Yeast surface display involves that the exogenous protein, which was fused with the yeast outer shell cell wall protein, was genetically anchored on the yeast cell surface. It has been widely used in expression and screening of functional protein. Here, we focused on the construction of lipase-displaying systems and its application in enzymatic biosynthesis, such as fatty acid methyl esters, short-chain flavour esters and sugar esters applications, and so on.
Candida ; enzymology ; genetics ; Lipase ; biosynthesis ; genetics ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Solvents ; Yeasts ; enzymology ; genetics

D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.
Bacterial Proteins ; biosynthesis ; genetics ; Carrier Proteins ; biosynthesis ; genetics ; D-Amino-Acid Oxidase ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Maltose-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Truncated Hemoglobins ; biosynthesis ; genetics ; Yeasts ; enzymology ; genetics

Cytochrome P450 (CYP450) is a key element in the Ganoderma triterpenoid biosynthetic pathway. The catalytic reaction process for CYP450 requires NADPH / NADH for electron transfer. After searching the genome dataset of Ganoderma lucidum, the unique sequence encoding CYP450 and NADPH were discovered, separately. The open reading frames of GLCYP450 and GLNADPH were cloned separately using RT-PCR strategy from G lucidum. The appropriate restriction enzyme cutting sites were introduced at the 5' and 3' ends of gene sequence. The genes of GLCYP450 and GLNADPH were recombined into the yeast expression vector pESC-URA, leading to the formation of the yeast expression plasmid pESC-GLNADPH-GLCYP450. This study provides a foundation for researching Ganoderma triterpene biosynthesis using the approach of synthetic biology.
Amino Acid Sequence ; Cloning, Molecular ; Cytochrome P-450 Enzyme System ; genetics ; DNA, Complementary ; genetics ; metabolism ; Gene Expression Regulation, Fungal ; Genetic Vectors ; NADP ; genetics ; Open Reading Frames ; Plasmids ; Reishi ; enzymology ; genetics ; metabolism ; Synthetic Biology ; Triterpenes ; metabolism ; Yeasts ; genetics ; metabolism

The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions. Copyright 2000 Academic Press.
Amine Oxidoreductases/metabolism* ; Amine Oxidoreductases/genetics ; Amino Acid Motifs ; Amino Acid Sequence ; Escherichia coli/genetics ; Fungal Proteins/metabolism* ; Fungal Proteins/genetics ; Human ; Molecular Sequence Data ; NAD/metabolism ; Phosphorylation ; Promoter Regions (Genetics) ; Protein Kinase C/metabolism* ; Recombinant Proteins/metabolism ; Recombinant Proteins/genetics ; Sequence Deletion ; Sequence Homology, Amino Acid ; Threonine/metabolism ; Yeasts/enzymology