New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris

Abstract

Biopharmaceuticals are an integral part of modern medicine and pharmacy. Both, the development and the biotechnological production of biopharmaceuticals are highly cost-intensive and require suitable expression systems. In this review we discuss established and emerging tools for reengineering the methylotrophic yeast Pichia pastoris for biopharmaceutical production. Recent advancements of this industrial expression system through synthetic biology include synthetic promoters to avoid methanol induction and to fine-tune protein production. New platform strains and molecular cloning tools as well as in vivo glycoengineering to produce humanized glycoforms have made P. pastoris an important host for biopharmaceutical production.

Graphical abstract

Figure 1

Current synthetic biology approaches to improve biopharmaceutical yields and quality in P. pastoris. Glycoengineered strains provide humanized N-glycosylation patterns [14,15,16^•], synthetic promoters allow the fine-tuning of expression levels [41,42,43^•] and various tools for strain engineering [47–49,50^•] and metabolic modeling [55^•,56^•,57^•] are available.

Figure 2

Design strategies to create semi-synthetic glycosyltransferases and glycosidases for glycoengineering. On the left side, the general domain structure of glycosyltransferases and glycosidases is shown. These type II membrane proteins consist of an N-terminal cytosolic tail, a transmembrane domain (TMD), a stem region (these elements are referred to as CTS), and a C-terminal catalytic domain. In the middle and on the right side, design strategies for creating tailor-made enzymes with the desired catalytic activity and the proper localization in the sec pathway are shown. The combinatorial library approach involved the combination of large sets of catalytic domains with CTS fragments to fusion proteins, which were then screened for the desired activity [19,21,22]. Different lengths of the catalytic domains and the CTS fragments were tested (referred to as ‘s’ for short, ‘m’ for medium, ‘l’ for long and shown exemplarily for one catalytic domain and one CTS). Rational approaches were also used to design these chimeric enzymes [23–25]. The schematic for the domain architecture and the combinatorial libraries is based on Czlapinski et al. [20] and Nett et al. [26^••].

Figure 3

Recombinase based self-excisable knockout cassettes for marker regeneration (left side). Increased rates of homologous recombination in a P. pastoris Δku70 strain (right side). The knockout cassettes consist of a recombinase (Cre or FLP [48,49,50^•]) and a marker gene flanked by the respective recombinase recognition sites and are directed to the genome via the 5′ and 3′ homologous sequences to delete the desired target sequence. After integration via a double cross-over event, self-excision of the recombinase and the marker gene can be initiated by the expression of the recombinase from the methanol inducible AOX1 promoter (P_AOX1), leaving only the recombinase recognition site in the genome (notably Marx et al. [49] provided the recombinase transiently on a CEN/ARS plasmid). The initial integration in the genome is dependent on homologous recombination (HR). Exemplary frequencies of homologous recombination (in %) of the wildtype compared to the Δku70 strain are shown (right side). The length of the homologous sequence indicates the number of base pairs (bp) added on both sides of the cassette [50^•]. For 650 bp two different integration loci were tested, therefore two % values are given.