Abstract:
BACKGROUND:Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development. RESULTS:A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development. CONCLUSIONS:In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.
journal_name
Microb Cell Factjournal_title
Microbial cell factoriesauthors
Samazan F,Rokbi B,Seguin D,Telles F,Gautier V,Richarme G,Chevret D,Varela PF,Velours C,Poquet Idoi
10.1186/s12934-015-0271-zsubject
Has Abstractpub_date
2015-07-16 00:00:00pages
104issn
1475-2859pii
10.1186/s12934-015-0271-zjournal_volume
14pub_type
杂志文章abstract:BACKGROUND:Griseoviridin (GV) and viridogrisein (VG, also referred as etamycin), both biosynthesized by a distinct 105 kb biosynthetic gene cluster (BGC) in Streptomyces griseoviridis NRRL 2427, are a pair of synergistic streptogramin antibiotics and very important in treating infections of many multi-drug resistant mi...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0792-8
更新日期:2017-10-25 00:00:00
abstract::Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features...
journal_title:Microbial cell factories
pub_type: 杂志文章,评审
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abstract:BACKGROUND:Glucose is the main carbon source of E. coli and a typical substrate in production processes. The main glucose uptake system is the glucose specific phosphotransferase system (Glc-PTS). The PTS couples glucose uptake with its phosphorylation. This is achieved by the concomitant conversion of phosphoenolpyruv...
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abstract:BACKGROUND:Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyc...
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abstract:BACKGROUND:Rhodosporidium and Rhodotorula are two genera of oleaginous red yeast with great potential for industrial biotechnology. To date, there is no effective method for inducible expression of proteins and RNAs in these hosts. RESULTS:We have developed a luciferase gene reporter assay based on a new codon-optimiz...
journal_title:Microbial cell factories
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doi:10.1186/s12934-015-0357-7
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journal_title:Microbial cell factories
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更新日期:2018-11-16 00:00:00
abstract:BACKGROUND:L-Ornithine is a non-protein amino acid with extensive applications in medicine and the food industry. Currently, L-ornithine production is based on microbial fermentation, and few microbes are used for producing L-ornithine owing to unsatisfactory production titer. RESULTS:In this study, Corynebacterium gl...
journal_title:Microbial cell factories
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-019-1102-4
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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abstract:BACKGROUND:Activation of silent biosynthetic gene clusters (BGCs) in marine-derived actinomycete strains is a feasible strategy to discover bioactive natural products. Actinoalloteichus sp. AHMU CJ021, isolated from the seashore, was shown to contain an intact but silent caerulomycin A (CRM A) BGC-cam in its genome. Th...
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pub_type: 杂志文章
doi:10.1186/s12934-020-01418-w
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-020-01448-4
更新日期:2020-10-07 00:00:00
abstract:BACKGROUND:In the biopharmaceutical industry, Escherichia coli (E. coli) strains are among the most frequently used bacterial hosts for producing recombinant proteins because they allow a simple process set-up and they are Food and Drug Administration (FDA)-approved for human applications. Widespread use of E. coli in ...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-12-58
更新日期:2013-06-11 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0758-x
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abstract::This article describes the use of biofilm reactors for the production of various chemicals by fermentation and wastewater treatment. Biofilm formation is a natural process where microbial cells attach to the support (adsorbent) or form flocs/aggregates (also called granules) without use of chemicals and form thick lay...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-4-24
更新日期:2005-08-25 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-015-0378-2
更新日期:2015-11-25 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-015-0224-6
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journal_title:Microbial cell factories
pub_type: 杂志文章
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
pub_type: 杂志文章
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更新日期:2014-08-19 00:00:00
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-016-0486-7
更新日期:2016-05-20 00:00:00
abstract::L-arginine (ARG) is an important amino acid for both medicinal and industrial applications. For almost six decades, the research has been going on for its improved industrial level production using different microorganisms. While the initial approaches involved random mutagenesis for increased tolerance to ARG and con...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-014-0166-4
更新日期:2014-12-03 00:00:00
abstract:BACKGROUND:The Gram-positive bacterium Bacillus subtilis has been widely used as a cell factory for the production of proteins due to its generally regarded as safe (GRAS) nature and secretion capability. Of the known secretory pathways in B. subtilis, the majority of proteins are exported from the cytoplasm by Sec pat...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-016-0469-8
更新日期:2016-04-28 00:00:00
abstract:BACKGROUND:The oleaginous yeast Yarrowia lipolytica is an organism of choice for the tailored production of various compounds such as biofuels or biopolymers. When properly engineered, it is capable of producing medium-chain-length polyhydroxyalkanoate (mcl-PHA), a biobased and biodegradable polymer that can be used as...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-019-1140-y
更新日期:2019-05-31 00:00:00
abstract:BACKGROUND:Bacteriocin-producing Lactic acid bacteria (LAB) have vast applications in human and animal health, as well as in food industry. The structural, immunity, regulatory, export and modification genes are required for effective bacteriocin biosynthesis. Variations in gene sequence, composition and organisation w...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-015-0280-y
更新日期:2015-06-16 00:00:00
abstract:BACKGROUND:The methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-018-1032-6
更新日期:2018-11-24 00:00:00
abstract:BACKGROUND:Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether prot...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0688-7
更新日期:2017-04-24 00:00:00