Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

Abstract

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.

Engineering lipase B from Candida antarctica

“…26 Library construction could also be assisted by more efficient transformation techniques such as in vivo homologous recombination. 27 While new methods are important for constructing new and more diverse libraries, the research focus should remain on how to improve the functional properties of proteins in general and lipases such as CAL-B in particular. Three selected examples for such functional challenges will be discussed in more detail: (a) the expansion of lipase substrate specificity to tertiary alcohols; (b) the improvement of enantioselectivity, and (c) the variation of enantio-preference within the a/b-hydrolase framework.…”

Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production

“…Nevertheless, the HARS sequences do not guarantee stability for circular plasmids. Thus, the prolonged incubation in selective medium forces the plasmid integration usually in the respective subtelomeric locus (Kim et al, 2003). Hence, the integrative plasmids are most suitable for genetic manipulation of H. polymorpha .…”

“…The integrative plasmid pFPMT-MFα containing the coding region for the 1–34 fragment of PTH fused with MFα signal was inserted into Δ yps7 strain (deficient protease YPS7). The FMD promoter controlled the production of PTH, and the plasmid carried a HARS sequence for genomic integration (Kim et al, 2003). Positive clones for PTH production were selected for medium optimization on a micro-plate scale.…”

New yeast expression platforms based on methylotrophic and and on dimorphic and – A comparison

“…In H. polymorpha , host strains are either derived from a CBS4732 (MedHp and RB series) or a ATCC26012 (DL‐1 series) background; the plasmids applied for transformation are incorporated by homologous recombination and usually result in recombinant strains harbouring multiple tandemly repeated copies integrated into the target locus. For this purpose, plasmids harbouring one of a set of several cloned sub‐telomeric ARS sequences derived from the DL‐1 strain have been described [57,58]. A set of vectors has been used to target the heterologous DNA to the rDNA locus of H. polymorpha [20,59,60].…”