Mutant strain of asaia bogorensis and uses thereof

Abstract

The present application discloses a mutant Asaia bogorensis strain (Deposition number DSM 34962) as well as compositions, cell cultures and kits comprising said strain. The present application also discloses the use of said Asaia bogorensis strain, compositions, cell cultures or kits in methods of producing dihydroxyacetone (DHA).

Description

MUTANT STRAIN OF ASAIA BOGORENSIS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority benefit of Singapore Application No. 10202401277W, filed on 30 April 2024, which is hereby incorporated by reference in its entirety.FIELD OF INVENTION

[0002] The present invention relates generally to the field of microbiology. In particular, the present invention relates to bacterial strains and their uses in biosynthesis.BACKGROUND OF INVENTION

[0003] The global demand for chemicals is increasing exponentially each year, but production still relies predominantly on fossil resources and chemical synthesis methods that are harmful to the environment. The increasing needs of the industries for chemicals necessitate the development of more sustainable approaches to produce chemicals via greener and cleaner approaches. In this context, microbial fermentation has emerged as an attractive option for achieving sustainability in the production of commodity chemicals. One such example is the microbial production of dihydroxyacetone (DHA).

[0004] DHA is a valuable chemical widely used in the cosmetics industry and as a precursor in the synthesis of pharmaceuticals, biodegradable polymers, and fine chemicals. While DHA can be produced through chemical synthesis or microbial production, the latter is increasingly favoured due to its cost-effectiveness and sustainability, making it the preferred approach for large-scale industrial applications.

[0005] Glycerol, an abundant and low-cost by-product of the oleochemical industry, serves as an ideal feedstock for microbial DHA production. Certain microbes are capable of enzymatically oxidizing glycerol into DHA, offering a sustainable bioproduction route that not only reduces reliance on chemical synthesis but also contributes to a circular economy by utilizing industrial waste. However, the number of known microbial strains that can efficiently perform this conversion remains limited, presenting a challenge for achieving high-level DHA production suitable for commercial applications.

[0006] Given the increasing demand for DHA and the challenges associated with current microbial production methods, there remains a need for the development of microbial strains with enhanced DHA biosynthesis capabilities to support sustainable and scalable industrial production.SUMMARY OF INVENTION

[0007] In one aspect, the present disclosure refers to a bacterial strain of Asaia bogorensis deposited under deposition number DSM 34962.

[0008] In another aspect, the present disclosure refers to a cell culture or composition comprising the bacterial strain of Asaia bogorensis deposited under deposition number DSM 34962.

[0009] In another aspect, the present disclosure refers to a kit comprising the bacterial strain of Asaia bogorensis deposited under deposition number DSM 34962, and instructions for use.

[0010] In a further aspect, the present disclosure refers to a method of producing dihydroxyacetone (DHA), the method comprises culturing the bacterial strain of Asaia bogorensis deposited under deposition number DSM 34962 in a medium comprising glycerol to produce DHADESCRIPTION OF FIGURES

[0011] The accompanying figures illustrate disclosed embodiments of the invention and serve to explain the principles of the disclosed embodiments, it is to be understood, however, that the figures are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0012] Figure 1 is a schematic representation of the overall workflow for dihydroxyacetone (DHA) production and analysis as described in the present disclosure.

[0013] Figure 2 shows the results of DHA production using the mutant Asaia bogorensis strain deposited under deposition number DSM 34962 (named as AB001 strain in the figure) described in the present disclosure. Figure 2A compares DHA production between the wild-type strain and the AB001 strain. The error bars represent the standard deviation from three independent measurements. Figure 2B shows the results of HPLC analysis of the crude biotransformation products obtained using the AB001 strain, with DHA indicated by an arrow. The results demonstrate that the AB001 strain shows improved DHA production compared to the wild-type Asaia bogorensis strain, and yields a high-purity DHA product with minimal by-products.

[0014] Figure 3 shows the results of the fitness evaluation of the AB001 strain. Figure 3A shows the relative growth of the wild-type Asaia bogorensis strain and the AB001 mutant strain under non-DHA- producing conditions. The error bars represent the standard deviation from three independent measurements. The results show no significant difference in cell growth between the wild-type strain and the AB001 strain, suggesting that the mutations in the high-DHA-producing AB001 strain do not have a deleterious effect on cell fitness. Figure 3B are histograms showing the cell viability of the AB001 strain under DHA-producing conditions, obtained from flow cytometry (FlowJo v10). The positive control histogram represents live cells, while the negative control histogram represents dead cells. The results demonstrate that the AB001 mutant strain remains highly viable even when producing high levels of DHA.DETAILED DESCRiPTiON OF INVENTION

[0015] Exemplary, non-limiting embodiments of the present disclosure will now be described.

[0016] The present disclosure addresses the need for novel technologies for the cost-efficient production of valuable products from low-cost feedstocks. Specifically, the present inventors have addressed this need with the development of a novel microbial strain with enhanced capabilities for dihydroxyacetone (DHA) biosynthesis from glycerol.

[0022] The term “strain” as used herein in the context of a microbial strain refers to a microbe which remains genetically unchanged when grown or multiplied,

[0023] In some examples, the strain is a biologically pure strain. The term "biologically pure strain" as used herein refers to a strain containing no other microbial strains in quantities sufficient to interfere with replication of the strain or to be detectable by normal microbiological techniques.

[0024] in some examples, the strain is an isolated strain. The term "isolated” as used herein means that the strain is in a form or environment which does not occur in nature, i.e., the strain is at least partially removed from one or more or all of the naturally occurring constituents with which it may be associated in nature, in preferred examples, the isolated strain is a biologically pure strain.

[0025] The viability and activity of the strain as disclosed herein should be maintained using appropriate storage methods. The choice of preservation technique depends on the intended duration of storage, and such choices can be made by a person skilled in the art without undue experimentation, in some examples where short-term storage is desired for cultures that are used regularly, suitable methods include storing the strain or cultures containing the strain at about 4°C. In some examples where long-term storage is needed, more robust methods such as cryopreservation and lyophilization will be required.

[0026] Cryopreservation is a process in which the cells are preserved at subzero temperatures resulting in a radical decrease in the rate of metabolic processes, and the ability to store samples for extended periods. Cryopreservation is generally carried out at below ~80”C. For maximum preservation, cryopreservation is typically carried out at or near the temperature of liquid nitrogen (about -196°C). In some examples, a cryoprotectant is used in the cryopreservation process to protect the ceils from freezing damage. Examples of commonly used cryoprotectants include but are not limited to, glycerol, ethanediol, dimethyl sulfoxide, ethylene glycol, and propanediol.

[0027] Lyophilization, commonly known as freeze-drying, is a widely used method for the long-term preservation of bacterial strains. This technique involves the removal of wafer from bacterial cells through a combination of freezing and sublimation under vacuum conditions. By preserving bacteria strains in a freeze-dried state, lyophilization significantly extends their shelf life while maintaining their viability and genetic stability. Lyophilized bacteria can typically be stored at room temperature, making them easy to transport and handle. In some examples, the bacteria strains preserved using lyophilization can remain viable for more than 10, 20, 30, 40, or 50 years.

[0028] The strain of Asaia bogorensis deposited under deposition number DSM 34962 can enhance the production of DHA from glycerol as compared to, for example, other microbial strains known in the art. In some examples, the enhanced production is at least about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800. 850, 900, 950, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 times of the amount of DHA produced using other microbial strains known in the art, under the same or similar reaction conditions.

[0029] In another aspect, there is provided a cell culture or composition comprising the strain of Asaia bogorensis deposited under deposition number DSM 34962. In some examples, the cell culture further comprises a culture medium.

[0030] The culture medium used herein can be any suitable medium in the form of a liquid (e.g. broth), a solid (e.g. agar plates), or as a semi-solid (e.g. agar deeps). In some specific examples, the culture medium is a liquid culture medium, such as a lysogeny broth (LB), or other suitable liquid culture medium, such as the seed culture medium (SCM) as disclosed herein.

[0031] In another aspect, there is provided a kit comprising the strain of Asaia bogorensis deposited under deposition number DSM 34962. The kit can further comprise instructions for use.

[0032] The strain of Asaia bogorensis as disclosed herein can be used atone or in combination with other strains. Thus, in some examples, the cell culture, composition, or kit as disclosed herein further comprises one or more of other strains that can be used for the production of DHA.

[0033] In some examples, the ceil culture or composition comprising the strain of Asaia bogorensis as disclosed herein is an enriched culture or composition. The term "enriched culture'’ or “enriched composition" of a strain refers to a culture or composition which contains more than about 50%, 60%, 70%, 80%, 90%, or 95% of the strain.

[0034] In some examples, the cell culture or composition as disclosed herein comprises from about 105to about 1012CFU / g, such as from about 5 x 105to about 5 x 10", or from about 1 x 10sto about 1 x 10", or from about 5 x 106to about 5 x 10 % or from about 1 x 107to about 1 x 101°, or from about 5 x 107to about 5 x 10®, or from about 1 x 10sto about 1 x 10®, or at about 5 x 10sCFU / g of the strain of Asaia bogorensis deposited under deposition number DSM 34962.

[0035] The term "CFU / g" as used herein refers to the number of colony-forming units per gram weight of the cell culture or composition. The gram weight includes carriers and other components present in the composition or cell culture.

[0036] Bacterial strains can be provided in many different forms. For example, such a form can be an inactive, but viable form of the bacterial strains, in some examples, the strain of Asaia bogorensis as disclosed herein are provided as a dried formulation, such as a freeze-dried product. In some other examples, the strain of Asaia bogorensis as disclosed herein are provided in a liquid formulation, such as a liquid culture product, or a liquid broth. A person skilled in the art will appreciate that the form of the strain can be tailored to the required applications, so long as the strain is viable upon receipt and application.

[0037] in a further aspect, there is provided a method oi producing DHA, the method comprises culturing the strain of Asaia bogorensis as disclosed herein under conditions in which DHA is produced, in some examples, the culture conditions include fermentation conditions. In some examples, the culture conditions include culturing in a medium comprising glycerol to produce DHA. The method of producing DHA as disclosed herein is efficient, energy-saving, and easy to operate.peptones also contain inorganic saits and / or vitamins. In some examples, peptones contain lipids and / or sugars, while in some other examples, peptones do not contain lipids and / or sugars. Peptones generally serve as a source of nitrogen in culture media, in particular serum-free culture media, to promote growth of micro-organisms. Examples of peptones include but are not limited to, soy peptone, casein peptone, meat peptone.

[0049] A person skilled in the art can determine the amount or concentration of peptones used in the medium without undue experimentation. In some examples, the concentration of peptones in the medium is about 0.1 to about 30, or about 0.5 to about 29, or about 1 to about 28, or about 2 to about27, or about 3 to about 26, or about 4 io about 25, or about 5 to about 24, or about 6 to about 23, or about 7 to about 22, or about 8 to about 21 , or about 9 to about 20, or about 10 to about 19, or about 1 1 to about 18, or about 12 to about 17, or about 13 to about 16, or about 14 to about 15 g / L, or at about 0.1 , 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 , 11 .5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 1 7.5, 18, 18.5, 19, 19.5, 20, 20.5, 21 , 21 .5, 22, 22.5, 23,23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, or 30 g / L. In some specifically examples, the concentration of peptones in the medium is about 10 g / L.

[0050] in some examples, the medium is supplemented with one or more carbon sources. Carbon sources are important components in enhancing the growth and metabolism of the strain as disclosed herein. A person skilled in the art can determine the amount or concentration of the carbon source(s) used in the medium without undue experimentation, in some examples, the concentration of the carbon source(s) in the medium is about 0.1 to about 50, or about 0.5 to about 48, or about 1 io about 46, or about 2 to about 44, or about 3 to about 42, or about 4 to about 40, or about 5 to about 38, or about 6 to about 36, or about 7 to about 34, or about 8 to about 32, or about 9 to about 30, or about 10 to about28, or about 12 to about 26, or about 14 to about 24, or about 16 to about 22, or about 18 to about 20 g / L, or at about 0.1 , 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 g / L. In some specific examples, the concentration of the carbon source(s) in the medium is about 20 g / L.

[0051] Examples of suitable carbon sources include but are not limited to, sorbitol, sucrose, glucose, lactose, galactose, dextrose, maltose, xylose, ribose, and mannitol. In some specific examples, the carbon source is sorbitol.

[0052] Sorbitol, also known as D-glucitol, is a sugar alcohoi. A person skilled in the art can determine the amount or concentration of sorbitol used in the medium without undue experimentation. In some examples, the concentration of sorbitol in the medium is about 0.1 to about 50, or about 0.5 to about 48, or about 1 to about 46, or about 2 to about 44, or about 3 to about 42, or about 4 to about 40, or about 5 to about 38, or about 6 to about 36, or about 7 to about 34, or about 8 to about 32, or about 9 to about 30, or about 10 to about 28, or about 12 to about 26, or about 14 to about 24, or about 16 to about 22, or about 18 to about 20 g / L, or at about 0.1 , 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 g / L. In some specific examples, the concentration of sorbitol in the medium is about 20 g / L.to about 4, or about 0.5 to about 3.8, or about 0.6 to about 3.6, or about 0.7 to about 3.4, or about 0.8 to about 3.2, or about 0.9 to about 3, or about 1 to about 2.8, or about 1 .2 to about 2.6, or about 1 .4 to about 2.4, or about 1.6 to about 2.2, or about 1.8 to about 2 g / L, or at about 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, .2.8, 2.9, 3, 3.1 , 3.2, 3 3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 g / L. In some specific examples, the concentration of the source(s) of phosphorus and / or potassium in the medium is about 2 g / L.

[0058] Examples of suitable sources of phosphorus and / or potassium include but are not limited to, inorganic phosphates such as dipotassium phosphate (K2HPO4), monopotassium phosphate (KH2PO4), disodium phosphate (Na2HPCU), monosodium phosphate (NaEEPCU), ammonium phosphate dibasic ((NH-^HPCU), calcium phosphate (CaafPO^), magnesium phosphate (MgafPCU)?), and inorganic potassium salts such as potassium chloride (KCI), potassium sulfate (K2SO4), potassium nitrate (KNO3), potassium acetate (CH3COOK) and potassium bicarbonate (KHCO3). In some specific examples, the source of phosphorus and potassium is dipotassium phosphate.

[0059] Dipotassium phosphate, also known as dipotassium hydrogen phosphate or potassium phosphate dibasic, is an inorganic compound with the chemical formula K2HPO4. it appears as a white, odorless, and highly water-soluble solid. In microbiological and fermentation applications, K2HPO4 is a crucial component of culture media, it provides essentia! phosphorus and potassium, vita! nutrients that support various cellular processes. Additionally, K2HPO4 acts as a buffering agent, helping to stabilize the pH of the culture medium, which is critical for optimal microbial growth and metabolism.

[0060] A person skilled in the art can determine the amount or concentration of KzHPCb used in the medium without undue experimentation In some examples, the concentration of K2HPO4 in the medium is about 0.01 to about 5, or about 0.05 to about 4.8, or about 0.1 to about 4.6, or about 0.2 to about 4.4. or about 0.3 to about 4.2, or about 0.4 to about 4, or about 0.5 to about 3.8, or about 0.6 to about 3.6, or about 0.7 to about 3.4, or about 0.8 to about 3.2, or about 0.9 to about 3, or about 1 to about 2.8, or about 1 .2 to about 2.6, or about 1 .4 to about 2.4, or about 1 6 to about 2.2, or about 1 .8 to about 2 g / L, or at about 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 .8, 1 .9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1 , 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 g / L. In some specific examples, the concentration of K2HPO4 in the medium is about 2 g / L.

[0061] In some examples, the medium is supplemented with one or more sources of nitrogen. Nitrogen is a fundamental component of amino acids, nucleic acids, and other cellular constituents, making its availability crucial for supporting the growth and metabolism of various microorganisms. A person skilled in the art can determine the amount or concentration of the source(s) of nitrogen used in the medium without undue experimentation, in some examples, the concentration of the source(s) of nitrogen in the medium is about 0.05 to about 10, or about 0.1 to about 9.5, or about 0.5 to about 9, or about 1 to about8.5, or about 1 .5 to about 8, or about 2 to about 7.5, or about 2.5 to about 7, or about 3 to about 6.5, or about 3.5 to about 6, or about 4 to about 5.5, or about 4.5 to about 5 g / L, or at about 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .2, 1 .4, 1 .6, 1 .8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.5, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8,or 10 g / L. In some specific examples, the concentration of the source(s) of nitrogen in the medium is about 3 g / L.

[0062] Examples of suitable nitrogen source include but are not limited to, ammonium sulfate ((NH^SCU), ammonium chloride (NH4CI), ammonium phosphate ((NH^zHPCk, NH4H2PO4), ammonium nitrate (NH4NO3), ammonium carbonate ((NEk^COs), potassium nitrate (KNOs), sodium nitrate (NaNOs), calcium nitrate (Ca(NOs)2), and sodium nitrite (NaNOz). In some specific examples, the nitrogen source is ammonium sulfate.

[0063] Ammonium sulfate, with the chemical formula (NH4I2SO4, is an inorganic salt that appears as a white, odorless solid, highly soluble in water. A person skilled in the art can determine the amount or concentration of (NH^zSOi used in the medium without undue experimentation, in some examples, the concentration of (NH^zSCk m ths medium is about 0.05 to about 10, or about 0.1 to about 9.5, or about 0.5 to about 9, or about 1 to about 8.5, or about 1 .5 to about 8, or about 2 to about 7.5, or about 2.5 to about 7, or about 3 to about 6.5, or about 3 5 to about 6, or about 4 to about 5.5, or about 4.5 to about 5 g / L, or at about 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .2, 1 .4, 1 .6, 1 .8, 2, 2.2, 2.4, 2.S, 2.8, 3, 3.2, 3-4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.8, 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8, or 10 g / L. In some specific examples, the concentration of (NH4)2SO4 in the medium is about 3 g / L.

[0064] In some examples, the medium is supplemented with one or more sources of calcium ions. Calcium ions play a crucial rale in maintaining ceii wall stability, enzyme activation, sporulation, biofiim formation, and transport mechanisms, making them essential for growth and division. A person skilled in the art can determine the amount or concentration of the source(s) of calcium ions used in the medium without undue experimentation. In some examples, the concentration of the source(s) of calcium ions in the medium is about 0.01 to about 5, or about 0.05 to about 4.8, or about 0.1 to about 4.6, or about 0.2 to about 4.4, or about 0.3 to about 4.2, or about 0.4 to about 4, or about 0.5 to about 3.8, or about 0.6 to about 3.6, or about 0.7 to about 3.4, or about 0.8 to about 3.2, or about 0.9 to about 3, or about 1 to about 2.8, or about 1 .2 to about 2.6, or about 1 .4 to about 2.4, or about 1 .6 to about 2.2, or about 1.8 to about 2 g / L, or at about 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1 .2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2 5, 2.6, 2.7, 2.8, 2.9, 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3 7, 3.8, 3.9, 4, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 g / L. In some specific examples, the concentration of the source(s) of calcium ions in the medium is about 2 g / L. In some other specific examples, the concentration of the source(s) of calcium ions in the medium is about 1 g / L,

[0065] Examples of suitable sources of calcium ions include but are not limited to, calcium carbonate (CaCOa), calcium chloride (CaCIs), calcium phosphate (CasCPO^z), calcium sulfate (CaSCu), calcium nitrate (CafNOsJs), calcium acetate (CafCzHsOzJz), calcium lactate (CeHioCaOs), calcium gluconate (CizHzzCaOu), calcium glycerophosphate (CafCaHzOsPJz). In some specific examples, the sources of calcium ions are calcium carbonate and calcium chloride.

[0066] Calcium carbonate (CaCOs) is an inorganic compound that appears as a white, odorless powder or crystalline substance and is sparingly soluble in water. CaCOs can also act as a buffering agent, helping to maintain a stable pH in the culture medium. A person skilled in the art can determine the amount or concentration of CaCOs used in the medium without undue experimentation. In someexamples, this time is the point at which the concentration of the product of interest has reached a predetermined level.

[0077] A person skilled in the art can determine the appropriate duration of culturing without undue experimentation, in some examples, the method of producing DHA comprises culturing the strain of Asaia bogorensis as disclosed herein in a medium containing glycerol for a time period of about 6 hours to about 10 days, or about 12 hours to about 9 days, or about 18 hours to about 8 days, or about 24 hours to about 7 days, or about 2 days to about 6 days, or about 3 days to about 5 days, or about 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22 or 24 hours, or about 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9,9.5, or 10 days. In some specific examples, the method of producing DHA comprises culturing the strain of Asaia bogorensis as disclosed herein in a medium containing glycerol for a time period of about 5 days.

[0078] The method of producing DHA using the mutated strain of Asaia bogorensis as disclosed herein, i.e., the strain deposited under deposition number DSM 34962, can increase the production ievei of DHA by at least about 5, at least about 10, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1200, at least about 1400, at least about 1600, at least about 1800, or at least about 2000 fold, as compared to the wild-type strain of Asaia bogorensis.

[0079] The method of producing DHA as disclosed herein can achieve a high percentage yield (% yield) as compared to other chemical or biochemical production methods. The term "percentage yield" as used herein refers to the actual yield divided by the theoretical yield and multiplied by 100%. Specifically, the percentage yield of DHA refers to the actual yield of DHA divided by the theoretical yield of DHA from glycerol and multiplied by 100%. In some examples, the method of producing DHA as disclosed herein can achieve a percentage yield (% yield) of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% of the maximum theoretical yield of production of DHA from glycerol. The molecular weight of glycerol is 92 09382 g / mol, and the molecular weight of DHA is 90.078 g / mol. These values can be used when calculating the percentage yield of DHA from glycerol using the method as disclosed herein. In one specific example, about 0.96 grams of DHA is produced per gram of glycerol used, i.e., about 0.98 mole of DHA is produced per mole of glycerol used. This achieves a percentage yield of about 98%.

[0080] in some examples, DHA is produced at a rate of between about 0.5 g / L / hour to about 20 g / L / hour, or between about 1 g / L / hour to about 15 g / L / hour, or between about 1 g / L / hour to about 10 g / L / hour, or between about 1 g / L / hour to about 5 g / L / hour, or at least about 0.1 , 0.5, 1 , 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 g / L / hour.

[0081] The method of producing DHA from glycerol as disclosed herein can achieve a high conversion rate of glycerol as compared to other chemical or biochemical production methods. The term "conversion rate" as used herein refers to the percentage of a reactant (e.g. glycerol) that has been

[0086] in some examples of industrial scale productions, the volume of the bacterial culture can be 100 mL or more, 500 mL or more, 1 L or more, 5 L or more, 10 L or more, 50 l_ or more, 100 L or more, 500 L or more, 1000 L or more, 5000 L or more, 10000 L or more, or 50000 L or more.

[0087] in industrial scale productions, the method as disclosed herein can be carried out in a bioreactor. Generally, the control of growth conditions, including control of culture media components and concentrations, temperature, and pH, is facilitated in a bioreactor.

[0088] in some examples of industrial scale productions, solid fermentation is used instead ol liquid fermentation, or used in combination of liquid fermentation. The term "solid state fermentation" refers to a fermentation process in which cells of the bacterial strain required for fermentation are grown on a solid substrate. By immobilizing bacterial cells on the solid substrate, the bacterial ceils can be easily re-used, enhancing the efficiency and sustainability of the fermentation process. The selection of an appropriate substrate depends on factors like nutrient content, availability, and cost, etc. Commonly used substrates include but are not limited to agar-, agarose-, polyacrylamide- based substrates, derivatives thereof, and combinations thereof. In some examples, the surface area of the solid substrate can be increased using methods known in the art, such as forming layered, porous, hollow, tubular, branched structures and / or three-dimensional (3D) scaffolds. In some examples, while the bacterial cells are immobilized on the solid substrate, the nutrients and / or reactants used in the fermentation process to produce DHA are supplied in liquid medium.

[0089] in industrial scale productions, in particular productions using liquid cultures, the method as disclosed herein can be carried out in a batch fermentation process, a continuous fermentation process, or a combination of both.

[0090] The term “batch fermentation" refers to a fermentation mode wherein the ceils are cultured in the initially present fermentation medium without any change in medium composition or the volume of the medium. Thus, m batch fermentation, no substantial or significant amount of fresh liquid culture medium is added to the ceil culture and no substantial or significant amount of liquid culture medium is removed from the cell culture during culturing. Batch fermentation begins with all ingredients combined at the start, allowing the reactions to proceed without further input. The process moves through distinct phases, starting with the lag phase, where bacterial cells adjust to their environment, followed by the exponential growth phase, during which rapid cell proliferation occurs as nutrients are abundant. As nutrients are gradually depleted, growth slows down and transitions into a stationary phase, during which the production of secondary metabolites, such as antibiotics and enzymes, accelerates. When most nutrients are exhausted, fermentation concludes, and product isolation begins. Depending on the desired culture conditions, batch fermentation can last from several hours to a week.

[0091] The term “continuous fermentation" refers to a fermentation mode wherein the cells are cultured in the initially present fermentation medium and new fermentation medium is continuously fed to the fermenter and ferment is removed from the fermenter. Fresh nutrients and reactants are continually supplied while the final products are continuously removed, maintaining steady-state conditions and prolonging the exponential growth phase. This method minimizes the accumulation of inhibitory byproducts. A typical continuous fermentation process starts with an initial batch growth phase to establish the desired cell density. Once the carbon source or other nutrients are exhausted, fresh medium of thesame composition is supplied at a controlled rate while fermentation liquid is withdrawn at preferably the same rate. This system can operate for weeks or even months, ensuring consistent product quality. During continuous fermentation, the fermentation contents are monitored periodically, including sampling up to every day, as desired, to ensure consistency of cell density, product concentration and / or product quality.

[0092] Fed-batch fermentation integrates aspects of both batch fermentation and continuous fermentation. The culture is initially grown in batch mode, and as cells proliferate and consume the carbon source, additional nutrients are periodically fed into the bioreactor at a rate that balances consumption. This approach optimizes cell density and product concentration while avoiding substrate inhibition.

[0093] In some examples, the strain of Asaia bogorensis as disclosed herein can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. In general, anaerobic conditions refer to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also include growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 % oxygen, it is highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process.

[0094] During fermentation, the growth rate of the bacteria! cells can be determined by measuring optical density using a spectrophotometer (e.g., at 600 nm).

[0095] At the end of the fermentation process, the fermenter contents can be collected for further processing.

[0096] in some examples, the method of producing DHA as disclosed herein further comprises the step(s) of separation and / or purification of the DHA produced.

[0097] in some examples, the DHA produced is secreted out of the bacterial cells into the culture medium. Culture media may be drawn from the culture system, and the DHA produced may then be extracted from the culture media. In such examples, the culture is centrifuged at a speed sufficient to pellet the cells but not disrupt the cells and allow extraction of the culture medium, as known in the art. The DHA produced is mainly present in the soluble fraction or the supernatant.

[0098] in some other examples, the DHA produced in mainly retained within the bacterial strain after the culture process is completed. In such examples, the DHA produced is extracted from the cultured bacteria! ceil using methods known in the art. For example, the cultured bacterial ceils may be physically disrupted (e.g., using shear force, sonication) or chemically disrupted (e.g., by contacting with detergent or other iysing agent(s)) to release the DHA produced into the soluble fraction or the supernatant. The soluble fraction or the supernatant may then be separated from the solid fraction (comprising mainly the ceil debris) by centrifugation or other method known in the art.

[0099] The DHA present in the soluble fraction or the supernatant can then be further separated and / or purified. The separation and purification can be carried out according to separation and purification steps conventionally applicable to DHA in the art, such as filtration, centrifugation, precipitation.crystallization, recrystallization, concentration, drying, freeze-drying, adsorption, ion exchange, chromatography and the like.[001001 In some examples, the method of producing DHA as disclosed herein can be carried out without the need of supplementing with other enzymes or catalysts.

[0101] In one aspect, there is provided the DHA produced using the methods as disclosed herein. In a further aspect, there is provided compositions or products comprising the DHA produced using the methods as disclosed herein.[001021 In some examples, commercial grade DHA is provided in substantially pure form (e.g., about 90% pure or greater, about 95% pure or greater, about 99% pure or greater, or about 99.5% pure or greater).[001031 DHA is a valuable chemical widely used in the cosmetics industry. For example, DHA is the main ingredient in sunless tanning products like self-tanners, bronzers, and spray tans. It reacts with amino acids in the skin to create a temporary tan. DHA is also used in creams, serums, and masks to give skin a healthy glow. DHA is also used in hair lotions, mists, oils, and dyes.

[0104] Thus, in some examples, the DHA produced may be modified into any one of a number of downstream products. In some examples, the present disclosure provides tanning products, in particular sunless tanning products, skin care products including but not limited to, creams, serums, and masks, hair care products such as hair lotions, hair mists, hair oils, and hair dyes comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% of the DHA produced using the methods as disclosed herein. It is understood that methods for producing such products comprising DHA. are well known in the art.

[0105] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0106] As used in this application, the singular form “a”, “an", and “the” include plural references unless the context clearly dictates otherwise.

[0107] As used herein, the term “about", in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1 % of the stated value, and even more typically + / - 0.5% of the stated value.

[0108] As used herein, the percentage (%) can refer to “% (w / vC (percent weight to volume), “% (v / v)" (percent volume to volume), or “% (w / w)” (percent weight to weight), unless specified otherwise.

[0109] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc , as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.[001 10J The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.[001 111 Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.REFERENCE TO DEPOSITED BIOLOGICAL MATERIAL[001 12] The isolated strain of Asaia bogorensis DSM 34962 was deposited under the terms of the Budapest Treaty with Leibniz institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (herein "DSMZ", German name Leibniz-lnsitut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), on 18 January 2024, by National University of Singapore, with co-depositor, Wilmar International Limited, Singapore Accession number DSM 34962 has been assigned.[001 131 The deposited strain is provided merely as convenience to those of skill in the art, and is not an admission that a deposit is required for enablement.EXAMPLES[001 14] Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.[001 15] Example 1. Materials and Methods[001 16] Strain, chemicals and culture media[001 17] Asaia bogorensis was used as the DHA-producing strain in the present disclosure. Yeast extract, peptone and agar were procured from Becton Dickinson (NJ, USA), and other chemicals were obtained from Sigma-Aldrich unless otherwise specified.

[0131] By screening a mutant library of Asaia bogorensis, the inventors identified a mutant strain, AB001 , with high DHA-producing capabilities. Using the AB001 strain as the DHA producer, the inventors established the conditions for efficient DHA production.

[0132] Remarkably, the production level of DHA using the AB001 strain was improved more than 1000- fold compared to the wild-type strain (Figure 2A), and achieved conversion of 94 g / L glycerol to 90 g / L DHA in 5 days with a yield of 98%. Importantly, besides the high DHA production, the DHA was produced at a high purity of >85% (Figure 2B).

[0133] These results were reproducible when scaled up to 3.5-L in a 7.5-L fermenter, demonstrating the robustness and scalability of the AB001 strain. Overall, the AB001 strain developed in the present disclosure is highly efficient and has achieved the highest DHA production levels reported to date.

[0134] Determining the fitness of the high DHA-producing mutant ABO01 strain

[0135] Viability of the microbial cell factories under production conditions is an important prerequisite for selecting ideal host strains to achieve cost-effective biochemical production through whole-cell biocatalysis. However, reduced cell viability during biotransformation is a common issue and poses a major challenge in the large-scale in vivo production of commodity chemicals.

[0136] To assess the fitness of the mutant AB001 strain, the inventors investigated its growth profile and viability under DHA-producing conditions. The growth of the mutant AB001 strain was compared to that of the wild-type strain under non-DHA-producing conditions by measuring cell density after 48 hours of cultivation in GM. No significant difference in cell growth was observed between the two strains (Figure 3A), suggesting that the mutations in the high DHA-producing mutant AB001 strain do not have a deleterious effect on cell fitness.

[0137] Subsequently, the inventors analysed the cell viability of the mutant AB001 strain under DHA- producing conditions using flow cytometry. The histograms for the AB001 cells (production sample) and the live cell control (positive control) were highly similar. In contrast, the dead cell control (negative control) displayed a distinctly different histogram from both the AB001 cells and the live cell control (Figure 3B).

[0138] Taken together, these results indicate that the mutant AB001 strain remained highly viable even while producing high levels of DHA. This suggests that AB001 is a highly robust strain, potentially suitable for DHA production at an industrially relevant scale.

Claims

1 . A bacteria; strain of Asaia bogorensis deposited under deposition number DSM 34962.

2. The bacteria! strain of claim 1 wherein the bacterial strain is freeze-dried.

3. The bacteria! strain of claim 1 , wherein the bacterial strain is present in a liquid culture.

4. A cell culture or composition comprising the bacterial strain of any one of claims 1 to 3.

5. A kit comprising the bacterial strain of any one of claims 1 to 3, and instructions for use.

6. A method of producing dihydroxyacetone (DHA), the method comprises culturing the bacterial strain of any one of claims 1 to 3 in a medium comprising glycerol to produce DHA.

7. The method of claim 6, wherein the medium further comprises yeast extract, peptone, sorbitol. MgSCh' / HsO, KzHPOz, ammonium sulphate, CaCOa and / or CaCIz.

8. The method of claim 6 or 7, wherein the method is carried out at a temperature between 25 to 40°C, preferably at about 30°C.

9. The method of any one of claims 6 to 8, wherein the medium has a pH value of 4.5 to 7.0, preferably at about 6.0.

10. The method of any one of claims 6 to 9, wherein the amount of the bacterial strain to be cultured is 1 .0 x 106to 1 .0 x 107CFU / ml.1 1 . The method of any one of claims 6 to 10, wherein the concentration of glycerol is 1 to 300 g / L, preferably 90 to 100 g / L.

12. The method of any one of claims 6 to 1 1 , wherein the concentration of yeast extract is 0.1 to 30 g / L, preferably about 10 g / L.

13. The method of any one of claims 6 to 12, wherein the concentration of peptone is 0.1 to 30 g / L. preferably about 10 g / L.

14. The method of any one of claims 6 to 13, wherein the concentration of sorbitol is 0.1 to 50 g / L, preferably about 20 g / 'L.

15. The method of any one of claims 6 to 14, wherein the concentration of MgSOi-THzO is 0.01 to 3g / L, preferably about 1 g / L16. The method of any one of claims 6 to 15, wherein the concentration of K2HPO4 is 0.01 to 5 g / L, preferably about 2 g / L17 The method of any one of claims 6 to 16, wherein the concentration of ammonium sulphate is 0 05 to 10 g / L, preferably about 3 g / L.

18. The method of any one of claims 6 to 17, wherein the concentration of CaCOs is 0.01 to t> g / L, preferably about 2 g / L.

19. The method of any one of ciaims 6 to 18, wherein the concentration of CaCIz is 0.01 to 3 g / L, preferably about 1 g / L.

20. The method of any one of claims 6 to 19, wherein the duration of culturing is 6 hours to 10 days, preferably about 5 days

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