A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5'-/3'-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST-CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 microg recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150-200 microg/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.
The capsule polysaccharide (CPS) of Escherichia coli K4 (K4CPS) is identical to fructosylated chondroitin, which can be modified to chondroitin sulfate, a commercially valuable biopolymer commonly used in pharmaceutical applications. In this study, we homologously overexpressed the transcriptional regulator SlyA to enhance the expression of K4 capsule gene cluster and production of CPS. The iTRAQ quantificaton of proteomics revealed 77 up-regulated proteins and 143 down-regulated proteins in E. coli THslyA. Most enzymes of glycolysis and citrate cycle pathway were weakened, while proteins associated with K4CPS synthesis were up-regulated, showing a shift of carbon flux from cell growth to K4CPS production. Further, the production of K4CPS by the recombinant strain was 1 and 2.6 g/L in a shake flask and 7-L batch bioreactor, which was 1.85- and 1.53-fold higher than that of the wild-type strain, respectively. Thus, this study provides a viable strategy for improving the production of K4CPS through a transcriptional-level manipulation.
Escherichia coli K4 synthesizes a capsular polysaccharide (CPS) consisting of a fructose-branched chondroitin (GalNAc-GlcA(fructose)n), which is a biosynthetic precursor of chondroitin sulfate. Here, the role of kfoE in the modification of the chondroitin backbone was investigated using knock-out and recombinant complementation experiments. kfoE disruption and complementation had no significant effect on cell growth. CPS production was increased by 15 % in the knock-out strain, and decreased by 21 % in the knock-out strain complemented with recombinant kfoE. CPS extracted from the knock-out strain was chondroitin, whereas CPS extracted from the complemented strain was a fructose-branched chondroitin. The results demonstrated that the kfoE gene product altered the fructose group at the C3 position of the GlcA residue during production of K4CPS.
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