this mutation: sensitivity to hygromycin B and reduced glycosylation of extracellular chitinase. GONST1 and GONST2 complemented both phenotypes, indicating that GONST2, like the previously characterized GONST1, is a GDP-mannose transporter. GONST3, 4 and 5 also rescued the antibiotic sensitivity, but not the chitinase glycosylation defect, suggesting that they can also transport GDP-mannose across the yeast Golgi membrane but with a lower efficiency. RT-PCR and analysis of Affymetrix data revealed partially overlapping patterns of expression of GONST1–5 in a variety of organs. Because of the differences in ability to rescue the vrg4 -2 phenotype, and the different expression patterns in plant organs, we speculate that GONST1 and GONST2 are both GDP-mannose transporters, whereas GONST3, GONST4 and GONST5 may transport other nucleotide-sugars in planta.Communicated by G. JürgensThe first two authors contributed equally to this work     

The <Emphasis Type="Italic">Vitis vinifera</Emphasis> sugar transporter gene family: phylogenetic overview and macroarray expression profiling

Background  

In higher plants, sugars are not only nutrients but also important signal molecules. They are distributed through the plant via sugar transporters, which are involved not only in sugar long-distance transport via the loading and the unloading of the conducting complex, but also in sugar allocation into source and sink cells. The availability of the recently released grapevine genome sequence offers the opportunity to identify sucrose and monosaccharide transporter gene families in a woody species and to compare them with those of the herbaceous Arabidopsis thaliana using a phylogenetic analysis.     

SIS8, a putative mitogen‐activated protein kinase kinase kinase,regulates sugar‐resistant seedling development in Arabidopsis

Sugar signaling pathways have been evolutionarily conserved among eukaryotes and are postulated to help regulate plant growth, development and responses to environmental cues. Forward genetic screens have identified sugar signaling or response mutants. Here we report the identification and characterization of Arabidopsis thaliana sugar insensitive8 (sis8) mutants, which display a sugar‐resistant seedling development phenotype. Unlike many other sugar insensitive mutants, sis8 mutants exhibit wild‐type responses to the inhibitory effects of abscisic acid and paclobutrazol (an inhibitor of gibberellin biosynthesis) on seed germination. Positional cloning of the SIS8 gene revealed that it encodes a putative mitogen‐activated protein kinase kinase kinase (MAPKKK; At1g73660). SIS8mRNA is expressed ubiquitously among Arabidopsis organs. A UDP‐glucosyltransferase, UGT72E1 (At3g50740), was identified as an interacting partner of SIS8 based on a yeast two‐hybrid screen and in planta bimolecular fluorescence complementation. Both SIS8–yellow fluorescent protein (YFP) and UGT72E1–YFP fusion proteins localize to the nucleus when transiently expressed in tobacco leaf cells. T‐DNA insertions in At3g50740 cause a sugar‐insensitive phenotype. These results indicate that SIS8, a putative MAPKKK, is a regulator of sugar response in Arabidopsis and interacts with a UDP‐glucosyltransferase in the nucleus.     

NDP-sugars, floridoside and floridean starch levels in relation to activities of UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase in Solieria chorda l is (Rhodophyceae) under experimental conditions

Under limited nutrient availability (i.e. unenriched sea‐water) and under 75 mol photons m^–2 s^–1 irradiance 12:12 LD, thalli of Solieria chordalis J. Agardh accumulated floridean starch and floridoside. When they were transferred into nutrient‐enriched seawater (150 umol L^?1 NO3¹‐ and 7 umol L^?1 P0₄³i at 35 umol photons m^?2 s^?1 in irradiance 12:12 LD, starch and floridoside levels decreased. The main nucleotide diphosphate (NDP) sugars (i.e. UDP‐glucose, UDP‐galactose and ADP‐glucose) and the activities of UDP‐glucose pyrophosphorylase [Enzyme Code (EC) 2.7.7.9] and UDP‐glucose‐4‐epimerase (EC 5.1.3.2) were measured under these controlled culture conditions. Both UDP‐glucose and UDP‐galactose in the thal l i increased under conditions known to favor the accumulation of floridean starch and floridoside, whereas they decreased under conditions leading to floridean starch and floridoside breakdown. On the other hand, ADP‐glucose level only varied slightly. Although UDP‐glucose pyrophosphorylase activity rose under conditions of floridean starch synthesis, little variation was observed in UDP‐glucose‐4‐epimerase activity. These results suggest a possible enzymatic regulation of the NDP‐sugar and carbohydrate pool in which UDP‐glucose pyrophosphorylase would play a major role.     

BRITTLE PLANT1 is required for normal cell wall composition and mechanical strength in rice

A series of nucleotide sugar interconversion enzymes (NSEs) generate the activated sugar donors required for biosynthesis of cell wall matrix polysaccharides and glycoproteins. UDP‐glucose 4‐epimerases (UGEs) are NSEs that function in the interconversion of UDP‐glucose (UDP‐Glc) and UDP‐galactose (UDP‐Gal). The roles of UDP‐glucose 4‐epimerases in monocots remain unclear due to redundancy in the pathways. Here, we report a brittle plant (bp1) rice mutant that exhibits brittle leaves and culms at all growth stages. The mutant culms had reduced levels of rhamnogalacturonan I, homogalacturonan, and arabinogalactan proteins. Moreover, the mutant had altered contents of uronic acids, neutral noncellulosic monosaccharides, and cellulose. Map‐based cloning demonstrated that OsBP1 encodes a UDP‐glucose 4‐epimerase (OsUGE2), a cytosolic protein. We also show that BP1 can form homo‐ and hetero‐protein complexes with other UGE family members and with UDP‐galactose transporters 2 (OsUGT2) and 3 (OsUGT3), which may facilitate the channeling of Gal to polysaccharides and proteoglycans. Our results demonstrate that BP1 participates in regulating the sugar composition and structure of rice cell walls.     

Role of α-phosphoglucomutase and phosphoglucose isomerase activities at the branching point between sugar catabolism and anabolism in Lactobacillus casei

Aims: To evaluate the role of α‐phosphoglucomutase (α‐Pgm) and phosphoglucose isomerase (Pgi) activities in growth rate, sugar‐phosphates, UDP‐sugars and lactate biosynthesis in Lactobacillus casei. Methods and Results: The pgm and pgi genes coding for α‐Pgm and Pgi activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous overexpression. In MRS fermentation medium with glucose, overexpression of pgm gene in L. casei resulted in a growth rate reduced to 75% and glucose‐6P levels reduced to 47%. By contrast, with lactose, the growth rate was raised to 119%. An increment of α‐Pgm activity had no significant effect on UDP‐sugar levels. Remarkably, Pgi overexpression in L. casei grown in lactose or galactose resulted in almost a double growth rate with respect to the control strain. The increased Pgi activity also resulted in glucose‐6P levels reduced to 25 and 59% of control strain cultured in glucose and lactose, respectively, and the fructose‐6P levels were increased to 128% on glucose. UDP‐glucose and UDP‐galactose levels were reduced to 66 and 55%, respectively, of control strain levels cultured in galactose. In addition, the lactate yield increased to 115% in the strain overproducing Pgi grown in galactose. Conclusions: The physiological amount of α‐Pgm and Pgi activities is limited for L. casei growth on lactose, and lactose and galactose, respectively, and that limitation was overcome by pgm and pgi gene overexpression. The increment of α‐Pgm and Pgi activities, respectively, resulted in modified levels of sugar‐phosphates, sugar‐nucleotides and lactate showing the modulation capacity of the carbon fluxes in L. casei at the level of the glycolytic intermediate glucose‐6P. Significance and Impact of the Study: Knowledge of the role of key enzymes in metabolic fluxes at the branching point between anabolic and catabolic pathways would allow a rational design of engineering strategies in L. casei.