Paving the way for the production of secretory proteins by yeast cell factories

Humans have used yeasts for the production of food and beverages since ancient times. Currently, Saccharomyces cerevisiae is also a key platform for the production of molecules with biopharmaceutical and industrial relevance. Yeasts are excellent choices as cell factories for recombinant proteins due to several reasons, including: (i) rapid growth, (ii) easy genetic manipulation, (iii) complete genome sequences usually available, plus (iv) their eukayotic machinery for posttranscriptional and post-translational modifications. But yeasts are much more than S. cerevisiae. The ‘other’ yeasts, so-called ‘non-conventional yeasts’, include a wide spectrum of species, barely exploited, with multiple peculiar physiological and molecular characteristics. Lately, methylotrophic yeast Pichia pastoris (a.k.a Komagataella phaffi or K. pastori) stands out as the ideal host for heterologous expression, basically due to three properties: its capacity to grow until very high densities, its ability to secrete fully proper folded and functional proteins, and its AOX1 (Alcohol oxidase I) promoter, that is both strongly repressed in the presence of glucose, glycerol or ethanol, and fully induced by methanol (Ahmad et al., 2014; Looser et al., 2015; Yang et al., 2018). Nevertheless, using non-conventional yeasts for biotechnological applications usually clashes with a main bottleneck, as the process for cloning the desired genes in not an easy and direct procedure but sometimes a long and winding road. Furthermore, in the last years a large amount of data about previously unknown genes or proteins has been generated by the ‘omics’ techniques. A significant step to study the biological functions of these proteins is its purification what needs a suitable system for their heterologous expression. This hindrance has been addressed in a recent report for Microbial Biotechnology where Gonz alez and coworkers describe a tool that allows the high-throughput expression of proteins in S. cerevisiae and P. pastoris. in a simple and fast way. The protocol involves the expression of heterologous proteins by transformation of S. cerevisiae with a PCR product that carries the gene of interest, obtained from the original cDNA, and its integration either into the S. cerevisiae genome or in an autonomous replicative plasmid (pYEDIS) that contains the P. pastoris AOX1 promoter. In the last procedure, a cloning step can be avoided as the genetic engineered plasmid is generated by homologous recombination between two fragments that are co-transformed into the yeast cell: one with the PCR product, and the other with a linearized vector containing different elements that drive expression and secretion of the recombinant protein. This plasmid can then be isolated from S. cerevisiae and directly used to transform P. pastoris to express the target proteins, as pYEDIS vector includes the elements necessary for expression in P. pastoris. Also, this tool could be used for other applications as expression of intracellular protein or chimeric proteins, only requiring PCR reactions with the corresponding primers, and a one step transformation in S. cerevisiae. However, to take full advantage of these cell factories, other limitations constraining product yields need to be addressed. Further major restrictions are related to the differences in post-translational modifications between yeasts and other eukaryotes, and in the extra effort required for the production of a high amount of protein products. These processes encompass multiple steps and thousands of genes but their imbalance usually leads to the induction of the unfolded protein response and to protein degradation which Received 2 November, 2018; accepted 6 November, 2018. *For correspondence. E-mail cmichan@uco.es; Tel. +34 957 218082; Fax +34 957 218688. Microbial Biotechnology (2019) 12(6), 1095–1096 doi:10.1111/1751-7915.13342 Funding Information No funding information provided.