Dulermo Rémi, Brunel François, Dulermo Thierry, Ledesma-Amaro Rodrigo, Vion Jérémy, Trassaert Marion, Thomas Stéphane, Nicaud Jean-Marc, Leplat Christophe
Micalis Institute, INRA-AgroParisTech, UMR1319, Team BIMLip: Integrative Metabolism of Microbial Lipids, Université Paris-Saclay, domaine de Vilvert, 78350, Jouy-en-Josas, France.
Microb Cell Fact. 2017 Feb 17;16(1):31. doi: 10.1186/s12934-017-0647-3.
The yeast Yarrowia lipolytica is an increasingly common biofactory. To enhance protein expression, several promoters have been developed, including the constitutive TEF promoter, the inducible POX2 promotor, and the hybrid hp4d promoter. Recently, new hp4d-inspired promoters have been created that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter. Three different protein-secretion signaling sequences can be used: preLip2, preXpr2, and preSuc2.
To our knowledge, our study is the first to use a set of vectors with promoters of variable strength to produce proteins of industrial interest. We used the more conventional TEF and hp4d promoters along with five new hybrid promoters: 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, 8UAS1-pTEF, and hp8d. We compared the production of RedStar2, glucoamylase, and xylanase C when strains were grown on three media. As expected, levels of RedStar2 and glucoamylase were greatest in the strain with the 8UAS1-pTEF promoter, which was stronger. However, surprisingly, the 2UAS1-pTEF promoter was associated with the greatest xylanase C production and activity. This finding underscored that stronger promoters are not always better when it comes to protein production. We therefore developed a method for easily identifying the best promoter for a given protein of interest. In this gateway method, genes for YFP and α-amylase were transferred into a pool of vectors containing different promoters and gene expression was then analyzed. We observed that, in most cases, protein production and activity were correlated with promoter strength, although this pattern was protein dependent.
Protein expression depends on more than just promoter strength. Indeed, promoter suitability appears to be protein dependent; in some cases, optimal expression and activity was obtained using a weaker promoter. We showed that using a vector pool containing promoters of variable strength can be a powerful tool for rapidly identifying the best producer for a given protein of interest.
解脂耶氏酵母是一种越来越常见的生物工厂。为了提高蛋白质表达，已经开发了几种启动子，包括组成型TEF启动子、诱导型POX2启动子和杂交hp4d启动子。最近，人们创建了新的受hp4d启发的启动子，这些启动子将不同数量的UAS1串联元件与最小的LEU2启动子或TEF启动子相结合。可以使用三种不同的蛋白质分泌信号序列：preLip2、preXpr2和preSuc2。
据我们所知，我们的研究是首次使用一组具有可变强度启动子的载体来生产具有工业价值的蛋白质。我们使用了更传统的TEF和hp4d启动子以及五个新的杂交启动子：2UAS1-pTEF、3UAS1-pTEF、4UAS1-pTEF、8UAS1-pTEF和hp8d。我们比较了菌株在三种培养基上生长时红星2、糖化酶和木聚糖酶C的产量。正如预期的那样，具有更强的8UAS1-pTEF启动子的菌株中红星2和糖化酶的水平最高。然而，令人惊讶的是，2UAS1-pTEF启动子与最高的木聚糖酶C产量和活性相关。这一发现强调，在蛋白质生产方面，更强的启动子并不总是更好。因此，我们开发了一种方法，用于轻松识别给定目标蛋白质的最佳启动子。在这种网关方法中，将YFP和α-淀粉酶的基因转移到一组包含不同启动子的载体中，然后分析基因表达。我们观察到，在大多数情况下，蛋白质产量和活性与启动子强度相关，尽管这种模式因蛋白质而异。
蛋白质表达不仅仅取决于启动子强度。事实上，启动子的适用性似乎取决于蛋白质；在某些情况下，使用较弱的启动子可获得最佳表达和活性。我们表明，使用一组包含可变强度启动子的载体可以成为快速识别给定目标蛋白质最佳生产者的有力工具。
