Engineered organism for utilizing sucrose

Abstract

The present disclosure relates to an engineered organism comprising at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene, such that said engineered organism utilizes sucrose, wherein said engineered organism has ≥ 90% homology to Cupriavidus genus The present disclosure further relates to a method for carrying out fermentation using the engineered organism in the presence of sucrose, such that sucrose is utilized.

Description

[0001]TITLE OF THE INVENTION ENGINEERED ORGANISM FOR UTILIZING SUCROSE CROSS-REFERENCETORELATEDAPPLICATIONSANDPRIORITYThe present application claims priority from Indian patent application number (202421041635) filed on (29 / 05 / 2024), incorporated herein by a reference. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 10,229 bytes ASCII (Text) file named "Sequence Listing.txt," created on May 27, 2024. TECHNICAL FIELD The present subject matter, in general, relates to the field of process chemistry. More particularly, the present subject matter relates to an engineered organism and method for utilizing sucrose using the same. BACKGROUND Microbial fermentation is a complex and diverse process that involves the use of microorganisms to transform organic compounds into valuable products across various industries. This process has been utilized for centuries to produce a wide range of products, including ethanol, lactic acid, acetic acid, citric acid, butanol, hydrogen, polyhydroxy alkanoates (PHA), enzymes, and antibiotics, among others. The versatility of microbial fermentation lies in the ability to manipulate various factors such as temperature, pH, nutrient supply, and oxygen availability, to yield different products. One of the key advantages of microbial fermentation is its scalability. This makes it a cost-effective and sustainable option compared to traditional chemical processes that rely on non-renewable resources. Sucrose is a critical ingredient in many industrial processes that plays a pivotal role in the fermentation industry. Sucrose fermentation is a highly beneficial process due to its abundance, versatility, ease of fermentation, and renewability. It is a cost- effective carbohydrate that is readily available in various plant sources and can be efficiently metabolized to produce a diverse range of products, including biofuels, organic acids, and other valuable chemicals. Compared to other complex carbohydrates, sucrose is relatively easy to ferment, making it ideal for large-scale industrial fermentation processes. Additionally, it can be sustainably produced, contributing to the development of a bio-based economy, and reducing reliance on fossil fuels. Microbial fermentation of sucrose is a significant process in microbial metabolism, where various organisms, including bacteria, yeast, and fungi, utilize sucrose to produce energy and precursors for growth and biosynthesis. The utilization of sucrose by microorganisms finds its application in food and beverage production, biofuel production, and the production of valuable chemicals and pharmaceuticals. Cupriavidus necator is a type of bacteria that has gained noteworthy attention in the field of biotechnology owing to its remarkable adaptability and ease of genetic manipulations. One of the primary advantages of Cupriavidus necator is its natural competence, which refers to its ability to take up exogenous DNA from the environment without the need for artificial transformation methods. This simplifies the process of introducing foreign DNA into the bacterial cells for genetic manipulation. Cupriavidus necator is also relatively easy to genetically modify, which allows researchers to selectively remove or introduce specific genes to study their functions or engineer strains with desired characteristics. Cupriavidus necator has a well-characterized metabolism, particularly with respect to its ability to accumulate polyhydroxyalkanoates (PHA). This makes it an excellent candidate for metabolic engineering approaches aimed at optimizing PHA production or redirecting metabolic pathways for the synthesis of other valuable compounds. Cupriavidus necator also harbors a variety of regulatory elements that govern gene expression and metabolic pathways, enabling fine-tuning of gene expression levels and metabolic fluxes to optimize desired phenotypes. Another significant advantage of Cupriavidus necator is its scalability and industrial relevance, which is particularly advantageous for industrial applications where scalable production of bio-based products is desired. Naturally occurring strains and mutants of C. necator cannot metabolize sucrose due to the absence of sucrose utilization pathways. However, different hydrolytic or phosphorolysis enzymes have been reported in bacteria to metabolize sucrose intracellularly. In recent studies (Hisashi Arikawa et.al.), a method has been developed to produce polyhydroxyalkanoates (PHA) from C. necator by introducing nucleotide sequences into the bacterium's genetic makeup that enable the sucrose hydrolysis pathway. This approach involves exploiting the capabilities of C. necator to produce PHA from sucrose using a sucrose hydrolysis pathway. However, as compared to the hydrolysis pathway, sucrose utilization by the phosphorolysis pathway is considered an energy-efficient pathway since it does not require additional ATP and relies on phosphorylation using inorganic phosphate. In pursuance of this, the present disclosure aims to place an engineered C. necator for utilizing sucrose using the sucrose phosphorylase pathway. SUMMARY An embodiment of the present disclosure relates to an engineered organism comprising at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene, such that said engineered organism utilizes sucrose, wherein said engineered organism has ≥ 90% homology to Cupriavidus genus. Another embodiment of the present disclosure relates to a method for utilizing sucrose comprising engineering an organism having, at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene; and carrying out fermentation using said engineered organism in the presence of sucrose, such that sucrose is utilized. This summary is not intended to identify all the essential features of the claimed subject matter, nor is it intended to be used in determining or limiting the scope of the claimed subject matter. BRIEF DESCRIPTION OF DRAWINGS The detailed description of the drawings is outlined with reference to the accompanying figures. In the figures, the left-most digit (s) of a reference number identifies the Figure in which the reference number first appears. The same numbers are used throughout the drawings to refer to like features and components. Fig. 1 illustrates A. sucrose utilization pathway consisting of phosphotransferase system (PTS), and sucrose-6-P hydrolase; B. Non-PTS sucrose utilization pathway consisting of sucrose permease and sucrase; and C. Non-PTS sucrose utilization pathway consisting of sucrose permease and sucrose phosphorylase. Fig.2 demonstrates the cell growth kinetics of recombinant / engineered and control strains in a (shake) flask culture. Fig. 3 demonstrates sucrose fermentation: sucrose analysis by High-performance liquid chromatography (HPLC) in a (shake) flask culture. Fig. 4 demonstrates the batch fermentation of sucrose by engineered strain CN- SPrv in a fermentor. Figs. 5-8 demonstrate plasmid constructed for sucrose phosphorylase pathway genes (Fig. 5 - pBBR1MCS2-SPrv-cscB-pgm comprising codon optimised sequence of sucrose phosphorylase of Rhizobium vitis (SPrv); Fig. 6 - pBBR1MCS2-SPlm-cscB-pgm comprising codon optimised sequence of sucrose phosphorylase of Leuconostoc mesenteroides (SPlm); Fig. 7 - pBBR1MCS2- SPsm-cscB-pgm comprising codon optimised sequence of sucrose phosphorylase of Streptococcus mutans (SPsm); and Fig. 8 - p BBR1MCS2-SPba-cscB-pgm comprising codon optimised sequence of sucrose phosphorylase of Bifidobacterium adolescentis (SPba)). Fig. 9 demonstrates pBBR1MCS2 empty plasmid. DETAILEDDESCRIPTIONReference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” “alternate embodiment”, or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” or “in an embodiment”, “in an alternate embodiment”, or “in a related embodiment” in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments. Reference throughout the specification to “components”, “component”, “features”, or “feature” means a constituent or group of constituents embodying the process. Before the present process is described, it is to be understood that this disclosure is not limited to the particular process as described, as there can be multiple possible embodiments which are not expressly illustrated in the present disclosure but may still be practicable within the scope of the present disclosure. Also, the technical solutions offered by the present disclosure are clearly and completely described below. Examples in which specific conditions may not have been specified, have been conducted under conventional conditions or in a manner recommended by the manufacturer. The present disclosure relates to an engineered organism and method for utilizing sucrose using the same. An aspect of the instant disclosure relates to an organism engineered for comprising at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene, such that said engineered organism utilizes sucrose, wherein said engineered organism has ≥ 90% homology to Cupriavidus genus. For the purpose of instant disclosure and as is perceivable to a person skilled in the art, the term “engineered”, or “engineering” pertains to “genetically engineered / engineering” and involves the process of using recombinant DNA (rDNA) technology to alter the genetic makeup of an organism. Further, for the purpose of instant disclosure, the term “engineered organism” also pertains to “host”, “host organism” or “to-be-engineered organism”. In an embodiment, the engineered organism has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to Cupriavidus genus. For the purpose of instant disclosure, the Cupriavidus genus comprises at least one of Cupriavidus necator, Cupriavidus metallidurans, Cupriavidus taiwanensis, Cupriavidus basilensis, Cupriavidus gilardii, or Cupriavidus taiwanensis. In another related embodiment, the engineered organism has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to Cupriavidus necator. In another embodiment, the organism is engineered to comprise at least one sucrose phosphorylase encoding gene. It is known that sucrose phosphorylase (sucP) catalyzes the reversible phosphorolysis of sucrose into α-D-glucose 1-phosphate (Glc1P) and D-fructose. The enzyme is a glucosyl transferase that can transfer glucosyl group of sucrose to inorganic phosphate (Pi) forming glucose 1-P and D-fructose. In a related embodiment, the sucrose phosphorylase encoding gene belongs to at least one of bacterial, fungal, archaeal, or yeast source, perceivable to a person skilled in the art; and preferably belongs to at least one of Leuconostoc mesenteroides, Streptococcus mutans, Bifidobacterium adolescentis, or Rhizobium vitis. In another related embodiment, the sucrose phosphorylase encoding gene has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to sucrose phosphorylase gene of least one of Rhizobium vitis, Leuconostoc mesenteroides, Streptococcus mutans, or Bifidobacterium adolescentis. In an embodiment, the sucrose phosphorylase encoding gene is at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In yet another embodiment, the organism is engineered to comprise at least one sucrose permease encoding gene. It is known that sucrose permease (cscB / scrA) is a sucrose / H+ symporter which belongs to the oligosaccharide / H+ symporter sub-family of the Major Facilitator Superfamily and catalyzes sugar / H+ symport across the cytoplasmic membrane. Bacteria with cscB gene are able to take up sucrose by utilizing the H+ gradient across plasma membranes. In a related embodiment, the sucrose permease encoding gene belongs to at least one of bacterial, yeast, or plant source, perceivable to a person skilled in the art; and preferably, belongs to Escherichia coli. In another related embodiment, the gene has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to sucrose permease gene of E.coli. In an embodiment, the sucrose permease encoding gene is SEQ ID NO: 5. In a further embodiment, the organism is engineered to comprise at least one phosphoglucomutase encoding gene. It is known that phosphoglucomutase (pgm) catalyzes the interconversion of glucose-1-phosphate and glucose-6-phosphate and thus plays an important role in the regulation of carbohydrate metabolism. In a related embodiment, the phosphoglucomutase encoding gene belongs to at least one of bacterial, archaeal, yeast, plant, or animal source, perceivable to a person skilled in the art; and preferably belongs to Escherichia coli. In another related embodiment, the gene has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to phosphoglucomutase gene of E.coli. In an embodiment, the phosphoglucomutase encoding gene is SEQ ID NO: 6. Another aspect of the instant disclosure relates to a method for utilizing sucrose comprising engineering an organism having, at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene; and carrying out fermentation using said engineered organism in the presence of sucrose, such that sucrose is utilized. An embodiment relates to engineering an organism having at least one sucrose phosphorylase encoding gene as described above. A related embodiment relates to engineering an organism having at least one sucrose permease encoding gene as described above. Another related embodiment relates to engineering an organism having at least one phosphoglucomutase encoding gene as described above. In a related embodiment, at least one vector, such as a plasmid, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), viral vectors, phage vectors, transposon vectors, or cosmid vectors is implemented as per requirement. In another related embodiment, transformation of the host organism is carried out using at least one of chemical transformation, electroporation, heat shock transformation, microinjection or conjugation known to a person skilled in the art. An embodiment relates to carrying out fermentation using the engineered organism. In a related embodiment, the fermentation is carried out in the presence of a synthetic medium. For the purpose of instant disclosure, synthetic medium is a “defined medium” known to a person skilled in the art. The synthetic medium of instant disclosure comprises at least one of disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), diazanium sulfate (NH4SO4), magnesium sulfate (MgSO4), calcium chloride (CaCl2), and trace element solution. In a related embodiment, trace element comprises at least one of Ferrous sulfate heptahydrate (FeSO4·7H2O), Manganese (2+) sulfate pentahydrate (MnSO4·5H2O), Sodium tetraborate decahydrate (Na2B4O7·10H2O), Zinc sulphate heptahydrate (ZnSO4·7H2O), Copper sulfate pentahydrate (CuSO4·5H2O), Calcium chloride dihydrate (CaCl2·2H2O), Ammonium molybdate ((NH4)6Mo7O24), and HCl, as per requirement. In another related embodiment, the synthetic medium comprises sucrose. In yet another embodiment, the synthetic medium comprises citric acid. The concentration of the above-described components comprised in the synthetic medium may be perceivable to a person skilled in the art as per requirement. It is known that in comparison to the phosphotransferase (PTS) dependent system of sucrose utilization via the sucrose hydrolysis pathway, the phosphorolysis pathway consumes lesser ATP for the conversion of one molecule of sucrose into fructose-6-P and glucose-6-P. Figure 1 shows sucrose metabolism (including transport and utilization) in bacteria can be categorized into three types. The phosphotransferase system (PTS) includes the phosphoenolpyruvate (PEP)- dependent sucrose-specific PTS, sucrose 6-phosphate hydrolase, and fructokinase, and is found in both gram-positive and gram-negative bacteria. Some micro- organisms employ a non-PTS system including sucrose permease and sucrase (sucrose hydrolase or invertase). While few of the microbes have the ability to use non-PTS system involving sucrose permease and sucrose phosphorylase. Pathway based on sucrose phosphorylase is an energy-conserving sucrose utilization pathway as it employs inorganic phosphate to convert sucrose into glucose 1- phosphate resulting in the saving of additional ATP molecule required for conversion of glucose to glucose 6-P by glucokinase enzyme. In an embodiment, the engineered organism utilizes sucrose; preferably through the phosphorolysis pathway. Referring to Fig. 1, in an embodiment of the instant disclosure, an engineered organism using heterologous nucleic acids coding for key enzymes of the sucrose phosphorylase pathway is achieved. Expression of genes coding for sucrose phosphorylase (sucP) and sucrose transport (cscB) allows sucrose utilization via reversible phosphorolysis of sucrose in the presence of inorganic phosphate, to yield glucose-1-P and fructose. The fructose is catalyzed by ATP dependent fructokinase to generate fructose-6-P. The glucose-1-P can be converted to glucose- 6-P by the enzyme phosphoglucomutase (pgm). Both the products (glucose-6-P and fructose-6-P), as intermediates, then enter the glycolytic pathway, that eventually can be implemented for producing organic acids, alcohols, or other valuable compounds perceivable to a skilled person in the art. In a related embodiment, the sucrose utilization is carried out in the presence of inorganic phosphate, perceivable to a skilled person in the art; and preferably, in the presence of at least one of Na2HPO4, and KH2PO4. In another related embodiment, sucrose utilization is carried out for producing at least one of organic acids, alcohols, or other valuable compounds known to a person skilled in the art; and preferably, producing polyhydroxyalkanoate (PHA). In an embodiment, the sucrose utilization is carried out for producing at least one polyhydroxyalkanoate (PHA ) such as poly-3-hydroxybutyrate (PHB), poly-3- hydroxybutyrate-co-4-hydroxybutyrate (P(3-HB-co-4-HB)), poly-3- hydroxybutyrate-co-valerate (PHBV), and polyhydroxybutyrate-co-hexanoate (PHBH). In a related embodiment, the maximum cell density of the engineered organism using OD600 is ≥ 18. In another related embodiment, the dry cell weight of the engineered organism after fermentation is ≥ 4 g / L. In yet another related embodiment, the PHB content achieved is ≥ 59.42%. In a further embodiment, the PHB titer achieved is ≥ 2.26 g / L. The instant disclosure finds application in bioplastics, packaging, medical, agricultural, cosmetics, textile, food packaging, food service, and environmental remediation, as is perceivable to a person skilled in the art. Various modifications to the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. However, one of ordinary skill in the art will readily recognize that the present disclosure is not intended to be limited to the embodiments illustrated but is to be accorded the widest scope consistent with the principles and features described herein. The foregoing description shall be interpreted as illustrative and not in any limiting sense. A person of ordinary skill in the art would understand that certain modifications could come within the scope of this disclosure. The features and properties of the present disclosure are described in further detail below with reference to examples. Example 1 Engineered organism for utilizing sucrose Engineering an organism having at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene. For this, Cupriavidus necator (DSM 545) (C. necator) with identification No. MTCC 25480 was implemented (as a host strain / to-be-engineered organism). Cupriavidus necator (Ralsotonia eutropha) is a well-known bacterial platform that has wide metabolic diversity. However, naturally occurring strains as well as its mutants are not capable of utilizing sucrose as a carbon source. Selection of sucrose phosphorylase pathway genes For this experiment, different variants of sucrose phosphorylase (SucP) encoding genes, including well-characterized SucP enzymes from Leuconostoc mesenteroides, Streptococcus mutans, and Bifidobacterium adolescentis were implemented. The gene encoding for SucP for one of the non-characterized enzymes of Rhizobium vitis was also implemented. The selection was done on the basis of its less than 70% homology with SucP from other well-characterized categories like Bifidobacterium adolescentis. Escherichia coli, and specifically, Escherichia coli strain W was implemented for sucrose permease (cscB), and phosphoglucomutase (pgm) encoding genes. Construction of Plasmid for sucrose phosphorylase pathway genes: (See Figs. 5-9) Codon-optimized genes of sucrose phosphorylase (sucP), sucrose permease (cscB) and phosphoglucomutase (pgm) were designed. All three genes including sucP, cscB, and pgm were linked by a linker sequence derived from the PHA operon. To improve the expression ribosomal binding site (RBS) of the PHA synthase, gene (phaC1) was introduced next to the lac promotor. The synthetic construct was cloned at nucleotide position 3147-3514 by replacing lacZα ORF in the pBBR1MSC2 expression vector to obtain constructed plasmids (See Figs. 5-8). pBBR1MCS2 (see Fig. 9) is a low copy number shuttle vector of about 5148 bp size which replicates in many gram-negative bacteria. Cloned genes are driven by a derepressed lac promoter and contain a kanamycin selection marker. pBBR1MSC2 vector can be mobilizable via RP4 / RK2 mating system, e.g. using E.coli strains S17 or SM10 through conjugation. Table 1 provides details of the genes implemented in the instant case. [Table 1] SEQ Gene-Enzyme Microorganism UniPort / E.C. NCBI Nucleo ID Name Name Protein Numb Gene tide NO. Accessio er Accessio Length n n Number Number 1 Sucrose Rhizobium vitis CAA8042 2.4.1. Z22734.1 1467 Phosphorylase (Agrobacterium 4.1 7 vitis) 2 Sucrose Leuconostoc BAA1434 2.4.1.7 D90314.1 1473 Phosphorylase mesenteroides(A 4.1 TCC 12291) 3 Sucrose Streptococcus CAA3084 2.4.1.7 X08057.1 1464 Phosphorylase mutans strain 6.1 GS5 4 Sucrose Bifidobacterium AAO338 2.4.1.7 CP02834 1515 Phosphorylase adolescentis 21.1 1.1 strain: 1-11 5 cscB-Sucrose Escherichia coli ADT760 DQ7777 1248 Permease strain W 24.1 2.7.1.2 70.1 11 6 pgm- Escherichia coli AAA570 5.4.2.2 NC_0009 1641 Phosphogluco strain W 67.1 13.3 mutase Transformation of C. necator: The constructed plasmids were transformed into the host organism, C. necator by electroporation to obtain engineered C. necator. For this, C. necator electrocompetent cells were prepared by inoculating glycerol stock in about 5 ml of nutrient broth (NB) supplemented with gentamicin for about 38 – 42 h at about 30°C to obtain seed, which was then subcultured in fresh NB with gentamicin (1:50 dilution) at 30°C. When an optical density of about 0.4–0.6 was achieved at about 600 nm (OD600), the C.necator broth was transferred to ice and chilled for about 5 – 10 min. Chilled cells were then transferred to about 50 ml centrifuge tube and centrifuged at about 6000 × g, about 4°C for about 1.5 min. The supernatant was decanted, and cells were resuspended in about 25 ml (about 50 mM) CaCl2and incubated for about 15 min on ice. The cells were then centrifuged at about 6500 × g, about 4°C for about 2 min and the supernatant was decanted. Cells were washed twice using about 25 ml and about 15 ml of ice-cold (about 0.2 M) sucrose, respectively. At the end of each wash, cells were centrifuged at about 6500 × g, about 4°C for about 2–3 min, and the supernatant was decanted. The cell pellet was finally resuspended in about 500 µl of ice-cold transformation buffer containing about 0.2 M sucrose solution. Aliquots of C. necator electrocompetent cells (about 100 µl) were transferred into about 1.5 ml centrifuge tubes. C. necator electrocompetent cells were transformed using the constructed plasmids shown in Table 2 via the electroporation method of transformation. For this, the constructed plasmid (DNA) (about ~0.25 µg) was added to the cells and gently mixed before transferring the cells and DNA into a chilled about 2-mm electroporation cuvette. Electroporation was performed at about 2.3 to 2.5 kV and about 1ml of NB was added immediately. The cells were then transferred to about 2 ml centrifuge tube for outgrowth at about 30°C for about 2 h. Following outgrowth, the cells were diluted, and depending on the initial cell concentration, about 1–9% (v / v) of the transformed cells / engineered organism (cells) were plated on NB agar plates with about 250 µg /  ml kanamycin and incubated for about 40– 48 h. The same transformed cells / engineered organism (cells) after outgrowth were diluted about 10-6times before plating on NB agar plates without antibiotics to obtain the corresponding number of survivor cells (viability Control). The transformant colonies / engineered organism (cells) were repatched on NB agar plates with about 300 µg  / ml kanamycin. Plates were incubated at about 30°C for about 48-72 h to allow the growth of transformants / engineered organism (cells) and tested for the desired traits. Glycerol stocks of the transformants / engineered organism (cells) were prepared and stored at about -80°C ultra-deep freezers for future studies. Alternatively, the constructed plasmids can be transferred into C. necator via conjugation using E.coli S17 donor cells. Tables 2 and 3 provide information on the engineered organisms / transformant strains. Table 2: Details of the C. necator host strain and transformants Sr.No. C.necator (host / Constructed Plasmid Source of the sucrose Transformant strains / Details Phosphorylase (SucP) engineered organism) 1 CN-545 - Control No plasmid NA 2 CN-CTR – Control pBBR1MCS2 Empty NA Plasmid 3 CN-SPrv pBBR1MCS2-SPrv- Rhizobium vitis cscB-pgm (Agrobacterium vitis) 4 CN-SPlm pBBR1MCS2-SPlm- Leuconostoc mesenteroides cscB-pgm 5 CN-SPsm pBBR1MCS2-SPsm- Streptococcus mutans cscB-pgm 6 CN-SPba pBBR1MCS2-SPba- Bifidobacterium adolescentis cscB-pgm [Table 3] Information of biological material (engineered organisms / transformants): Name of the depository institution: National Centre For Cell Science (NCCS) Address of the depository institution: NCCS Complex, P. B. No. 40, Ganeshkhind P.O, Pune – 411007, Maharashtra, India. Date of deposit: May 27, 2024 Sr. No. C.necator (host / Accession Strain Transformant strains / Number Designation engineered organism) 1 CN-SPba MCC 0278 PMCC / B / 70 2 CN-SPsm MCC 0279 PMCC / B / 69 3 CN-SPlm MCC 0280 PMCC / B / 68 4 CN-SPrv MCC 0281 PMCC / B / 67 Carrying out fermentation using the engineered organism in the presence of sucrose Sucrose utilization for producing at least one PHA using the engineered organism. All recombinant / engineered strains of C. necator and C. necator empty plasmid control strains (CN-CTR) were evaluated for sucrose utilization and PHB production. Recombinant / engineered C. necator strains were inoculated from glycerol stocks in NB comprising about 300 µg / ml kanamycin along with CN-CTR strain. All the cultures were incubated in a rotary shaking incubator at about 30°C / about 150 rpm for overnight growth. The synthetic medium used for the fermentation evaluation comprised about 4.47 g / L Na2HPO4, about 1.5 g / L KH2PO4, about 1.0 g / L NH4SO4, about 2 ml / L MgSO4(about 10% stock), about 1 ml / L CaCl2(about 1% stock) and about 1ml / L trace element solution. Stocks including MgSO4, CaCl2 and trace elements were autoclaved separately. Sterilization was carried out at about 121°C for about 20 min. Trace element stock solution was prepared using about 10 g / L FeSO4·7H2O, about 0.5 g / L MnSO4·5H2O, about 0.23 g / L Na2B4O7·10H2O (Sodium tetraborate decahydrate), about 2.25 g / L ZnSO4·7H2O, about 1g / L CuSO4·5H2O, about 2 g / L CaCl2·2H2O, about 0.1 g / L (NH4)6Mo7O24 (Ammonium molybdate), and about 10 ml of 35% HCl. The seed cultures were washed and normalized to initial OD in the range of about 0.05-0.09 for inoculation in about 100 ml synthetic media containing about 2% sucrose as a carbon source and about 300 µg / ml kanamycin. The CN-CTR strain fermentation was also evaluated in separate flasks containing about 2% glucose, about 2% sucrose, 2% fructose, and about 300 µg / ml kanamycin as controls. Table 4 demonstrates details of microbial cultures and media. All the flasks were incubated in a rotary shaking incubator at about 30°C / about 150 rpm for up to 6 days. Growth was monitored by OD600and sugar utilization was analysed using HPLC. Dry cell weight (DCW) was measured from lyophilized cell mass and PHB analysis was carried out using gas chromatography. All fermentation evaluations were carried out in duplicate using biological replicates. [Table 4] Sr.No. Culture Details Sugars and Antibiotic Details in synthetic media 1 CN-CTR (pBBR1MCS2 Empty Plasmid) 2% glucose+ 300 µg / ml (control) kanamycin 2 CN-CTR (pBBR1MCS2 Empty Plasmid) 2% fructose+ 300 µg / ml (control) kanamycin 3 CN-CTR (pBBR1MCS2 Empty Plasmid) 2% sucrose+ 300 µg / ml (control) kanamycin 4 CN-SPrv (pBBR1MCS2-SPrv-cscB-pgm) 2% sucrose+ 300 µg / ml kanamycin 5 CN-SPlm (pBBR1MCS2-SPlm-cscB- 2% sucrose+ 300 µg / ml pgm) kanamycin 6 CN-SPsm (pBBR1MCS2-SPsm-cscB- 2% sucrose+ 300 µg / ml pgm) kanamycin 4 CN-SPba (pBBR1MCS2-SPba-cscB- 2% sucrose+ 300 µg / ml pgm) kanamycin Results: Utilization of sucrose using engineered C. necator strains: Sucrose fermentation was carried out using different recombinant / engineered and CN-CTR strains and cell growth was analysed by measuring OD600. Fig. 2 demonstrates the cell growth kinetics of recombinant / engineered as well as control strains. [Table 5] Cell Growth Kinetics (OD600) on Sucrose Fermentation Time in h Sr.No.SDaemtapillse0 22.3 44.3 69.3 93 129 1CN-CTR-Glucose0.069 2.689 19.458 24.703 25.625 29.600 2CFNru-CctTosRe- 0.077 1.026 11.860 19.068 24.475 32.835 3CN-CTR-Sucrose0.079 0.829 0.730 0.777 0.692 1.187 4 CN-SPrv 0.062 1.895 20.210 25.460 28.170 32.305 5 CN-SPlm 0.067 0.010 0.165 0.002 0.936 0.094 6 CN-SPsm 0.070 0.005 1.191 1.365 8.101 10.040 7 CN-SPba 0.056 0.012 0.249 0.014 5.524 0.550 As per Table 5, engineered C. necator (transformant) CN-SPrv containing sucrose phosphorylase (SucP) derived from Rhizobium vitis showed maximum cell density (OD600) of up to 32.305 at about 129 h. Whereas the control strain, CN-CTR showed maximum cell density up to (OD600) 1.287 at about 129 h. Engineered C. necator (transformant) containing other sucrose phosphorylase encoding genes from L. mesenteroides , S. mutans and B. adolescentis showed maximum cell density (OD600) of up to 26.660, 30.710, and 18.555, respectively. Fig.3 shows the profile of sugar consumption during fermentation with control and recombinant / engineered strains. Estimation of Dry Cell weight (DCW) and PHB: At the end of fermentation (after about 129 h), cells were harvested by centrifugation and cell mass was lyophilized for estimation of DCW and analysis of PHB content by gas chromatography. Table 6: DCW, PHB content and PHB Titer of recombinant and CN-CTR strain grown on Sucrose. Sr.No. Culture Details DCW in %PHB PHB g / L Titer g / L Average Average Average 1 CN-CTR (Glucose) 5.23 63.35 3.30 2 CN-CTR (Fructose) 5.34 71.21 3.80 3 CN-CTR (Sucrose) 0.54 1.44 0.01 4 CN-SPrv (pBBR1MCS2-SPrv-cscB-pgm) 6.20 77.71 4.82 5 CN-SPlm (pBBR1MCS2-SPlm-cscB-pgm) 6.14 66.90 4.07 6 CN-SPsm (pBBR1MCS2-SPsm-cscB-pgm) 5.36 70.82 3.81 7 CN-SPba (pBBR1MCS2-SPba-cscB-pgm) 4.13 59.42 2.26 As per Table 6, engineered C. necator (transformant) CN-SPrv containing sucP gene derived from Rhizobium vitis showed maximum DCW of 6.20 g / L, PHB content up to 77.71 (%) and PHB titer of up to 4.82±0.04 g / L. In comparison, the control strain, CN-CTR showed maximum DCW up to 0.54g / L, PHB content less than 2% and PHB titer of up to 0.01 g / L. Engineered C. necator (transformant) containing other sucrose phosphorylase encoding genes from L. mesenteroides , S. mutans and B. adolescentis showed DCW of 6.14, 5.36, and 4.13g / L, PHB content up to 66.90, 70.82, and 59.42 (%) and PHB titer of up to 4.07, 3.81, and 2.26 g / L, respectively. Sugar Analysis: Table 7: Sugar Analysis (% w / w) by HPLC Fermentation Time in h Sr. Sample Sugar No. Name Type 0 22.3 44.3 69.3 93 129 Avg Avg Avg Avg Avg Avg CN- Sucrose 2.055 2.025 2.02 2.04 2.075 2.035 1 CTR(2% Glucose 0 0 0 0 0 0.05 Sucrose) Fructose 0 0 0 0.05 0 0.01 Sucrose 2.035 0.92 0.895 0.435 0.24 0.16 2 CN-SPrv Glucose 0 0.01 0 0.01 0.01 0.02 Fructose 0 0.48 0.11 0.7 0.03 0.02 Sucrose 1.97 1.965 1.595 0.555 0.435 0.32 3 CN-SPlm Glucose 0 0.02 0.02 0.03 0.03 0.05 Fructose 0 0.05 0.205 0.17 0.035 0.03 Sucrose 2.005 1.205 0.94 0.445 0.3 0.19 4 CN-SPsm Glucose 0 0.01 0 0.01 0.01 0.02 Fructose 0 0.345 0.1 0.08 0.03 0.02 Sucrose 1.96 1.715 1.11 0.675 0.61 0.56 5 CN-SPba Glucose 0 0.03 0 0.02 0.03 0.03 Fructose 0 0.425 0.15 0.05 0.01 0 As per Table 7, engineered C. necator (transformant) CN-SPrv containing sucrose phosphorylase (SucP) derived from Rhizobium vitis showed maximum sucrose utilization rate in the first 24 h with minimum residual sucrose concentration of about 0.16% sucrose in 129 h. Whereas the control strain, CN-CTR showed negligible sucrose utilization with residual sucrose same as the initial concentration of about 2% in 129 h. Engineered C. necator (transformant) containing other sucrose phosphorylase encoding genes from L. mesenteroides , S. mutans and B. adolescentis showed lesser sucrose utilization rate as compared to CN-SPrv strain containing sucrose phosphorylase (SucP) derived from Rhizobium vitis in first 24 h. The residual sucrose concentrations in CN-SPlm, CN-SPsm, and CN-SPba strains were found to be 0.32%, 0.19%, and 0.56% respectively. Example 2 Sucrose utilization for producing at least one PHA using the engineered organism. The engineered strain of C. necator, CN-SPrv was evaluated for sucrose utilization and PHB production in a fermenter. For this, the engineered strain of C. necator, CN-SPrv was inoculated from glycerol stocks in NB comprising about 300 µg / ml kanamycin. The resulting seed was then incubated in a rotary shaking incubator at about 30°C / about 150 rpm for overnight growth, as described above. Seed culture was prepared by inoculating about 5% of washed CN-SPrv seed (above obtained) in the synthetic medium comprising about 4.47 g / L Na2HPO4, about 6.65 g / L KH2PO4, about 2.0 g / L NH4SO4, about 2 ml / L MgSO4 (about 10% stock), about 1 ml / L CaCl2 (about 1% stock), about 1ml / L trace element solution, about 0.935 g / L citric acid, and about 20 g / L sucrose. The pH of the medium was adjusted to about 6.5-6.7 and stocks including MgSO4, CaCl2, and trace elements were autoclaved separately. Sterilization was carried out at about 121°C for about 20 min. After sterilization, the stock solutions (MgSO4, CaCl2, and trace elements ) along with about 300 µg / ml kanamycin were added to the medium. The seed culture prepared previously was incubated in a rotary shaking incubator at about 30°C / about 150 rpm for overnight growth. About 14 L fermenter contained about 7.0 L of the synthetic medium comprising about 4.0 g / L Na2HPO4, about 13.3 g / L KH2PO4, about 2.0 g / L NH4SO4, about 2 ml / L MgSO4 (about 10% stock), about 1 ml / L CaCl2 (about 1% stock), about 1ml / L trace element solution, about 1.87 g / L citric acid, and about 40 g / L sucrose. Synthetic medium, stocks including MgSO4, CaCl2,and trace elements were autoclaved separately at about 121°C for about 20 min. After sterilization, the stock solutions (MgSO4, CaCl2, and trace elements) along with about 300 µg / ml kanamycin were added to the medium. Fermenter was inoculated with about 10% of seed culture. The fermentation was carried out at about 30°C for about 72 h with aeration of about 1.0 vvm and agitation at about 600 rpm. The pH was adjusted to about 6.5-6.6 in auto mode. The sucrose batch fermentation of CN-SPrv can be evaluated in Fig. 4. Sampling was done every about 12 h to analyze culture purity, sugar profile, DCW and PHB (Table 8) [Table 8] Culture Details Time (h) DCW (g / L) Sucrose (g / L) PHB% CN-SPrv 0 0.35 40 0 12 2.55 30.6 37 24 7.4 10.5 53.88 36 10.25 7.7 63.99 48 11.1 6.2 67.09 60 12.65 3.5 69.33 72 13.55 2.1 71.35 As per Table 8, The engineered CN-SPrv utilized about 40 g / L of sucrose in about 72 h resulting in about 13.55 g / L of DCW and about 71.35 % PHB accumulation. The embodiments, examples and alternatives of the preceding paragraphs or the description and drawings, including any of their various aspects or respective individual features, may be taken independently or in any combination. Features described in connection with one embodiment are applicable to all embodiments, unless such features are incompatible. The preferred embodiments of the present invention are described in detail above. It should be understood that ordinary technologies in the field can make many modifications and changes according to the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

We claim:

1. An organism engineered for comprising: at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene, such that said engineered organism utilizes sucrose, wherein said engineered organism has ≥ 90% homology to Cupriavidus genus.

2. The engineered organism as claimed in claim 1, wherein the engineered organism has ≥ 90% homology to Cupriavidus necator.

3. The engineered organism as claimed in claim 1, wherein the sucrose phosphorylase encoding gene belongs to at least one of Leuconostoc mesenteroides, Streptococcus mutans, Bifidobacterium adolescentis, or Rhizobium vitis.

4. The engineered organism as claimed in claim 1, wherein the sucrose phosphorylase encoding gene belongs to Rhizobium vitis.

5. The engineered organism as claimed in claim 1, wherein the sucrose permease encoding gene belongs to Escherichia coli.

6. The engineered organism as claimed in claim 1, wherein the phosphoglucomutase encoding gene belongs to Escherichia coli.

7. The engineered organism as claimed in claim 1, wherein the sucrose utilization is carried out in the presence of inorganic phosphate.

8. The engineered organism as claimed in claim 1, wherein the sucrose utilization is carried out for producing at least one polyhydroxyalkanoate (PHA).

9. A method for utilizing sucrose comprising: engineering an organism having at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene; and carrying out fermentation using said engineered organism in the presence of sucrose, such that sucrose is utilized.

10. The method as claimed in claim 9, wherein the engineered organism has ≥ 90% homology to Cupriavidus genus.

11. The method as claimed in claim 9, wherein the engineered organism has ≥ 90% homology to Cupriavidus necator.

12. The method as claimed in claim 9, wherein the sucrose phosphorylase encoding gene belongs to at least one of Leuconostoc mesenteroides, Streptococcus mutans, Bifidobacterium adolescentis, or Rhizobium vitis.

13. The method as claimed in claim 9, wherein the sucrose phosphorylase encoding gene belongs to Rhizobium vitis.

14. The method as claimed in claim 9, wherein the sucrose permease encoding gene belongs to Escherichia coli.

15. The method as claimed in claim 9, wherein the phosphoglucomutase encoding gene belongs to Escherichia coli.

16. The method as claimed in claim 9, wherein the sucrose utilization is carried out in the presence of inorganic phosphate.

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