Construction of Genetically Engineered Noval Cellulolytic Yeast Strain for Single Step conversion of Biomass Production on Saline Land for Ethanol

Abstract

Summary Genes for B-glucose (Bgl) isolated from a genomic library of the cellulolytic bacterium, Cellulomonas biazotan, were cloned in pUC16 in its Sac 1cloning site and transformed to E.coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, liquid culture and on native polyacrylamide gel electrophorese (PAGE) activity gels. They fell into three distinct groups. Three representatives E.coli clones carried recombinant plasmids designd pRM 54, pMR 1and pRM 17. The genes were located on 5.6-, 3.7 and 1.84-kb fragments. Their location was obtained by deletion analysis which revealed that 5.5. 3.2 And 1.8 intrcellular production of B- glucosides in the three clones. Secretion occurred into the periplasmic fractions. The E.coli recombinant’s shoed higher substrate uptake (0.442+- 0.15 g/l/h) cell mass biosynthesis rate (o.23+- 0.013 g cells /l/lh) and products formation rate 8.83 IU/l/h) compared with donor (0.25, 0.235 and 6.2 respective values. Three inserts carrying bgl genes from representative recombinant E.coli were isolated with sac 1, ligated in the shuttle vector pYES 2.o in its Sac site and transformed to E.coli and S.cerevisiae. The recombinant plasmids were redesigned pRPG1, pRPG2 and pGR3 coding for BglAl, Bg1A1 and Bg1C! the recombinant plasmids were redesigned pRPG1 and PRPG2 and pPg3 coding for bg1A1, Bg1B1 and Bg1C1. The cloned genes conferred extracellular production of B- glycosidase on S. cerevisiae. And enabled it to grow on cellobiose and salicin. Gal 1- promotor of shuttle vector pYES 2.0 enabled the organism to produce two-fold greater B glucosodase than that supported by Lac Z-premotor of PUC 18 plasmid in E.coli. These E.coli recombinants showed higher substrate uptake rate (0.442+-0.015 g/1/h) cell mas biosynthesis rate (0.231=- 0.013 gcells/1/h) and product formation rate ( compared with donor). Gal1- premotor of shuttle vector enabled E.coli recombinants (18.17=-4.1Lu/1/h) and yeast recombinants organism (13.02=-6.89IUI/1/h) to producer 2.13- and 1.5 fold greater B- glycosidase respectively than that supported by lac promoter of pUC 18 plasmid in wild strains of S. cerevisae. The enzymes produced by bgl+ yeast and E.coli recombinants resemble that of the donor with respect to tehtempertaure and PH requirements for the maximum activity. Other enzymes properties of the B glucosudes from S.cerevivise were substantially the same as those from C. biazotea.