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WO2019003180A1 - Production et utilisations améliorées d'une protéine à auto-assemblage sécrétée par une souche native de yarrowia lipolytica - Google Patents

Production et utilisations améliorées d'une protéine à auto-assemblage sécrétée par une souche native de yarrowia lipolytica Download PDF

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Publication number
WO2019003180A1
WO2019003180A1 PCT/IB2018/054808 IB2018054808W WO2019003180A1 WO 2019003180 A1 WO2019003180 A1 WO 2019003180A1 IB 2018054808 W IB2018054808 W IB 2018054808W WO 2019003180 A1 WO2019003180 A1 WO 2019003180A1
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WIPO (PCT)
Prior art keywords
protein
self assembling
native self
assembling protein
liquid culture
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Ceased
Application number
PCT/IB2018/054808
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English (en)
Inventor
Ameeta RAVIKUMAR
Swanand Joshi
Prashant Gaikwad
Smita ZINJARDE
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Savitribai Phule Pune University
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Savitribai Phule Pune University
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Publication of WO2019003180A1 publication Critical patent/WO2019003180A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi

Definitions

  • This invention belongs to the field of biotechnology, and in that, relates generally to the methods, techniques and systems employing microorganisms for production of industrially applicable products.
  • the present invention specifically relates to underlying aspects, mainly media and growth conditions, for conditioning the enhanced secretion of a self-assembling native protein having surfactant and emulsifier properties through liquid culture of a Yarrowia lipolytica strain.
  • Microbial cells have been widely recognized as efficient cell factories capable of outputting molecules of interest should their biochemical flux be directed and managed in the right manner.
  • State-of-art today refers various techniques including physical, chemical, and genetic interventions for aforesaid objective.
  • technical complexities, high demands as to costs, resources and skills remain considerations that limit their global applicability to a large extent.
  • Biosurfactants are generally low molecular weight microbial products composed of sugars, amino acids, fatty acids and functional groups such as carboxylic acids. These molecules are amphiphilic in nature and this property allows them to dissolve in both polar and non-polar solvents. Biosurfactants are known for their excellent surface activity which involves lowering the surface and interfacial tension between different phases. They can act as wetting, foaming and solubilizing agents in different industrial processes (Source: . Perfumo A., Smyth T. J. P., Marchant R., Banat I. M. (2009).
  • bioemulsifiers are higher in molecular weight than biosurfactants as they are complex mixtures of heteropolysaccharides, lipopolysaccharides, lipoproteins and proteins.
  • the isolated protein is a surface active biomolecule which is categorized into surfactants and emulsifiers, while surfactants play the role of surface tension reduction, emulsifiers are involved in formation and stabilization of emulsions.
  • some biomolecules possess both surfactant and emulsifying properties which contributes to their unique functions and broad industrial uses.
  • Yarrowia Iipolytica is one of the most extensively studied nonpathogenic yeasts having GRAS status, which is currently used as one of the models for the study of secretion studies, dimorphism, and degradation of hydrophobic substrates.
  • Yarrowia Iipolytica with its rich potential is considered to be a promising candidate that can be utilized in various biotechnological industries. It has known to produce large number of biomolecules such as bio-surfactant, bioemulsifier growth factors, acids, enzymes etc. The earlier reports explains that the characterized bioemulsifier from Y. Iipolytica is the complex mixture of protein-lipid-carbohydrate, which shows the surfactant and emulsifier activity.
  • FIGURE 1 is a micro-photograph of foam produced by HBN obtained as per the present invention.
  • FIGURE 2 is a photograph of test for foaming undertaken using HBN obtained as per the present invention.
  • FIGURE 3 showcases surfactant properties as determined by hanging drop method undertaken using HBN obtained as per the present invention.
  • FIGURE 4 includes microphotographs, particularly of various substrates modified via coating with HBN obtained as per the present invention.
  • FIGURE 5 is a comparative of water droplets on various substrates in untreated versus when treated with HBN obtained as per the present invention.
  • FIGURE 6 is a graph illustrating vortexing time required to achieve a stable emulsion with
  • FIGURE 7 showcases concentration dependent variation in the particle size with HBN obtained as per the present invention.
  • the present invention attempts to resolve the wants of art, by meeting the objectives stated hereinabove. Specifically, principles of the present invention are generally directed to optimization of media and growth conditions, for the enhanced secretion of a self-assembling extracellular self assembling native protein from a Yarrowia lipolytics strain through liquid culture. Further disclosed are applications of said protein based on its surfactant and emulsifier properties.
  • the disclosures herein are directed towards an inventive method of achieving enhanced production of a self-assembling protein having surfactant and emulsifier properties secreted by a native Yarrowia lipolytica strain arranged to so manifest in liquid culture.
  • the resultant product, its downstream processing and applications form integrative aspects of the present invention.
  • Modified protein purification protocol- 1000 ml cell free supernatant is subjected to ultrafiltration (3 kDa AMICON concentrate ultrafiltration under positive nitrogen pressure of 2 bar). Frothing usually destroys protein content, but as the protein of interest (HBN) is resilient, frothing is purposefully allowed in this step so that other proteins get degraded, and also the HBN is dissociated from extracellular polysaccharides. Resultant supernatant is lyophilized and the dry powder so obtained is re-dissolved in 200ml Milli-Q water. Protein content is then estimated using BCA to therefore decide volume of TFA later in the procedure. The dry powder re-dissolved in 200ml milli-Q water is lyophilized again to remove water content.
  • ultrafiltration 3 kDa AMICON concentrate ultrafiltration under positive nitrogen pressure of 2 bar. Frothing usually destroys protein content, but as the protein of interest (HBN) is resilient, frothing is purposefully allowed in this step so that other proteins get degraded, and also the HBN is dissoci
  • Resultant powder is subjected to TFA extraction using appropriate amount of TFA as determined above, (maintaining 5ml 100% TFA per 1 mg protein) in chilled conditions ( ⁇ 4°C).
  • the resultant solution is sonicated at 53 Hz for 10 min and centrifuged at 30000 G for 60 minutes. Pellet is discarded, and supernatant is collected and lyophilized to remove water content.
  • the resultant powder is dissolved in buffer (phosphate buffer of physiological pH having 0.8M ammonium sulphate) and subjected to HIC (Phenyl sepharose 6 FF, where sample volume is 5 times bed volume), causing elution by reduction in salt concentration till water phase.
  • buffer phosphate buffer of physiological pH having 0.8M ammonium sulphate
  • Aim of media optimization was to improve the yields for protein production.
  • a statistical experimental designs and surface response methodologies were used to improve the protein yield.
  • Response surface methodology is a collection of statistical and mathematical techniques useful for developing, improving and optimizing processes, which also helps in understanding the effects of individual variables and their interactions in the final response.
  • the influence of system aeration, agitation speed , temperature and carbon and nitrogen sources were evaluated on the production of protein.
  • Plackett-Burman design was adopted to determine most important medium components that affect protein production In this optimization step, a Plackett-Burman design was used to determine the likely effects of different media components on protein production. Plackett-Burman design was created using Design Expert (from Stat Ease Inc, USA).
  • PBD Plackett-Burman design
  • Table 2 The actual experimental design is given in Table 3 (table is split into three sections a, b, and c due to space constraints of this document - these three sections should be read together in order of rows representing the 12 trail runs) accompanying this document as generated by the software.
  • the factors chosen for PBD are glycerol as the carbon source, Yeast extract and ammonium sulfate as nitrogen sources, NaCI, salt solution along with five other physical parameter viz., incubation time, aeration, inoculum size, temperature and surface /volume ratio of the media. Parameter optimization was undertaken across 12 runs as under.
  • Table 4 represents the effect of each variable along with the mean squares, F-values, and p- values.
  • the observed protein yield varied from 20 mg/l to 60 mg/l, reflecting the importance of medium optimization to attain higher yields. Variables having a probability value (p-value) less than 0.05 were considered significant.
  • the analyzed data in Table 3 suggests that protein production was affected by yeast extract surface to volume ratio and temperature which had p-values of 0.0004, 0.0353 and 0.0006, respectively.
  • yeast extract There is significant effect of yeast extract as a nitrogen source for protein production by Y. lipolytica.
  • Yeast extract and temperature were chosen and their possible interactive effects on protein production were evaluated.
  • Table 5 Statistical analysis of above results is shown in Table 5 below.
  • the Model F-value of 33.71 implies the model is significant. There is only a 0.01 % chance that an F-value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. mln this case A, G, H, AH are significant model terms. [053] Values greater than 0.1000 indicate the model terms are not significant. Table 5 shows the R- squared for the model is 0.9506. The "Pred R-Squared" of 0.8337 is in reasonable agreement with the "Adj R-Squared” of 0.9224; i.e. the difference is less than 0.2. “Adeq Precision” measures the signal to noise ratio. A ratio greater than 4 is desirable. The ratio of 15.473 indicates an adequate signal.
  • Production level of self assembling protein secreted in the culture medium is usually low.
  • the crude protein yield obtained using standard medium was observed to be 20 mgl "1 .
  • response surface methodology that is, a statistical model based on predicted growth and media parameters, by which yield of crude protein with the modified media and growth conditions was increased three-fold to 60 mgl "1 , and yield of purified protein obtained after the standardization of the purification procedure was 20 mgl "1 . It shall be appreciated that this increase in the production of the protein was achieved without using traditional genetic amplification tools and techniques.
  • the small molecular weight protein (8 to 9 kDa) obtained as above is a natural, surface active, bio-compatible molecule with dual properties of bioemulsifier as well as surfactant.
  • FIGURE 1 is a microphotograph of foam produced by HBN.
  • FIGURE 2 is a photograph showing difference in foaming obtained using blank water (control), 10% SDS (positive control / next peer for comparison) and 50u.gml "1 HBN (test).
  • FIGURE 3 showcases surfactant properties as determined by hanging drop method using blank water (control), 1 % SDS (positive control / next peer for comparison) and 0.05mgml "1 HBN (test), thereby prove excellent surfactant properties.
  • the accompanying FIGURE 4 includes microphotographs, particularly in which is (a) glass coated with 50u.gml "1 HBN seen via light microscopy; (b) glass coated with 50u.gml "1 HBN seen via SEM; (c) silicon coated with 50u.gml "1 HBN seen via light microscopy; and (d) teflon coated with 50u.gml "1 HBN seen via light microscopy. c) Assembly on solid surfaces - Hydrophobicity of the surfaces was reversed by change in water contact angle by 30° upon coating with HBN.
  • the accompanying FIGURE 5 is a comparative of water droplets on various substrates in untreated versus when treated with HBN. These results are listed in Table 6 below.
  • FIGURE 6 is a graph illustrating minimal time required for vortexing to achieve a stable emulsion (observed time was 10 minutes, which emulsion was stable till at least 144 hours). Stability of emulsions were as illustrated in the Table 7 below.
  • FIGURE 7 showcases concentration dependent variation in the particle size, particularly (a) is a graph showing particle size distribution in electrophoresis run where lane 1 has 7.5 ⁇ 9 HBN and Lane 2 has 5 ⁇ 9 HBN; and (b) is a compilation of graphs showing vortexing dependent variation in the particle size.
  • the present invention has been reduced to practice by the present inventors, which has been successful in production of the target self-assembling protein (that is, HBN) which has been so isolated and purified from the crude bioemulsifier and characterized for its bio-material property. It shall be appreciated that said protein is isolated from cell free supernatant of the organism and not extracted from cell lysate or cell wall extract, which makes it more suitable for large scale applications in industries by minimizing the downstream processing cost.
  • HBN target self-assembling protein
  • Yarrowia lipolytics is identified as a GRAS resource, and ability of the protein, being secreted by it as per foregoing narration to change the surface property upon adsorption and its assembly at the interface it is evident that said protein is a promising molecule as a surface active bio-material in food, environmental, cosmetic industry, in biosensors and as scaffolds in tissue engineering applications as well as an efficient drug delivery vehicle (independent trials conducted by the present inventors confirmed proof of concept by able imbibing of curcumin in HBN micelles with paraffin oil and ethanol as co- solvent).

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Abstract

L'invention concerne des aspects sous-jacents, dont des conditions optimisées de milieu et de croissance, permettant la sécrétion améliorée d'une protéine extracellulaire native à auto-assemblage à partir d'une souche de Yarrowia lipolytica par culture liquide. Des applications de ladite protéine sur la base de ses propriétés tensioactives et émulsifiantes sont en outre décrites.
PCT/IB2018/054808 2017-06-29 2018-06-28 Production et utilisations améliorées d'une protéine à auto-assemblage sécrétée par une souche native de yarrowia lipolytica Ceased WO2019003180A1 (fr)

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IN201621044801 2017-06-29

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WO2019003180A1 true WO2019003180A1 (fr) 2019-01-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103155A (zh) * 2023-02-09 2023-05-12 华东理工大学 响应面法优化毕赤酵母胞内蛋白提取工艺

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040115790A1 (en) * 2001-02-13 2004-06-17 Tiina Pakula Method for production of secreted proteins in fungi
US20130137140A1 (en) * 2010-06-17 2013-05-30 Universiteit Gent Increased protein expression through increased membrane formation
US20140127748A1 (en) * 2011-04-12 2014-05-08 Csir Production of Heterologous Extracellular Polypeptides in Yarrowia Lipolytica
US20170044557A1 (en) * 2014-04-17 2017-02-16 Boehringer Ingelheim Rcv Gmbh & Co Kg Recombinant host cell for expressing proteins of interest
US20170174745A1 (en) * 2011-03-03 2017-06-22 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040115790A1 (en) * 2001-02-13 2004-06-17 Tiina Pakula Method for production of secreted proteins in fungi
US20130137140A1 (en) * 2010-06-17 2013-05-30 Universiteit Gent Increased protein expression through increased membrane formation
US20170174745A1 (en) * 2011-03-03 2017-06-22 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs
US20140127748A1 (en) * 2011-04-12 2014-05-08 Csir Production of Heterologous Extracellular Polypeptides in Yarrowia Lipolytica
US20170044557A1 (en) * 2014-04-17 2017-02-16 Boehringer Ingelheim Rcv Gmbh & Co Kg Recombinant host cell for expressing proteins of interest

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DOS SANTOS EO, MICHELON M, FURLONG EB, BURKERT JF, KALIL SJ, BURKERT CA.: "Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis.", BRAZ J MICROBIOL., vol. 43, no. 2, 1 June 2012 (2012-06-01), pages 432 - 440, XP055670109, DOI: 10.1590/S1517-83822012000200002 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103155A (zh) * 2023-02-09 2023-05-12 华东理工大学 响应面法优化毕赤酵母胞内蛋白提取工艺

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