Petroleum is a limited, natural resource found in the earth in liquid, gaseous, or solid forms. However, petroleum products are developed at considerable costs, both financial and environmental. In its natural form, crude petroleum extracted from the Earth has few commercial uses. It is a mixture of hydrocarbons, e.g., paraffins (or alkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes), aliphatic compounds, aromatic compounds, etc. of varying length and complexity. In addition, crude petroleum contains other organic compounds (e.g., organic compounds containing nitrogen, oxygen, sulfur, etc.) and impurities (e.g., sulfur, salt, acid, metals, etc.). Due to its high energy density and its easy transportability, most petroleum is refined into fuels, such as transportation fuels (e.g., gasoline, diesel, aviation fuel, etc.), heating oil, liquefied petroleum gas, etc.
Petrochemicals can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, or gels. Specialty chemicals have many commercial uses. Examples of specialty chemicals which can be produced from petrochemical raw materials include fatty acids, hydrocarbons (e.g., long chain hydrocarbons, branched chain hydrocarbons, saturated hydrocarbons, unsaturated hydrocarbons, etc.), fatty alcohols, fatty esters, fatty aldehydes, ketones, lubricants, etc. Fatty acids are used commercially as surfactants. Surfactants can be found, for example, in detergents and soaps. Fatty acids can also be used as additives in fuels, lubricating oils, paints, lacquers, candles, shortenings, cosmetics, and emulsifiers. In addition, fatty acids are used as accelerator activators in rubber products. Fatty acids can also be used as a feedstock to produce methyl esters, amides, amines, acid chlorides, anhydrides, ketene dimers, peroxy acids and esters.
Fatty esters have many commercial uses. For example, biodiesel, an alternative fuel, is comprised of esters (e.g., fatty acid methyl ester (FAME), fatty acid ethyl esters (FAEE), etc.). Some low molecular weight esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are comprised of esters. Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.
Similarly, fatty alcohols have numerous commercial uses. For example, worldwide annual sales of fatty alcohols and their derivatives are in excess of US$1 billion. The shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal care and household products, for example, detergents. In addition, fatty alcohols are used in waxes, gums, resins, pharmaceutical lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats.
Acetyl CoA carboxylase (ACC) plays an important role in regulating fatty acid synthesis and degradation. It is a biotin-dependent enzyme complex that catalyzes the first committed step of fatty acid biosynthesis, i.e., the irreversible carboxylation of acetyl-CoA to malonyl-CoA. ACC produces malonyl-CoA via its two catalytic activities, i.e., biotin carboxylase (BC) and carboxyltransferase (CT). In most prokaryotes, ACC is a multi-subunit enzyme that includes four polypeptides (subunits), encoded by distinct genes whose coordinate expression is controlled through multiple levels of regulation (Cronan et al. (2002) Progress in Lipid Research 41:407-435; James et al. (2004) Journal of Biological Chemistry 279(4):2520-2527). The four polypeptides of ACC assemble into a complex at a fixed ratio (Broussard et al. (2013) Structure 21:650-657). More specifically, the ACC reaction requires four proteins, i.e., biotin carboxylase (BC), biotinoyl (or biotin) carboxyl carrier protein (BCCP), and two proteins that form the carboxyltransferase (CT). The overall ACC reaction can be assayed by the ATP-dependent conversion of the acid-labile NaH14CO3 to the acid-stable malonic acid. There are similarities and differences between the ACC subunits of bacteria and plant plastids. But despite the complexity of the plant proteins, the sequences that are essential for ACC activity are not significantly different from the bacterial homologues (Cronan et al., supra).
It has been reported that the E. coli ACC is the least stable of the known ACC enzymes. The overall activity can be measured only when all four subunits are present at high concentrations, although two partial reactions can be measured in dilute protein solutions. The stable complexes are believed to be the BC complex and the CT alpha2 beta2 complex. The full length BCCP has been purified as a dimer and there are hints of the presence of an unstable BC2-BCCP2 complex. Other bacterial ACCs seem more stable than that of E. coli and ACC activity can be measured in dilute extracts of Helicobacter pylori and Pseudomonas citronellolis. In addition, the plant plastid ACCs seem more stable than E. coli ACCs. However, as in E. coli further purification of the intact enzyme results in dissociation and loss of the ACC activity that can be restored by mixing fractions containing the partial reaction activities. The subcomplexes are BC-BCCP and CT with no evidence for free intact BCCP or free CT beta, suggesting that BCCP and CT beta are degraded when free in solution (Cronan et al., supra).
The identification of the E. coli acc genes including accA, accB, accC, and accD has facilitated the study of the ACC proteins. Radiation suicide selections have been used to isolate mutants in fatty acid synthesis including in genes accB and accD that encode ACC subunits BCCP and CT beta, respectively. The accB mutant has been studied more extensively and the mutation G133S is responsible for temperature sensitive growth. This mutation results in a steric clash within the biotinoyl domain. This resultant mutant protein is easily denatured at higher temperatures and is thus sensitive to intracellular proteases. The mutant BCCP strain has only about 25 percent of the normal level of BCCP when it is grown at 30° C., yet the rates of growth and fatty acid synthesis are normal (Cronan et al., supra). It is, however, known that increasing the concentration of all four proteins of ACC can improve the flux through fatty acid biosynthesis to a certain degree (Davis et al. (2000) Journal of Biological Chemistry 275(37):28593-28598). Conversely, it has been shown that E. coli ACC can be inhibited by acylated derivatives of ACP while ACP lacking an acyl moiety cannot inhibit ACC (Davies et al. (2001) Journal of Bacteriology 183(4): 1499-1503).
There is a need for alternative routes to create both fuels and products currently derived from petroleum. As such, microbial systems offer the potential for the biological production of numerous types of biofuels and chemicals. Renewable fuels and chemicals can be derived from genetically engineered organisms (such as bacteria, yeast and algae). Naturally occurring biosynthetic pathways can be genetically altered to enable engineered organisms to synthesize renewable fuel and chemical products. In addition, microbes can be tailored, or metabolically engineered, to utilize various carbon sources as feedstock for the production of fuel and chemical products. Thus, it would be desirable to engineer an ACC to produce higher yields of malonyl-derived compounds (e.g., fatty esters, fatty alcohols and other fatty acid derivatives as well as non-fatty acid compounds) when expressed in a recombinant host cell.
Notwithstanding the advances in the field, there remains a need for improvements in genetically modified enzymes, recombinant host cells, methods and systems in order to achieve robust and cost-effective production of fuels and chemicals through fermentation of recombinant host cells. The present disclosure addresses this need by providing ACC variants that increase the yield and titer of malonyl-derived compounds.
USPTO Backgrounds
USPTO Backgrounds