Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

Abstract

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.

Codon Optimization Increases Steady-State mRNA Levels in Aspergillus oryzae Heterologous Gene Expression

“…Using the native Der f 7 gene and the synthesized, codon-optimized Der f 7 gene, we constructed Der f 7 expression vectors pDer/ntv and pDer/opt. These contained the Der f 7 gene that was inserted downstream of the improved glaA promoter (46). In addition, we constructed the expression vectors pGlaDer/ntv (for native Der f 7) and pGlaDer/opt (for codon-optimized Der f 7) to evaluate the effect of codon optimization in the fusion construct.…”

“…Der f 7 without a signal sequence was fused to the catalytic domain of A. oryzae GlaA at the N terminus through a KexB cleavage site. Both types of expression vectors were constructed based on a fungal highlevel-expression vector, pNGA142 (46).…”

“…The Der f 7 gene with native codons and an original signal sequence was amplified by PCR on pGEX-Der f 7 (a gift from S. Kawamoto, Hiroshima University), using primers 5Ј-CCCGTCGACATGAT GAAATTTTTGTTGAT-3Ј (the SalI restriction site is underlined) and 5Ј-CCC TCTAGATTAATTTTTTTCCAATTCAC-3Ј (the XbaI restriction site is underlined). An amplified fragment was inserted between the improved glaA promoter and the agdA terminator of an expression vector, pNGA142 (46), which contains the niaD gene as a selectable marker, yielding pDer/ntv. The Der f 7 gene fragment with optimized codons, obtained by digestion of pTDopt with XbaI and SalI, was also cloned into XbaI/SalI-digested pNGA142, yielding pDer/opt.…”

Biodiesel: visão crítica do status atual e perspectivas na academia e na indústria

“…188 os resultados descritos até o presente mostram interessantes potencialidades de uso, em função da facilidade de recuperação e reuso, a baixas temperaturas requeridas, condições reacionais suaves com baixo consumo energético e pH não agressivo. Porém, é necessário realizar uma série de estudos que resultem na melhoria da velocidade de reação, reduzindo o tempo de transesterificação e fornecendo os produtos em rendimentos mais altos, com transformações mais eficientes, de forma a possibilitar aplicações industriais práticas e competitivas.…”

ENZYMATIC GLYCEROLYSIS OF METHYL LAURATE UTILIZING Candida antarctica Lipase b

“…Aspergillus oryzae is a filamentous fungus that has ability to secrete large amounts of hydrolytic enzymes [15]. It is used as a host because it has high protein productivity in the expression of heterogeneous gene using improved promoters [16].…”