Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

Abstract

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications.

Optimization of chondroitin production inE. coliusing genome scale models

“…The OD530nm was then read in a microplate reader. Protein expression in cultured E. coli K-12 MG1655 (DE3) carrying pETDuet-1, pETDuet_glmU, pETDuet_sodA, pCDFDuet-1, or pCDFDuet_mltB, was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 42 (4% stacking gel and 12% running gel). Samples (soluble and insoluble fractions of lysates) were mixed with 2x Laemmli Sample Buffer (65.8 mM Tris-HCl pH 6.8, 2.1% SDS, 26.3% glycerol, 0.01% bromophenol blue and 5% β-mercaptoethanol, from Fisher Scientific, JMGS, Sigma-Aldrich and AppliChem, respectively) and denatured at 95°C for 5 min.…”

“…The genes kfoA and kfoC (encoding UAE and CHSY, respectively) present in the pETM6 plasmid, were kindly provided by Dr. Matheos Koffas (Rensselaer Polytechnic Institute, Troy, NY) 32 and were cloned in pRSFDuet-1 plasmid (Novagen, Madison, USA) in multiple cloning site 1 (MCS1). Also, since the UDP-glucose dehydrogenase (UGD) overexpression has been determined to be crucial for glycosaminoglycan production [39][40][41][42] , Zymomonas mobilis UGD gene (Zmugd) 42 was also cloned in the same plasmid in MCS2. The three genes were cloned in pseudooperon configuration, i.e., the DNA sequence of each gene follows its own lac operator, T7 promoter and RBS, and a single T7 terminator exists in the end of all genes.…”

Optimization of chondroitin production in E. coli using genome scale models

“…42 The genes kfoA and kfoC (encoding UAE and CHSY, respectively) present in the pETM6 plasmid were kindly provided by Dr. Matheos Koffas (Rensselaer Polytechnic Institute, Troy, NY) 36 and were cloned in pRSFDuet-1 plasmid (Novagen, Madison, USA) in multiple cloning site 1 (MCS1). Also, since the UGD overexpression has been determined to be crucial for glycosaminoglycan production, [43][44][45][46] Zymomonas mobilis UGD gene (Zmugd) 46 was also cloned in the same plasmid in MCS2. The three genes were cloned in pseudo-operon configuration, i.e., the DNA sequence of each gene follows its own lac operator, T7 promoter and RBS, and a single T7 terminator exists in the end of all genes.…”

Synthetic Genetic Elements, Devices, and Systems

“…The step catalyzed by UGD is considered to be the limiting factor in the heterologous production of glucosaminoglycans. Couto et al have characterized three new UGDs and propose new enzyme options for industrial use [ 10 ].…”