Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags

Abstract

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-8966-9) contains supplementary material, which is available to authorized users.

Filamentous fungi-like secretory pathway strayed in a yeast system: peculiarities of Yarrowia lipolytica secretory pathway underlying its extraordinary performance

“…Since it was earlier reported that hydrophobicity and secondary structure are the key factors determining efficiency of the pre-sequence in driving the nascent polypeptide translocation and specifying the translocation mode (Yaver et al 1992; Matoba and Ogrydziak 1998), we used a panel of computational tools to study these parameters in the analyzed secretory elements. However, we could not see any straightforward correlation between the average hydrophobicity and the final extracellular abundance of the reporters (Celińska et al 2018). Correspondingly, neither calculated D-score values (reflecting probability of being recognized and processed by signal peptidase) nor predicted secondary structure could provide the key or support the rules governing the observed results.…”

“…Enhanced production of the targeted, secretory protein may be achieved by several different strategies, including (i) engineering the DNA coding sequence itself, (ii) modification of a genetic construct structure, (iii) optimization of culturing conditions, and (iv) engineering molecular mechanisms of protein synthesis and secretion pathway (Graf et al 2009; Liu et al 2012). Within each of the proposed strategies, various solutions have been proposed, and some of them were conducted in Y. lipolytica , like (i) optimization of codon usage within the coding sequence (Celińska et al 2015; Celińska 2015; Dulermo et al 2017), (ii) amplification of the gene copy number, manipulation with regulatory elements contained in the genetic construction, mainly promoter (Le Dall et al 1994; Gasmi et al 2011b; Gasmi et al 2011a; Gasmi et al 2012; Dulermo et al 2017; Larroude et al 2018), (iii) bioprocess engineering, optimization of cultivation conditions (Chang et al 1998; Kim et al 2000; Nicaud et al 2002; Celińska et al 2017a), and (iv) co-synthesis of chaperones, manipulation with protein folding, and maturation mechanisms, engineering secretory tags (De Pourcq et al 2012; Celińska et al 2018). Nevertheless, the vast majority of studies on enhanced production of secretory proteins in Y. lipolytica was focused on strategies from (i) to (iii), while the approaches directing secretory pathway were largely neglected.…”

“…We observed high efficiency of secretion upon transcriptional fusion of the mature SoAMY polypeptide with pre-AEP, and unsuitability of native pre-LIP2 in this regard. In our recent study (Celińska et al 2018), we analyzed the potential of ten different pre-domains towards targeting of two model polypeptides to the secretory pathway. The pre-sequences under study covered those well-known, like pre-LIP2 or pre-AEP, some previously described, like the hybrid pre-LIP2 or the insect-derived pre-SoAMY, but most importantly novel pre-sequences, previously undescribed in the context of recombinant protein secretion in Y. lipolytica .…”

Rapid micro-assays for amylolytic activities determination: customization and validation of the tests

“…Y. lipolytica strains, bearing separately either SoAMY or TlGAMY genes under the control of strong promoter, secreted the recombinant proteins to the cultivation medium (Celińska et al 2018). In this experiment, crude medium supernatants were directly used as enzymatic preparations for hydrolysis of starch, without any purification procedures.…”

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

“…The example presented here demonstrates that top-performing transformants can be screened in one step. Previously, we used a smaller version of this toolkit to illustrate the assembly of an entire pathway, notably the carotenoid pathway (Celinska et al, 2017;Celi nska et al, 2018;Larroude et al, 2018), and the construction of a set of expression cassettes containing multiple secretion signal sequences, which allows the optimization of protein secretion (Celi nska et al, 2018;Soudier et al, 2019). Here, we have greatly expanded the capacity of our system, and we now provide strong as well as inducible promoters.…”