title: Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications.
abstract: BACKGROUND
  Recombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome. Recently, heterologous and endogenous autonomously replicating sequences (ARS) were identified in P. pastoris by genome mining, opening the possibility of expanding the available toolbox to include efficient episomal plasmids. The aim of this technical report is to validate a 452-bp sequence ("panARS") in context of P. pastoris expression vectors, and to compare their performance to classical integrative plasmids. Moreover, we aimed to test if such episomal vectors would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells.
  
  
  RESULTS
  A panARS-based episomal vector was evaluated using blue fluorescent protein (BFP) as a reporter gene. Normalized fluorescence from colonies carrying panARS-BFP outperformed the level of signal obtained from integrative controls by several-fold, whereas endogenous sequences, identified from the P. pastoris genome, were not as efficient in terms of protein production. At the single cell level, panARS-BFP clones showed lower interclonal variability but higher intraclonal variation compared to their integrative counterparts, supporting the idea that heterologous protein production could benefit from episomal plasmids. Finally, efficiency of 2-fragment and 3-fragment in vivo recombination was tested using varying lengths of overlapping regions and molar ratios between fragments. Upon optimization, minimal background was obtained for in vivo assembled vectors, suggesting this could be a quick and efficient method to generate of episomal plasmids of interest.
  
  
  CONCLUSIONS
  An expression vector based on the panARS sequence was shown to outperform its integrative counterparts in terms of protein productivity and interclonal variability, facilitating recombinant protein expression and screening. Using optimized fragment lengths and ratios, it was possible to perform reliable in vivo recombination of fragments in P. pastoris. Taken together, these results support the applicability of panARS episomal vectors for synthetic biology approaches.
authors: Bioprocessing Technology Institute (A-STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore. andrea_camattari@bti.a-star.edu.sg.
authors: Andrea
authors: http://orcid.org/0000-0001-9560-8294
authors: A
authors: Camattari
authors: Bioprocessing Technology Institute (A-STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors: Amelia
authors: A
authors: Goh
authors: Bioprocessing Technology Institute (A-STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors: Lian Yee
authors: LY
authors: Yip
authors: Bioprocessing Technology Institute (A-STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors: Andy Hee Meng
authors: AH
authors: Tan
authors: Bioprocessing Technology Institute (A-STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors: Sze Wai
authors: SW
authors: Ng
authors: Institute of Medical Biology (A-STAR), 8a Biomedical Grove, #06-06, Singapore, 138648, Singapore.
authors: Anthony
authors: A
authors: Tran
authors: Institute of Medical Biology (A-STAR), 8a Biomedical Grove, #06-06, Singapore, 138648, Singapore.
authors: Gaowen
authors: G
authors: Liu
authors: Department of Genome Sciences, University of Washington, Seattle, WA, USA.
authors: Ivan
authors: I
authors: Liachko
authors: Department of Genome Sciences, University of Washington, Seattle, WA, USA.
authors: Maitreya J
authors: MJ
authors: Dunham
authors: Institute of Medical Biology (A-STAR), 8a Biomedical Grove, #06-06, Singapore, 138648, Singapore.
authors: Giulia
authors: G
authors: Rancati
mesh_terms: D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis*; D011995:Recombination, Genetic; D058615:Synthetic Biology / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D000432:Methanol
keywords: BFP; Digital droplet PCR; Episomal plasmid; In vivo recombination; Interclonal variability; P. pastoris; Synthetic biology; panARS