Increasing energy costs and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals. Fatty acids are composed of long alkyl chains and represent nature's “petroleum,” being a primary metabolite used by cells for both chemical and energy storage functions. These energy-rich molecules are today isolated from plant and animal oils for a diverse set of products ranging from fuels to oleochemicals.
Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methylesters of fatty acids) is the major long chain product produced biologically, and it is almost exclusively derived from plant oils today. However, slow cycle times for engineering oil seed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria do produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels and large quantities are not made. Thus, the production of fatty acids from bacteria has not yet reached the point where it is cost effective.
Our laboratory has already had considerable success in engineering bacteria to produce more free fatty acids than are normally found in native bacteria. WO2011116279 for example, describes a recombinant bacterium comprising at least one overexpressed acyl-ACP thioesterase gene, and wherein at least one gene from the tricarboxylic acid cycle or glycolysis or both is inactivated to drive carbon in the direction of fat production. For example, an ACP thioesterase was combined with deletions in native fadD, and sucC. These bacteria significantly increased overall fat levels, as shown:
Free%Yield%FAimprove-(g FA/gimprove-Strain nameRelevant genotype(g/l)ment*glucose)ment*ML103_18ΔfadD acyl-ACP3.12—0.21—thioesterase+MLK163_18ΔfadD, ΔsucC acyl-ACP3.96270.2729thioesterase+MLK211_18ΔfadD ΔfabR acyl-ACP3.73200.2519thioesterase+MLK211_18AΔfadD ΔfabR fabA+ acyl-ACP0.79−750.09−57thioesterase+MLK211_18ZΔfadD ΔfabR fabZ+ acyl-ACP3.62160.2414thioesterase+MLK225_18ΔfadD ΔfadR acyl-ACP2.57−180.17−19thioesterase+MLK225_18ZΔfadD ΔfadR fabZ+ acyl-ACP3.71190.2519thioesterase+MLK227_18ΔfadD ΔfadR ΔfabR acyl-ACP2.25−280.17−19thioesterase+ML103_18AΔfadD fabA+ acyl-ACP0.44−860.07−67thioesterase+ML103_18ZΔfadD fabZ+ acyl-ACP4.61480.3148thioesterase+ML103_18fadRΔfadD fadR+ acyl-ACP4.19340.2729thioesterase+MLK212_18ΔfadD ΔsucC ΔfabR acyl-ACP3.83230.2624thioesterase+MLK212_18AΔfadD ΔsucC ΔfabR fabA+ acyl-ACP1.58−490.10−52thioesterase+MLK212_18ZΔfadD ΔsucC ΔfabR fabZ+ acyl-ACP5.15650.3462thioesterase+MLK213_18ΔfadD ΔsucC ΔfadR acyl-ACP2.75−120.19−10thioesterase+MLK213_18ZΔfadD ΔsucC ΔfadR fabZ+ acyl-ACP0.32−900.06−71thioesterase+MLK228_18ΔfadD ΔsucC ΔfabR ΔfadR acyl-ACP3.2440.210thioesterase+MLK163_18AΔfadD ΔsucC fabA+ acyl-ACP2.03−350.17−19thioesterase+MLK163_18ZΔfadD ΔsucC fabZ+ acyl-ACP5.65810.3881thioesterase+MLK163_18fadRΔfadD ΔsucC fadR+ acyl-ACP1.49−520.225thioesterase+fabA+ = overexpression of FabA by plasmid, plus wild type gene present;fabZ+ = overexpression of FabZ by plasmid, plus wild type gene present;fadR+ = overexpression of FadR by plasmid, plus wild type gene present;acyl-ACP thioesterase+ = overexpression of castor bean acyl ACP TE, plus wild type present.*percentage improvement based on ML103_18
WO2013096665 describes the next step in our work, which was to engineer a microorganism for producing enhanced amounts of long chain fatty acids, having an overexpressed acyl-ACP thioesterase, and at least one mutated gene selected from the group consisting of fabR, fabZ, fadR, fabH and combinations thereof, and optionally including a inactivated sucC gene. Various bacteria in this category produce more long chain fats, some of which are shown below:
Bacteria:Fatty Acid Profile:ΔfadD ΔfabR TE+about 60% C16:1ΔfadD ΔfadR TE+about 60% C14ΔfadD ΔfadR FabZ+ TE+about 60% C14ΔfadD FabA+ TE+about 90% C16
The next step was to enable the production of odd chain length fatty acids. Odd chain fatty acids can be made as described in U.S. application Ser. No. 14/104,628, MICROBIAL ODD CHAIN FATTY ACIDS, filed Dec. 12, 2013. In that application, the starting material was manipulated to be a C3 molecule (propionyl-CoA) by overexpressing a propionyl-CoA synthase gene. We also replaced the native β-ketoacyl-acyl carrier protein synthase III gene with one having a greater substrate preference for propionyl-coA than acetyl-coA. With these three modifications, greater odd chain fats were produced that was heretofore possible. In fact, >80% of the fats produced by such strains were of odd chain lengths. Some of the genes used therein included:
StrainGeneGene IDProtein_IDSalmonella entericaprpE1251890AFD57404.1Escherichia colifabH946003AAC74175.1E. colipfkA948412AAC76898.1E. colifadD946327AAC74875.1Bacillus subtilisfabH1936392CAB12974.1Bacillus subtilisfabH2939306CAB12857.1Staphylococcus aureusfabH1120958BAB57145.1Streptomyces peucetiusdpsCL35560.1AAA65208.1Ricinus communisacyl-ACPXM002515518thioesteraseCuphea hookerianaacyl-ACPU17076thioesteraseUmbellularia californicaacyl-ACPAAC49001thioesterase
The above genetic manipulations provided significant improvements in fat levels, and the excretion of visible amounts of fats also provided an easy method of collecting fats, while keeping the culture active and undisturbed, churning out more fats.
However, one improvement would be to provide a bacterium that could preferentially provide short chain fats, and another improvement would be to remove any feedback inhibition so that overall yields of short chain fats are even further increased.