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  • VHb expression in recombinant E coli expressing P H

    2019-07-05

    VHb expression in recombinant E. coli expressing P4H improved Hyp production, presumably by enhancing oxygen transfer. VHb expression contributed to improving growth and extending the period of exponential growth in both shaker flask culture and bioreactor fermentation. A similar phenomenon was also observed in poly-l-glutamic Temafloxacin hydrochloride production (18). VHb expression enhanced Hyp concentration and cell concentration, indicating that the TCA cycle was maintained by regenerating succinate from α-ketoglutarate coupled with conversion of proline to Hyp. Acetic acid is the major metabolite resulting from metabolic overflow in E. coli when oxygen is limited (39), and VHb expression was able to reduce the concentration of acetic acid in the present study, suggesting the VHb-mediated improvement of oxygen transfer. Meanwhile, probably because VHb can deliver oxygen to terminal oxidases in the cells\' metabolic pathways 11, 12, 13, the conversion rate from glucose to Hyp was also increased in the strain expressing VHb by improving the TCA cycle. These results illustrate that VHb expression helps the TCA cycle to match up with the glycolysis pathway in E. coli WD3 (pTrc99a-p4h).
    Acknowledgments We gratefully thank Professor Huimin Yu of Tsinghua University for kindly providing the pRW plasmid containing vgb.
    Introduction Collagens, the most abundant proteins in animals, consists a family of 28 protein types encoded by 46 genes in the human genome [1]. The hallmark of collagens is the domain of the same name that features repeats of the three amino acids motif Gly-Xaa-Yaa, where Yaa is often occupied by proline. A typical collagen polypeptide includes hundreds of this motif, which confers a left-handed helical conformation to the collagen domain. After translation, three collagen polypeptides combine to form a right-handed triple helical coil stabilized through hydrogen bonds between the three subunits. Before formation of the triple helix, collagen subunits become extensively modified by hydroxylation of proline and lysine, and by glycosylation of selected hydroxylysine (Hyl) residues. The hydroxylation of proline increases the thermal stability of collagen, which already partially denatures at body temperature [2]. The functional significance of lysyl hydroxylation and Hyl glycosylation is less distinct, although these modifications are essential for collagen integrity and hence animal viability. In addition to hydroxylation and glycosylation, collagens undergo further modifications upon secretion into the extracellular matrix (Figure 1). The N-terminal and C-terminal propeptide regions are cleaved by dedicated proteinases [3], thereby promoting the assembly of collagen fibrils. These fibrils then undergo intramolecular and intermolecular cross-links after oxidation of selected lysine and Hyl side chains to aldehydes by the copper-dependent lysyl oxidase enzyme. Hydroxylation and glycosylation take place in the endoplasmic reticulum, before the formation of the collagen triple helix, whereas procollagen propeptide cleavage and cross-linking occur in the extracellular space.
    Structure The presence of glycans on collagens has been first described in 1935, when Grassmann and Schleich assigned up to 1% of the mass of collagen to carbohydrates [4]. Twenty years later, these carbohydrates were determined to be bound to the polypeptide chains through O-glycosidic linkages [5]. In the late 1960s, Robert and Mary Jane Spiro determined the structure of the collagen O-glycan [] and characterized the glycosyltransferase enzymes involved in its biosynthesis [7,8]. In their seminal work, they described the O-glycan as a simple disaccharide of glucose and galactose linked to Hyl. Despite its simplicity, the Glc(α1-2)Gal(β1-O) sequence (Figure 2) is unique among glycoconjugates. The collagen disaccharide is also remarkable in respect to its strict conservation among animals expressing collagens, from sponges to humans. The extent of glycosylation varies according to the types of collagen, with collagen type IV being more extensively glycosylated than collagen type II and collagen type I. In general, glycosylation of Hyl is more prominent in basement membrane collagens than in fibrillar collagens. The extent of collagen glycosylation probably depends on the speed of collagen folding, the expression levels of glycosyltransferases, and UDP-Gal availability in the ER compartment. Collagen type IV is the main constituent of basement membranes. It does not form fibrils but oligomerizes at its N-terminus with other collagen IV molecules for form tetramers and at its C-terminus to form dimers. Mouse collagen type IV isolated from Engelbreth-Holm-Swarm tumor cells was shown to contain 39 glycosylated Hyl residues, whereas additional 10 Hyl were detected without glycans [9]. Its C-terminus, which initiates the helical polymerization process, lacks post-translational modifications.