Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris

Sci Rep. 2017 Sep 11;7(1):11113. doi: 10.1038/s41598-017-11494-5.

Abstract

Pichia pastoris is a widely used heterologous protein production workhorse. However, with its multiple genetic modifications to solve bottlenecks for heterologous protein productivity, P. pastoris lacks selectable markers. Existing selectable marker recycling plasmids have drawbacks (e.g., slow growth and conditional lethality). Here, zeocin-resistance marker recycling vectors were constructed using the Cre/loxP recombination system. The vectors were used to (i) knock in heterologous phytase, xylanase and lipase expression cassettes, (ii) increase the phytase, xylanase and lipase gene copy number to 13, 5, and 5, respectively, with vector introduction and (iii) engineer the secretion pathway by co-overexpressing secretion helper factors (Sly1p and Sec1p) without introducing selectable markers, giving a phytase field of 0.833 g/L. The vectors allow selectable marker recycling and would be a useful tool to engineer P. pastoris for high heterologous protein productivity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 6-Phytase / genetics
  • 6-Phytase / metabolism
  • Gene Dosage
  • Gene Expression
  • Gene Order
  • Genetic Markers*
  • Genetic Vectors
  • Lipase / genetics
  • Lipase / metabolism
  • Pichia / genetics*
  • Pichia / metabolism
  • Plasmids / genetics*
  • Recombination, Genetic
  • Xylosidases / genetics
  • Xylosidases / metabolism

Substances

  • Genetic Markers
  • Lipase
  • 6-Phytase
  • Xylosidases