Xplor® 2—an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans

Abstract

Combining ease of genetic manipulation and fermentation with the ability to secrete and to glycosylate proteins in the basic eukaryotic manner, Arxula adeninivorans provides an attractive expression platform. Based on a redesign of the basic vector, a new Arxula vector system, Xplor 2, for heterologous gene expression was established, which allows (1) the construction of expression plasmids for supertransformation of A. adeninivorans strains secreting target proteins of biotechnological interest and (2) the integration of small vector cassettes consisting of yeast DNA sequences only. For this purpose, a set of modules including the ATRP1m selection-marker module, expression modules for constitutive expression of the genes phyK (Klebsiella-derived phytase) and IFNalpha2a (human interferon alpha), the HARS (Hansenula polymorpha autonomous replication sequence) for autonomous replication and the chaperone module AHSB4 promoter -HpCNE1 gene (calnexin) -PHO5 terminator to improve secretion efficiency were constructed and integrated in various combinations in the basic vector Xplor 2. After removal of the complete Escherichia coli-based plasmid parts (resistance marker, ColE1 ori and f1(-) origin), the remaining yeast-based linear vector fragment with or without rDNA targeting sequences were transformed as yeast rDNA integrative expression cassettes and yeast integrative expression cassettes (YICs), respectively, and the resulting strains were tested for their capacity to secrete PhyK or IFNalpha2a. Maximal expression levels were consistently obtained using YICs for transformation irrespective of whether or not they carry HARS and/or calnexin modules. It is recommended that at least 50 such transformants be analyzed to ensure selection of the best transformants.

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

“…The amplified gene fragments were inserted into the plasmid pBS-TEF1-PHO5-SS (flanked by SpeI and SacII restriction sites) between the A. adeninivorans-derived TEF1 promoter and the Saccharomyces cerevisiae-derived PHO5 terminator. After restriction of the resulting pBS-TEF1-ACUT1-(6H)-PHO5-SS, pBS-TEF1-ACUT2-(6H)-PHO5-SS, pBS-TEF1-ACUT3-(6H)-PHO5-SS, and pBS-TEF1-FSCUT-(6H)-PHO5-SS plasmids with SpeI-SacII, the fragments were cloned into the Xplor2 vector of the Xplor2 transformation/expression platform containing auxotrophic marker ATRP1m (31). Linearization with AscI produced fragments that were flanked by 25S ribosomal DNA (rDNA) sequences (yeast rDNA integrative cassettes [YRCs]) for homologous recombination or SbfI sequences (yeast integrative cassettes [YICs]) for nonhomologous integration.…”

Cloning, Production, and Functional Expression of the Bacteriocin Enterocin A, Produced by Enterococcus faecium T136, by the Yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans

“…Purified pBS-AYNI1P-MF-KR-EA-PHO5 was digested with SpeI-SacII, and the resulting 1,187-bp SpeI-SacII cleavage fragment (AYNI1P-MF-KR-EA-PHO5) was ligated into plasmid pB25S-ATRP1m to generate the final plasmid pBYEA. Plasmid pBYEA was further restricted with NcoI, and the resulting YRC was used to transform A. adeninivorans G1212 competent cells (7,8). The presence of the YRCs integrated into H. polymorpha KL8-1EA and A. adeninivorans G1212EA, respectively, was confirmed by a bacteriocinogenicity test, PCR, and sequencing of the inserts.…”

“…The heterologous production of EntA by bacterial hosts has been attained through the expression of its native biosynthetic genes (29), by exchange or replacement of the EntA leader peptide and/or dedicated processing and secretion systems (33), or by fusion of mature EntA to signal peptides that act as secretion signals (10,28,32). Recently, several yeast platforms have been developed for the large-scale expression of proteins (5,7,13,19). However, the heterologous production of bacteriocins by yeasts has not yet been fully exploited.…”

“…Purified pBS-TEF1-MF-KR-EA-PHO5 was digested with SpeI-SacII, and the resulting 894-bp SpeI-SacII cleavage fragment (TEF1-MF-KR-EA-PHO5) was ligated into plasmid pB25S-ALEU2m to generate plasmid pBTEA. Plasmid pBTEA was further restricted with AscI to remove the Escherichia coli part of the plasmid construct, and the yeast ribosomal DNA integrative cassette (YRC) with the selection marker and expression module, flanked by rRNA gene sequences, was used to transform H. polymorpha KL8-1 competent cells (7). Purified pBS-AYNI1P-MF-KR-EA-PHO5 was digested with SpeI-SacII, and the resulting 1,187-bp SpeI-SacII cleavage fragment (AYNI1P-MF-KR-EA-PHO5) was ligated into plasmid pB25S-ATRP1m to generate the final plasmid pBYEA.…”

<b><i>Arxula adeninivorans</i></b> Recombinant Urate Oxidase and Its Application in the Production of Food with Low Uric Acid Content

“…The Xplor ® 2 platform has been established as a routine transformation/expression platform in A. adeninivorans [Böer et al, 2009b]. The system, which allows construction of resistance marker-free yeast transformants, is based on a bacterial vector backbone into which yeast modules for selection and expression can be inserted between two 25S rDNA fragments that are arranged in the same orientation but in reverse order relative to the genomic rDNA.…”