Functional display of Pseudomonas and Burkholderia lipases using a translocator domain of EstA autotransporter on the cell surface of Escherichia coli.

Functional expression of the industrially important Pseudomonas and Burkholderia lipases, such as those from P. aeruginosa, B. cepacia and P.

Enhancement of the efficiency of secretion of heterologous lipase in Escherichia coli by directed evolution of the ABC transporter system.

The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay.

Use of Pseudomonas putida EstA as an anchoring motif for display of a periplasmic enzyme on the surface of Escherichia coli.

The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme beta-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain.

Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast.

The Schizosaccharomyces pombe pfh1+ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1+ gene suppress a temperature-sensitive allele of cdc24+, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism.

Cloning and functional characterization of the SUR2/SYR2 gene encoding sphinganine hydroxylase in Pichia ciferrii.

Saccharomyces cerevisiae sphinganine C4-hydroxylase encoded by the SUR2 gene catalyses the conversion of sphinganine to phytosphingosine. We isolated the SUR2 gene from Pichia ciferrii using nucleotide sequence homology to S. cerevisiae SUR2 to study hydroxylation of sphinganine in the sphingoid base overproducing yeast P.

Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast Pichia ciferrii.

We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P.

Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method: changing from a substrate specificity of phospholipase to that of lipase.

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer.