Invented by Derek L. Greenfield, Donald E. Trimbur, Andreas W. Schirmer, Cindy Chang, Behnaz Behrouzian, Jessica WINGER, Genomatica Inc

Acetyl CoA carboxylase (ACC) is an enzyme that plays a crucial role in fatty acid synthesis. It catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, which is the first step in the synthesis of long-chain fatty acids. ACC is an essential enzyme for cellular metabolism and is found in all living organisms, including plants, animals, and bacteria. The market for Acetyl CoA carboxylase has been steadily growing in recent years, driven by the increasing demand for fatty acids in various industries. Fatty acids are used in the production of a wide range of products, including cosmetics, pharmaceuticals, food additives, and biofuels. The growing awareness of the health benefits of omega-3 and omega-6 fatty acids has also contributed to the demand for ACC. One of the key drivers of the market is the rising prevalence of obesity and related metabolic disorders. Obesity is a major global health concern, and it is often associated with dysregulation of lipid metabolism. ACC inhibitors have shown promise in the treatment of obesity and other metabolic disorders by reducing fatty acid synthesis and promoting fat oxidation. As a result, pharmaceutical companies are investing heavily in the development of ACC inhibitors as potential therapeutics. The market for ACC is also driven by the increasing use of biofuels as a renewable energy source. Fatty acids derived from plants and algae are used to produce biodiesel, which is a cleaner alternative to conventional diesel fuel. ACC is a key enzyme in the biosynthesis of fatty acids, making it an important target for genetic engineering and metabolic engineering efforts to enhance the production of fatty acids for biofuel production. Furthermore, the cosmetic industry is another major consumer of fatty acids. Fatty acids are used in the formulation of skincare and haircare products due to their moisturizing and emollient properties. ACC inhibitors can be used to modulate the fatty acid composition of oils and fats used in cosmetic products, allowing manufacturers to create products with specific functional properties. In terms of geographical distribution, the market for ACC is expected to witness significant growth in Asia-Pacific, particularly in countries like China and India. These countries have a large population base and are experiencing rapid economic growth, leading to increased demand for products that utilize fatty acids. Additionally, the presence of a well-established pharmaceutical and cosmetic industry in these regions further contributes to the market growth. In conclusion, the market for Acetyl CoA carboxylase is witnessing steady growth due to the increasing demand for fatty acids in various industries. The rising prevalence of obesity and metabolic disorders, the growing use of biofuels, and the demand for fatty acids in the cosmetic industry are key factors driving the market. With ongoing research and development efforts, the market for ACC is expected to continue expanding in the coming years.

The Genomatica Inc invention works as follows

The disclosure relates to acetyl CoA carboxylase variants (ACC) and host cells expressing these for the production malonyl CoA derived compounds, including fatty acids derivatives. The disclosure also contemplates methods for producing higher amounts of malonyl CoA derived compounds, and related cell culture.

Background for Acetyl CoA carboxylase

Petroleum, a natural resource, is found on earth in solid, liquid or gaseous form. Petroleum products, however, are produced at a high cost, both financially and environmentally. Crude petroleum is a product of the Earth that has limited commercial applications in its natural state. The mixture is made up of hydrocarbons such as paraffins (or aromatic compounds), olefins or alkenes, alkynes and napthenes. The length and complexity of the hydrocarbons can vary. Crude petroleum also contains organic compounds (e.g. organic compounds containing oxygen, nitrogen, sulfur, etc.). Impurities such as sulfur, salts, acids, metals and others. Most petroleum is refined due to its high energy density, easy transportability and ability to be refined into fuels such as transportation fuels like gasoline, diesel, aviation, etc. ), heating oil, liquefied petroleum gas, etc.

Petrochemicals are used to produce specialty chemicals such as plastics and resins. They can also be made into lubricants or gels. Specialty chemicals are used in many different commercial applications. Specialty chemicals can be made from petrochemicals. Examples include hydrocarbons such as long-chain hydrocarbons and branched-chain hydrocarbons. They also include saturated hydrocarbons and unsaturated hydrocarbons. Fatty alcohols, esters, aldehydes and ketones are all specialty chemicals that can be produced from petrochemical raw materials. Commercially, fatty acids are used as surfactants. Surfactants are found in soaps and detergents. Fatty acids are also used in paints and lacquers as well as fuels, lubricating oil, cosmetics, candles, shortenings and emulsifiers. Fatty acids are also used in rubber products as activators. “Fatty acids are also used as a source of ketene dimers and peroxy acids, and can be used to make methyl esters.

Fatty esters are used in many different commercial applications. Biodiesel is an alternative fuel that contains esters. Some esters with low molecular mass are volatile and have a pleasant smell, making them suitable as flavoring or fragrance agents. Esters can also be used as solvents in lacquers and paints. Esters are also found in some natural substances such as oils, fats and waxes. Esters can also be used in plasticizers, flame-retardants, gasoline, and oil additives. Esters are also used to make polymers, textiles and dyes.

Fatty alcohols also have many commercial applications.” The annual global sales of fatty acids and their derivatives exceed US$1 billion. In the food and cosmetic industries, shorter-chain fatty alcohols can be used as thickeners, emulsifiers and emollients. fatty alcohols are nonionic surfactants that can be used in household and personal care products. “Fatty alcohols can also be found in cosmetics and industrial solvents. They are also used as lubricating oils, antistatic agents for textiles and finishes, waxes and gums.

Acetyl CoA Carboxylase (ACC), plays an important part in regulating fatty acids synthesis and degrada-tion. It is a complex enzyme that requires biotin to catalyze the first step in fatty acid synthesis, which is the irreversible conversion of acetyl CoA into malonyl CoA. ACC generates malonyl CoA through its two catalytic functions, i.e. biotin carboxylase and carboxyltransferase. In prokaryotes ACC is a multisubunit enzyme. It is made up of four polypeptides, or subunits. These are encoded by different genes whose coordinated expression is controlled at multiple levels (Cronan et. (2002) Progress in Lipid Research 41:407-435; James et al. Journal of Biological Chemistry, 279(4), 2520-2527 (2004). The four polypeptides in ACC assemble at a fixed rate into a complex (Broussard et. al. Structure 21:650-657 (2013). The ACC reaction is a four-protein process, requiring the biotin carboxylase, biotinoyl or biotin carboxyl carrier proteins (BCCP) and the two proteins that make up the carboxyltransferase. The ACC reaction is characterized by the ATP dependent conversion of acid-labile NaH14CO3 into acid-stable Malonic Acid. The ACC subunits in bacteria and plant plastids are similar and different. “Despite the complexity of plant proteins, sequences essential for ACC function are not significantly distinct from their bacterial homologues” (Cronan et. al., supra).

It has been reported by scientists that E. coli ACC was the least stable ACC enzyme. Only when all four subunits at high concentrations are present can the overall activity be measured. However, two partial reactions can still be measured with diluted protein solutions. It is believed that the stable complexes include the CT alpha2 Beta2 and BC complexes. There are hints that an unstable BC2-BCCP2 is present. The BCCP dimer has been purified and the full length BCCP can be measured as a dimer. Other bacterial ACCs appear to be more stable than E. coli, and ACC activity is measured in dilute extracts from Helicobacter pylori or Pseudomonas Citronellolis. ACCs from plant plastids also seem to be more stable than E. coli ACCs. As in E. coli, further purification results in loss of ACC activity. This can be recovered by mixing fractions containing partial reaction activities. There are two subcomplexes, BC-BCCP, and CT, with no evidence of intact BCCP, or free CT beta (Cronan and al., supra).

There are alternative ways to produce fuels and other products that are currently derived by petroleum. Microbial systems have the potential to produce biofuels and other chemicals. Genetically engineered organisms, such as bacteria and yeast, can produce renewable fuels and chemicals. Genetically altering naturally occurring biosynthetic pathway can enable engineered organisms synthesize renewable chemical and fuel products. Moreover, microbes may be metabolically engineered to use different carbon sources for fuel and chemical production. It would be desirable to engineer ACCs to produce higher yields when expressed in a recombinant cell.

The field has made significant progress, but there is still a need to improve genetically modified host cells, recombinant enzymes, and methods and systems for robust and cost-effective fermentation of recombinant cells in order to produce fuels and chemicals. This need is addressed by the present disclosure, which provides ACC variants to increase the yield and titer for malonyl derivative compounds.

The disclosure includes a variant of biotin carboxyl carriers protein (BCCP), which has at least one amino acid mutation. The disclosure includes a variant of biotin-carboxyl carrier proteins (BCCPs) that comprises at least one amino acid mutation. In one embodiment, a variant BCCP increases the production of malonyl CoA-derived compounds in a cell when compared with a wild type cell. In another embodiment, when expressed in a cellular system, the variant BCCP can confer increased acetyl CoA carboxylase activity, resulting to an increase in production of a malonyl CoA-derived compound compared to a wild type cell. The malonyl CoA-derived compounds include, but are not limited to a free-fatty-acid derivative, such as an acetyl-CoA carboxylase (ACC) activity, or a fatty-acid methyl ester, such as FAME (fatty-acid methyl ester), FAEE (fatty-acid ethylester), a fat-acid amine, beta-hydroxy-fatty-acid derivatives, bifunctional fatty-

Another aspect” of the disclosure is a variant Biotin carboxyl Carrier Protein (BCCP) with at least one amino acid mutation, the mutation being in the N terminal amino acid region. In one embodiment, a mutation occurs in amino acid position two of SEQID NO: 2. In another embodiment, a variant BCCP confers an increased production of malonyl CoA-derived compounds to a cell when compared with a wild type cell. When expressed in a cellular environment, the variant BCCP can confer an improved acetyl CoA carboxylase activity, resulting in a higher production of a malonyl CoA-derived compound.

Another aspect” of the disclosure is a variant BCCP having at least one amino acid mutation, wherein the BCCP variant is selected from SEQ NOS: 4, 5, 6, 8, 10, 12, 16, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60,62, 64,66,68, 70, 72, 75, 78, 80,82,84, In one embodiment, a variant BCCP increases the production of malonyl CoA-derived compounds in a cell when compared with a wild type cell. In another embodiment, when expressed in a cellular environment, the variant BCCP can confer an improved acetyl CoA carboxylase activity (ACC), resulting in a higher production of a malonyl CoA-derived compound than a wild type cell.

Still another feature of the disclosure is a variant BCCP encoded by variant accB genes or accB sequences, wherein the sequence is chosen from SEQ ID NoS: 3, 5, 7 9, 11, 15, 17, 19, 25, 27, 29, 31 and/or 35.

Another aspect” of the disclosure is a recombinant cellular or microorganism expressing a variant BCCP. The variant BCCP has a mutation at least in one amino acid sequence. In one embodiment, a cell is a microbial cell or microbial host cell. In another embodiment, a cell is a microbe or microbial cell. In another embodiment, a microorganism can be a microbial or microbial-host cell or microbe. In one embodiment, a mutation occurs in the amino acid N-terminal region. In another embodiment, a mutation occurs in the amino acid position 2 on SEQ ID No: 2. In another embodiment, there is a mutation. In different embodiments, aspartate is substituted for asparagine. In another embodiment, BCCP variant SEQ ID No: 6 encompasses a polypeptide containing a mutation including a substitution aspartate (D). In another embodiment, variant BCCP includes SEQ NO: 4 or 8 which encompasses a polypeptide containing a mutation including a substitution of histidine (H) for aspartate. In another embodiment, BCCP variant has SEQ NO: 10 or 12 which encompasses a polypeptide containing a mutation including a substitution of isoleucine to aspartate. In one embodiment, a variant BCCP with at least one amino acid mutation confers an increased production of malonyl CoA-derived compounds to a cell recombinant when compared to the wild type. In a second embodiment, the BCCP variant has at least one amino acid mutation and can confer increased acetyl CoA carboxylase activity (ACC) to a recombinant cellular, resulting in an increase in production of a malonyl CoA-derived compound compared to a wild type cellular. In another embodiment, the recombinant cell is a microorganism recombinant or recombinant cell which can be compared or contrasted to a wild-type microorganism recombinant cell. In another embodiment, a microbial cell is used.

The disclosure also provides a method for producing a malonyl CoA-derived compound. This includes culturing cells that express a variant BCCP within a fermentation broth that contains a carbon source. The malonyl CoA-derived compounds include a derivative of a fat acid such as, for instance, an fatty ester, a variant BCCP, or a fatty ester methyl ester. In one embodiment, a cell is a recombinant bacterium or host cell. This can be compared or contrasted to a wild-type bacterium or host cell. In another embodiment, a microbial cell is used.

The disclosure also contemplates a variant of operon that controls the expression a BCCP. In one embodiment, an operon causes a difference in BCCP expression between a wild-type cell and a recombinant. In one embodiment, a recombinant host cell, or recombinant organism, is used instead of a wild-type microbial cell host or wild-type microorganism. In another embodiment, an operon increases BCCP expression and improves acetylcoa carboxylase activity (ACC) in a recombinant cellular system. This results in increased production of a malonylcoa-derived compound compared to a wild type cell. The variant operon may also include a promoter in one aspect. The promoter can include, but not be limited to, heterologous, heterologous variant and synthetic promoters. In one embodiment, a promoter can be a genetically-modified accBC promoter or a naturally occurring E. coli Promoter. In a different embodiment, the promoter can be an accBC variant. In another embodiment, a T5 or T5 variant promoter. In one embodiment, a T5 promoter accBC is used. The accBC promoter can be selected from SEQ NOS 93, 94 95 or 96, or variations thereof.

The disclosure provides further a recombinant host cell or microorganism that contains a variant operon which controls the expression a BCCP. The operon can result in a change to the BCCP expression in one embodiment. In one embodiment, an operon increases the BCCP expression and improves the acetylcoa carboxylase activity of a recombinant cellular system. This results in a higher production of a malonylcoa-derived compound compared to a wild type cell. In another embodiment, a promoter is included in the variant operon.

Another aspect” of the disclosure relates to a method for producing a malonyl CoA-derived compound. This includes culturing microorganisms or host cells that express a variant operon within a fermentation broth that contains a carbon source. In one embodiment, a recombinant cell can be compared or contrasted to a wild-type microorganism. In one embodiment, the cell has a microbial nature. The malonyl CoA-derived compounds include a fat acid, an ethyl ester of a FAME or FAEE, a fatty alcohol (FAEE), and a fatty amino acid. They also include a beta-hydroxy fatty-acid derivative, bifunctional fatty-acid derivatives (e.g.? -hydroxy fatty-acids, diols? -hydroxy,??-hydroxy FAME and???-hydroxy FAEE),

Another aspect of the disclosure is a method for producing malonyl CoA-derived compounds, which includes culturing host cells expressing variant BCCPs and variant operons in a fermentation broth containing carbon sources. In one embodiment, the recombinant cell is either a recombinant bacterium or a recombinant cell host that can be compared or contrasted to a wild-type bacterium or host cell. In one embodiment, the cell has a microbial nature. The malonyl CoA-derived compounds include a fat acid, an fatty ester, such as a FAME or FAEE, a fatty alcohol (FAE), a saturated fatty compound, and a bifunctional fatty compound (such as? -hydroxy fatty acids and? -hydroxy diols), as well as non-fatty acids based compounds, including flavanones and flavonoids, polyketides, and 3-hydroxypropionic Acid.

The disclosure contemplates further a microorganism containing a variant of biotin carboxyl transporter protein (BCCP), which has at least one amino acid mutation. In one embodiment, a variant BCCP contains a mutation at an amino acid N-terminal region. In another embodiment, a substitution is the mutation. In different embodiments, aspartate is substituted for asparagine or histidine or isoleucine or threonine or serine or tyrosine or arginine or leucine or glutamine or glutamate. In another embodiment, BCCP variant has one or multiple mutations. These include substitutions such as aspartate to asparagine(N), aspartate to histidine(H), aspartate to isoleucine(I), aspartate to threonine/T, aspartate to serine/S), aspartate to tyrosine/Y; aspartate to arginine/R; aspartate to leucine/L; aspartate to glutamine In another embodiment of the variant BCCP, SEQ ID No: 6 encompasses a polypeptide that has a mutation including a substitution aspartate(D) to asparagine(N). In another embodiment, variant BCCP includes SEQ NO: 4 or 8 which encompasses a polypeptide containing a mutation including a substitution of histidine (H) for aspartate. In another embodiment, variant BCCP includes SEQ NO: 10 or 12 which encompasses a polypeptide containing a mutation including a substitution of isoleucine to aspartate. In one embodiment, expression of variant BCCP increases the production of malonyl CoA-derived compounds by the microorganism. In another embodiment, expression of the BCCP variant may result in improved acetyl CoA carboxylase activity (ACC) in the microorganism. This can lead to increased production of a Malonyl CoA-derived compound. The malonyl CoA-derived compounds include, but are not limited to a monofunctional fatty acids, a bifunctional fatty acids, an unsaturated fat acid derivatives, a 3-hydroxypropionic, flavanone or flavonoid. In one embodiment, a malonyl CoA-derived component is a fatty acid methyl ester (FAME), a fatty acid ethyl ester (FAEE), a fatty alcohol, a lipid amine or bifunctional acyl fatty acid derivative. In another embodiment, a fatty-alcohol is the malonyl CoA-derived compound. In another embodiment, a microorganism can be a microbial cellular. In another embodiment, a microbial cell can be a recombinant one. Cells from Escherichia include, but aren’t limited to: Bacillus; Cyanophyta; Lactobacillus; Zymomonas; Rhodococcus Pseudomonas Aspergillus Trichoderma Neurospora Fusarium Humicola Rhizomucor Kluyveromyces Pichia Mucor Mucor Myceliophtora Penicillium Phanerochaete Pleurot In one embodiment, a microbial cell comes from the Escherichia genus. In one embodiment, the cell of the microorganism is Escherichia. In another embodiment, a microbial cellular is from the genus Cyanophyta or a cyanobacteria. In another embodiment, a microbial cellular is from a Cyanophyta, such as, but not restricted to, Prochlorococcus or Synechococcus. In another embodiment, a microbial cellular is from a cyanobacterial specie including, but without limitation, Synechococcus, Synechocystis, and Synechococcus Elongatus PCC7942. In another embodiment, the microbial cell is from a specific cyanobacterial species including, but not limited to Synechococcus PCC7942; Synechocystis sp. PCC7001.

Another aspect” of the disclosure is a recombinant bacterium with altered expression of nucleic acids including accB, accC, or combinations thereof. This results in altered production by the microorganism of a malonyl CoA-derived compound. In one embodiment the altered expression is an increased expression. In another embodiment the altered expression is reduced expression. In another embodiment, altered expression occurs due to changes in one or several promoters driving expression of the nucleic sequence. The nucleic acids sequence of accB code for BCCP. In one embodiment, a variant nucleic sequence of accB code for the variant BCCP. In one embodiment, malonyl CoA-derived compounds include, but are not limited to: a monofunctional fatty acids derivative; a bifunctional fatty acids derivative; a polyketide and a 3-hydroxypropionic Acid. In one embodiment, a microorganism can be any of the following: Escherichia (but not limited to), Bacillus (but not restricted to), Cyanophyta (but including Lactobacillus), Rhodococcus (but excluding Pseudomonas), Aspergillus (3-hydroxypropionic acid), Neurospora (4-hydroxypropionic acid), Fusarium, Humicola (4, hydroxypropionic acid), Kluy In one embodiment, a microbial cell comes from the Escherichia genus. In one embodiment, a microbial coli cell is used. In another embodiment, a microbial cell from the genus Cyanophyta or a cyanobacteria is used. In another embodiment, the microbial cell is from a cyanobacteria, such as Prochlorococcus or Synechococcus or Synechocystis or Cyanothece or Nostoc Punctiforme. In one embodiment, a cyanobacterial sp. of Synechococcus Elongatus PCC7942 or Synechocystis Sp. is used. In one embodiment, the microorganism is a cyanobacterial species from Synechococcus PCC7942, Synechocystis sp. PCC7001.

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