Abstract
BackgroundKluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far.ResultsIn this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2–Ire1–Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells.ConclusionsOur results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.
Highlights
Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species
7.8% of genes in Neurospora crassa and 6.8% of genes in Saccharomyces cerevisiae were upregulated by both dithiothreitol (DTT) and tunicamycin (TM), while 6.8% of genes in Komagataella phaffii were upregulated by both DTT and the overexpression of HAC1 [3,4,5]
A proportion of ER stress response target genes (ESRTs) was induced through a signaling network called unfolded protein response (UPR), which is one of the best-characterized pathways to deal with endoplasmic reticulum (ER) stress [6]
Summary
Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). When the ER encounters a high flux of heterologous proteins, its folding capacity could be transiently saturated, leading to the accumulation of misfolded or unfolded proteins and causing ER stress [2]. A proportion of ESRTs was induced through a signaling network called unfolded protein response (UPR), which is one of the best-characterized pathways to deal with ER stress [6]. Some ESRTs were induced independently of UPR, as reported in N. crassa and S. cerevisiae [4, 7]
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