1.A Novel Potential Signal Peptide Sequence and Overexpression of ER-Resident Chaperones Enhance Heterologous Protein Secretion in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica.
Roongsawang N1,2, Puseenam A3, Kitikhun S4, Sae-Tang K3, Harnpicharnchai P3, Ohashi T5, Fujiyama K5, Tirasophon W4, Tanapongpipat S3. Appl Biochem Biotechnol. 2016 Feb;178(4):710-24. doi: 10.1007/s12010-015-1904-8. Epub 2015 Oct 30.
The thermotolerant methylotrophic yeast Ogataea thermomethanolica is a host for heterologous protein expression via secretion to the culture medium. Efficient secretion is a major bottleneck for heterologous protein production in this strain. To improve protein secretion, we explored whether the use of a native signal peptide sequence for directing heterologous protein secretion and overexpression of native ER-resident chaperone genes could improve heterologous protein secretion in O. thermomethanolica. We cloned and characterized genes encoding α-mating factor (Otα-MF) and ER-resident chaperones OtBiP, OtCNE1, and OtPDI. The pre and pre-pro sequences of Otα-MF were shown to promote higher secretion of heterologous endoxylanase comparing with the classical pre-pro sequence of Saccharomyces cerevisiae. However, in the case of heterologous glycosylated phytase, only the Otα-MF pre-pro sequence significantly enhanced protein secretion. The effect of chaperone overexpression on heterologous protein secretion was tested in cotransformant cells of O.
2.A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris.
Dagher SF1, Bruno-Bárcena JM1. Microbiology. 2016 Jan;162(1):23-34. doi: 10.1099/mic.0.000211. Epub 2015 Nov 9.
The β-hexosyltransferase (BHT) from Sporobolomyces singularis is a membrane-bound enzyme that catalyses transgalactosylation reactions to synthesize galacto-oligosaccharides (GOSs). To increase the secretion of the active soluble version of this protein, we examined the uncharacterized novel N-terminal region (amino acids 1-110), which included two predicted endogenous structural domains. The first domain (amino acids 1-22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23-110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant P. pastoris strains carrying combinations of the predicted structural domains and the α mating factor (MFα) from Saccharomyces cerevisiae as positive control. Upon replacement of the leader domain (amino acids 1-22) by MFα (MFα-rBht(23-594)), protein secretion increased and activity of both soluble and membrane-bound enzymes was improved 53- and 14-fold, respectively.
3.Cloning, expression, and molecular dynamics simulations of a xylosidase obtained from Thermomyces lanuginosus.
Gramany V1, Khan FI1,2, Govender A1, Bisetty K2, Singh S1, Permaul K1. J Biomol Struct Dyn. 2015 Oct 19:1-12. [Epub ahead of print]
The aim of this study was to clone, express, and characterize a β-xylosidase (Tlxyn1) from the thermophilic fungus Thermomyces lanuginosus SSBP in Pichia pastoris GS115 as well as analyze optimal activity and stability using computational and experimental methods. The enzyme was constitutively expressed using the GAP promoter and secreted into the medium due to the alpha-mating factor secretion signal present on the expression vector pBGPI. The 1276 bp gene consists of an open reading frame that does not contain introns. A 12% SDS-PAGE gel revealed a major protein band at an estimated molecular mass of 50 kDa which corresponded to zymogram analysis. The three-dimensional structure of β-xylosidase was predicted, and molecular dynamics simulations at different ranges of temperature and pH were performed in order to predict optimal activity and folding energy. The results suggested a strong conformational temperature and pH dependence. The recombinant enzyme exhibited optimal activity at pH 7 and 50°C and retained 80% activity at 50°C, pH 7 for about 45 min.
4.A Cellular System for Spatial Signal Decoding in Chemical Gradients.
Hegemann B1, Unger M2, Lee SS3, Stoffel-Studer I3, van den Heuvel J3, Pelet S4, Koeppl H5, Peter M6. Dev Cell. 2015 Nov 23;35(4):458-70. doi: 10.1016/j.devcel.2015.10.013. Epub 2015 Nov 12.
Directional cell growth requires that cells read and interpret shallow chemical gradients, but how the gradient directional information is identified remains elusive. We use single-cell analysis and mathematical modeling to define the cellular gradient decoding network in yeast. Our results demonstrate that the spatial information of the gradient signal is read locally within the polarity site complex using double-positive feedback between the GTPase Cdc42 and trafficking of the receptor Ste2. Spatial decoding critically depends on low Cdc42 activity, which is maintained by the MAPK Fus3 through sequestration of the Cdc42 activator Cdc24. Deregulated Cdc42 or Ste2 trafficking prevents gradient decoding and leads to mis-oriented growth. Our work discovers how a conserved set of components assembles a network integrating signal intensity and directionality to decode the spatial information contained in chemical gradients.
