A Novel Potential Signal Peptide Sequence and Overexpression of ER-Resident Chaperones Enhance Heterologous Protein Secretion in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica

2015 ◽  
Vol 178 (4) ◽  
pp. 710-724 ◽  
Author(s):  
Niran Roongsawang ◽  
Aekkachai Puseenam ◽  
Supattra Kitikhun ◽  
Kittapong Sae-Tang ◽  
Piyanun Harnpicharnchai ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Signal Peptide ◽  
Heterologous Protein ◽  
Methylotrophic Yeast ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Heterologous Protein Secretion ◽  
Potential Signal

scholarly journals Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0258005
Author(s):  
Worarat Kruasuwan ◽  
Aekkachai Puseenam ◽  
Chitwadee Phithakrotchanakoon ◽  
Sutipa Tanapongpipat ◽  
Niran Roongsawang
Keyword(s):  
Er Stress ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Pathway ◽  
Heterologous Protein ◽  
Methylotrophic Yeast ◽  
Heterologous Proteins ◽  
Loss Of Function ◽  
Heterologous Protein Secretion ◽  
Model Protein

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O. thermomethanolica and used it to activate SOD1, VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O. thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.

scholarly journals Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

2001 ◽  
Vol 67 (9) ◽  
pp. 4119-4127 ◽  
Author(s):  
Y. Le Loir ◽  
S. Nouaille ◽  
J. Commissaire ◽  
L. Brétigny ◽  
A. Gruss ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Heterologous Protein ◽  
Staphylococcal Nuclease ◽  
Heterologous Proteins ◽  
Heterologous Protein Secretion ◽  
Precursor Proteins ◽  
Secretion Efficiency

ABSTRACT Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SPNuc) by that of L. lactis protein Usp45 (SPUsp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SPUsp than when directed by SPNuc. This SPUsp effect on Nuc secretion is not due to a better antifolding activity, since SPUsp:Nuc precursor proteins display enzymatic activity in vitro, while SPNuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SPUsp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.

scholarly journals An optimized growth medium for increased recombinant protein secretion titer via the type III secretion system

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Lisa Ann Burdette ◽  
Han Teng Wong ◽  
Danielle Tullman-Ercek
Keyword(s):  
Protein Secretion ◽  
Salt Concentration ◽  
Type Iii Secretion ◽  
Heterologous Protein ◽  
Carbon Sources ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
Heterologous Protein Secretion ◽  
Extracellular Environment

Abstract Background Protein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica Typhimurium transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an “outside-in” approach to elucidate the effect of growth medium components on secretion titer. Results We dissected the individual and combined effects of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements. Conclusions We leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

Cassette mutagenic analysis of the yeast invertase signal peptide: effects on protein translocation

1989 ◽  
Vol 9 (8) ◽  
pp. 3400-3410
Author(s):  
J K Ngsee ◽  
W Hansen ◽  
P Walter ◽  
M Smith
Keyword(s):  
Signal Peptide ◽  
Protein Translocation ◽  
Plasmid Vector ◽  
Peptide Sequence ◽  
Secretory Process ◽  
Hydrophobic Core ◽  
Class Iii ◽  
Yeast Plasmid ◽  
Signal Peptide Sequence ◽  
Transcription Terminator

The coding sequence of the SUC2 locus was placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere-based yeast plasmid vector from which invertase is expressed in a Suc- strain of Saccharomyces cerevisiae. Mutants in the signal peptide sequence were produced by replacing this region of the gene with synthetic oligonucleotide cassettes containing mixtures of nucleotides at several positions. The mutants could be divided into three classes on the basis of the ability to secrete invertase. Class I mutants produced secreted invertase but in reduced amount. The class II mutant, 4-55B, also exhibited reduced a level of invertase, but a significant fraction of the enzyme was intracellular. Class III mutants were partially defective in translocation from the cytoplasm to the endoplasmic reticulum and produced enzymatically active, unglycosylated preinvertase in the cytoplasm. Class III mutant preinvertases were also defective in translocation across canine pancreas microsomes. These results suggested that the reduced level of invertase resulted from proteolytic degradation of inefficiently transported intermediates. Comparison of the sequences of the mutant signal peptides indicated that amino acids at the extreme amino terminus and adjacent to the cleavage site play a crucial role in the secretory process when combined with a mutation within the hydrophobic core.

scholarly journals Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Doreen A. Wüstenhagen ◽  
Phil Lukas ◽  
Christian Müller ◽  
Simone A. Aubele ◽  
Jan-Peter Hildebrandt ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Peptide Sequence ◽  
Bacterial Cells ◽  
Recombinant Hirudin ◽  
Signal Peptide Sequence ◽  
Cell Lysates ◽  
Blood Sucking ◽  
A Cell ◽  
Synthesis Approach ◽  
Cell Free Protein Synthesis

AbstractSynthesis and purification of peptide drugs for medical applications is a challenging task. The leech-derived factor hirudin is in clinical use as an alternative to heparin in anticoagulatory therapies. So far, recombinant hirudin is mainly produced in bacterial or yeast expression systems. We describe the successful development and application of an alternative protocol for the synthesis of active hirudin based on a cell-free protein synthesis approach. Three different cell lysates were compared, and the effects of two different signal peptide sequences on the synthesis of mature hirudin were determined. The combination of K562 cell lysates and the endogenous wild-type signal peptide sequence was most effective. Cell-free synthesized hirudin showed a considerably higher anti-thrombin activity compared to recombinant hirudin produced in bacterial cells.

Heterologous protein secretion by Candida utilis

2013 ◽  
Vol 97 (16) ◽  
pp. 7357-7368 ◽  
Author(s):  
Maya Kunigo ◽  
Christoph Buerth ◽  
Denis Tielker ◽  
Joachim F. Ernst
Keyword(s):  
Protein Secretion ◽  
Candida Utilis ◽  
Heterologous Protein ◽  
Heterologous Protein Secretion
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