A METHOD FOR PRODUCTION OF POLYHYDROXYALKANOATES USING GENETICALLY ENGINEERED ALGAE

Abstract

The present invention discloses a method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae. More particularly to production of PHA from lignocellulosic waste by using algal transformant with phaC gene from phaC gene bearing bacteria. The said method includes various steps such as isolation of polyhydroxyalkanoates (PHA) producing bacteria (110), preparation of lignocellulosic waste (120), genetic engineering of algae (130), cultivation of algal transformants (140), and polyhydroxyalkanoates (PHA) extraction and purification (150). PHAs are biodegradable polymers produced by various microorganisms. They are considered an environmentally friendly alternative to conventional plastics. The production of PHA from lignocellulosic waste, an abundant and renewable resource, presents a sustainable method to produce bioplastics. However, the efficiency of PHA production from such waste is often limited by the microorganism's metabolic capabilities. This invention addresses these limitations by using genetically modified algae.

Specification

Description:The following specification particularly describes the invention and the manner in which it is to be performed.
CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY
[001] The present application does not claim priority from any patent application.
PREAMBLE
[002] The following specification particularly describes the invention and the manner in which it is to be performed.
TECHNICAL FIELD
[003] The present subject matter described herein, in general, relates to a method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae, and more particularly, production of PHA from lignocellulosic waste by using algal transformant with phaC gene from phaC gene bearing bacteria.
BACKGROUND
[004] Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by various microorganisms. They are considered an environmentally friendly alternative to conventional plastics. The production of PHA from lignocellulosic waste, an abundant and renewable resource, presents a sustainable method to produce bioplastics. However, the efficiency of PHA production from such waste is often limited by the microorganism's metabolic capabilities. This invention addresses these limitations by using genetically modified algae.
[005] The prior art US9663791B2 discloses the organism that is genetically engineered to express an alcohol dehydrogenase and an aldehyde dehydrogenase and to convert ethanol to acetyl-CoA when grown on ethanol as a sole carbon. The organisms are genetically engineered to make useful products when grown on ethanol as a carbon source. The organisms are genetically engineered to produce various useful products such as polyhydroxyalkanoates, diols, diacids, higher alcohols, and other useful chemicals. However prior art fails to mention the production of PHA on lignocellulosic waste by genetically engineered algae.
[006] The prior art US20230235370A1 discloses a method for biosynthesis polyhydroxybutyrate by a yeast transformant which provides a way of cheaper, faster and flexibility in biotechnology metabolism to improve PHB production. The polyhydroxybutyrate biosynthesis related gene comprises at least one of PhaA gene, PhaB gene or PhaC gene. However prior art fails to mention the production of PHA on lignocellulosic waste by genetically engineered algae.
[007] The present invention addresses the above shortcomings of the prior art. However, the invention is entirely different from the prior art in terms of novelty and technological advancements.
OBJECT
[008] The main object of the present invention is to optimize PHA production from lignocellulosic waste in isolated bacteria, understanding gene expression and regulation of PHA biosynthetic pathways, selecting and engineering algal strains for enhanced PHA production, and assessing economic feasibility and scalability for industrial applications.
SUMMARY
[009] The disclosure of the present invention discloses a method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae. More particularly to production of PHA from lignocellulosic waste by using algal transformant with phaC gene from phaC gene bearing bacteria. PHAs are biodegradable polymers produced by various microorganisms as carbon and energy storage compounds. They are of significant interest due to their potential as environmentally friendly alternatives to petroleum-based plastics. PHAs are naturally occurring biodegradable polymers. PHAs are synthesized and stored as water-insoluble inclusions in the cytoplasm of several bacteria and used as carbon and energy reserve material. PHAs are intracellular microbial polyesters synthesized by many species of bacteria and archaea, generally under nutrient limitation and excess of carbon source as storage granules of energy and also conferring stress resistance to prokaryotes. PHAs are naturally occurring, and the choice of microbial strains and carbon sources for cell cultivations results in biopolymers with different physicochemical properties.
[0010] Embodiments may disclose the method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae. The phaC gene encodes the enzyme PHA synthase, which helps in the biosynthesis of polyhydroxyalkanoates (PHAs) in bacteria. The primary function of PHA synthase is to catalyze the polymerization of hydroxyalkanoate monomers (such as 3-hydroxybutyrate) into PHA polymers. This reaction involves the formation of ester bonds between monomers, resulting in the production of PHAs. PHA synthases can vary in their specificity for different hydroxyalkanoate monomers. The phaC gene from different bacterial species can produce synthases that preferentially use specific monomers, influencing the properties of the resulting PHA. PHAs serve as energy reserves for bacteria, allowing them to survive in conditions where other carbon sources are scarce. The phaC gene thus contributes to the organism's ability to store energy efficiently. The phaC gene is essential for the synthesis of PHAs, providing bacteria with a mechanism to store energy and adapt to changing nutrient availability. Its enzymatic function is vital for biotechnological applications aimed at producing sustainable materials.
[0011] In these implementations a method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae is described. The above-mentioned method comprises various steps such as a) isolation of polyhydroxyalkanoates (PHA) producing bacteria (110) wherein, PHA producing bacteria is isolated by serially diluting the sewage water and plating it on Nile blue minimal agar media, b) preparation of lignocellulosic waste (120) wherein, lignocellulosic waste is prepared by subjecting it to physical, chemical, or biological pre-treatment to release fermentable sugars and converting complex polysaccharides into a form that is utilized by algae, c) genetic engineering of algae (130) wherein, algae is genetically engineered to incorporate the phaC gene isolated from bacteria, which is responsible for the polymerization of PHA, d) cultivation of algal transformants (140) wherein, algal transformants are cultivated in a culture medium containing the pre-treated lignocellulosic waste and algae utilize the sugars derived from the lignocellulosic waste to produce PHA intracellularly, e) polyhydroxyalkanoates (PHA) extraction and purification (150) wherein, PHA is extracted from the algal biomass using a solvent extraction method and then purified by precipitation and washing to obtain a high-purity biopolymer.
BRIEF DESCRIPTION
[0012] The foregoing detailed description of embodiments is better understood when read in conjunction with the appended drawings. For the purpose of illustrating of the present subject matter, an example of construction of the present subject matter is provided as figures; however, the invention is not limited to the specific method disclosed in the document and the figures.
[0013] Figure. 1 is a block diagram illustrating the process, according to some embodiments of the present disclosure.
DETAILED DESCRIPTION
[0014] Some of the embodiments of this disclosure, illustrating all its features will now be discussed in detail. The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items or meant to be limited to only the listed item or items. It must also be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.
[0015] Before the present method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae is described, it is to be understood that this method is not limited to the particular system(s), and methodologies described, as there can be multiple possible embodiments that are not expressly illustrated in the present disclosure. It is also to be understood that the terminology used in the description is to describe the particular implementations or versions or embodiments only, and is not intended to limit the scope of the present application. This summary is provided to introduce aspects involved in the method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae. This summary is not intended to identify essential features of the claimed subject matter nor is it intended for use in determining or limiting the scope of the claimed subject matter.
[0016] In one embodiment, a method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae is disclosed. The phaC gene encodes the enzyme PHA synthase, which helps in the biosynthesis of polyhydroxyalkanoates (PHAs) in bacteria. The primary function of PHA synthase is to catalyze the polymerization of hydroxyalkanoate monomers (such as 3-hydroxybutyrate) into PHA polymers. This reaction involves the formation of ester bonds between monomers, resulting in the production of PHAs. PHA synthases can vary in their specificity for different hydroxyalkanoate monomers. The phaC gene from different bacterial species can produce synthases that preferentially use specific monomers, influencing the properties of the resulting PHA. PHAs serve as energy reserves for bacteria, allowing them to survive in conditions where other carbon sources are scarce. The phaC gene thus contributes to the organism's ability to store energy efficiently. The phaC gene is essential for the synthesis of PHAs, providing bacteria with a mechanism to store energy and adapt to changing nutrient availability. Its enzymatic function is vital for biotechnological applications aimed at producing sustainable materials.
[0017] In another embodiment, invention discloses a method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae. PHAs are biodegradable polymers produced by various microorganisms as carbon and energy storage compounds. They are of significant interest due to their potential as environmentally friendly alternatives to petroleum-based plastics. PHAs are naturally occurring biodegradable polymers. PHAs are synthesized and stored as water-insoluble inclusions in the cytoplasm of several bacteria and used as carbon and energy reserve material. PHAs are intracellular microbial polyesters synthesized by many species of bacteria and archaea, generally under nutrient limitation and excess of carbon source as storage granules of energy and also conferring stress resistance to prokaryotes. PHAs are naturally occurring, and the choice of microbial strains and carbon sources for cell cultivations results in biopolymers with different physicochemical properties.
[0018] In yet another embodiment, the efficacy of the PHA producing bacteria from lignocellulosic waste was evaluated using a Plackett-Burman design for screening significant factors and a Box-Behnken design for optimization. The significant factors identified include pre-treated lignocellulose concentration, pH, incubation period, inoculum size. The optimized conditions led to a significant increase in PHA yield compared to the standard glucose. The structural and functional integrity of the produced PHA was confirmed using UV spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), and Scanning Electron Microscopy (SEM), Nuclear Magnetic Resonance (NMR), Thermogravimetric analysis (TGA). Some of the advantages of above-mentioned invention are utilizes renewable lignocellulosic waste as a carbon source, enhanced PHA production through genetic engineering, produces biodegradable plastics, reducing reliance on petrochemical plastics, and potentially lowers production costs by utilizing waste materials.
[0019] In one embodiment, referring to figure 1, the method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae comprises various steps such as: a) Isolation of polyhydroxyalkanoates (PHA) producing bacteria (110) where sewage water is serially diluted and plated on Nile Blue minimal agar media to screen PHA bacteria, b) Preparation of lignocellulosic waste (120) where lignocellulosic waste, such as agricultural residues, is subjected to physical, chemical, or biological pretreatment to release fermentable sugars. This step is crucial for converting the complex polysaccharides into a form that can be readily utilized by the algae c) Genetic engineering of algae (130) where the algae are genetically engineered to incorporate the phaC gene isolated from bacteria, which is responsible for the polymerization of PHA. The transformation is carried out using standard molecular biology techniques. The phaC gene is integrated into the algal genome under the control of a suitable promoter to ensure high-level expression, d) Cultivation of algal transformants (140) where genetically modified algae are grown in a culture medium containing the pre-treated lignocellulosic waste. The concentration of pre-treated lignocellulosic extract is optimized to maximize PHA production. The algae utilize the sugars derived from the lignocellulosic waste to produce PHA intracellularly, e) Polyhydroxyalkanoates (PHA) extraction and purification (150) where, PHA is extracted from the algal biomass using a solvent extraction method. The extracted PHA is then purified by precipitation and washing steps to obtain a high-purity biopolymer suitable for various applications.
, Claims:1. A method for production of polyhydroxyalkanoates (PHA) using genetically engineered algae comprises:
a) isolation of polyhydroxyalkanoates (PHA) producing bacteria (110);
b) preparation of lignocellulosic waste (120);
c) genetic engineering of algae (130);
d) cultivation of algal transformants (140); and
e) polyhydroxyalkanoates (PHA) extraction and purification (150).
2. The method as claimed in claim 1, wherein PHA producing bacteria is isolated by serially diluting the sewage water and plating it on Nile blue minimal agar media.
3. The method as claimed in claim 1, wherein lignocellulosic waste is prepared by subjecting it to physical, chemical, or biological pre-treatment to release fermentable sugars and converting complex polysaccharides into a form that is utilized by algae.
4. The method as claimed in claim 1, wherein algae is genetically engineered to incorporate the phaC gene isolated from bacteria, which is responsible for the polymerization of PHA.
5. The method as claimed in claim 1, wherein algal transformants are cultivated in a culture medium containing the pre-treated lignocellulosic waste and algae utilize the sugars derived from the lignocellulosic waste to produce PHA intracellularly.
6. The method as claimed in claim 1, wherein PHA is extracted from the algal biomass using a solvent extraction method and then purified by precipitation and washing to obtain a high-purity biopolymer.

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