Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring

Abstract

Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50pg (4.91×10(3) copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300pg gDNA (2.95×10(4) copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600=80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600=80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard.

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

“…Consequently, we cultivated P. pastoris strain TK150 to high cell density and measured the level of bioconversion achieved. Figure details the typical growth profile observed during cultivation of P. pastoris cells in a 1 L Infors bioreactor, reaching wet cell weight (WCW) of 600 g/L using the cultivation regime reported by Templar et al Samples were taken immediately prior to the start of methanol feeding (indicated as M1 in Figure ) and then 33 h and 58 h after the start of methanol feeding.…”

Whole cell biosynthesis of 1-methyl-3-phenylpropylamine and 2-amino-1,3,4-butanetriol using Komagataella phaffii (Pichia pastoris) strain BG-10 engineered with a transgene encoding Chromobacterium violaceum ω-transaminase

“…Cultivation in bioreactor was performed using a Multifors 1L device (INFORS HT). The Multifors 1L device has been reported previously to achieve OD 600 in the range of 800–1000 and DCW of 150–200 g L −1 during K. phaffii cultivation by Templar et al. (2016) and Wei et al.…”