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 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 Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

2014 ◽  
Vol 81 (2) ◽  
pp. 533-543 ◽  
Author(s):  
Huy-Dung Hoang ◽  
Jun-ichi Maruyama ◽  
Katsuhiko Kitamoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Filamentous Fungi ◽  
Protein Secretion ◽  
Filamentous Fungus ◽  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Proteins ◽  
Content Type ◽  
Heterologous Protein Secretion ◽  
Golgi Compartment

ABSTRACTFilamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, usingAspergillus oryzaeas a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors,A. oryzaeVip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi.

scholarly journals Controlled Production of Stable Heterologous Proteins in Lactococcus lactis

2002 ◽  
Vol 68 (6) ◽  
pp. 3141-3146 ◽  
Author(s):  
A. Miyoshi ◽  
I. Poquet ◽  
V. Azevedo ◽  
J. Commissaire ◽  
L. Bermudez-Humaran ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Protein Production ◽  
Heterologous Protein ◽  
Nonstructural Protein ◽  
Inducible Promoter ◽  
Staphylococcal Nuclease ◽  
Poor Quality ◽  
Heterologous Proteins ◽  
Wild Type ◽  
Bovine Rotavirus

ABSTRACT The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P nisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.

Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2193-2201 ◽  
Author(s):  
Peter Ravn ◽  
José Arnau ◽  
Søren M. Madsen ◽  
Astrid Vrang ◽  
Hans Israelsen
Keyword(s):  
Lactococcus Lactis ◽  
Signal Peptide ◽  
Protein Production ◽  
Heterologous Protein ◽  
Site Directed Mutagenesis ◽  
Hydrophobic Core ◽  
Transposon Insertion ◽  
Charged Amino Acids ◽  
Secretion Efficiency ◽  
Positively Charged

The authors have previously reported the identification of novel signal peptides (SPs) from Lactococcus lactis using transposon insertion. Of these, SP310 caused the highest level of secretion. However, the levels were lower than those obtained using the signal peptide from Usp45 (SPUSP), the major secreted lactococcal protein. In this study, site-directed mutagenesis of signal peptide SP310 was used to investigate the effect of amino acid alterations on lactococcal secretion and to improve secretion efficiency. Several mutated SPs caused higher secretion. This increase in secretion was due to modifications in the cleavage region. In fermenter experiments, the signal peptide SP310mut2 resulted in an extracellular Staphylococcus aureus nuclease (Nuc) yield which was 45 % higher than that with the natural SP310. Surprisingly, increasing the hydrophobicity of the hydrophobic core or increasing the number of positively charged amino acids in the N-terminal region of SP310 decreased secretion. High extracellular yields of Nuc resulted from more efficient secretion, as strains with less efficient SPs accumulated more intracellular SP-Nuc precursor.

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