Bacteria and method for synthesizing fatty acids

Abstract

The present invention discloses a process for increasing the production of free fatty acids at high yield (close to maximum theoretical yield), with various fatty acid compositions and various percentage of fatty acids accumulated intracellularly. This invention will enable the efficient production of other products derived from free fatty acids and/or products that can be branched out from the fatty acid synthesis pathways.

Claims

1. A genetically engineered bacteria for producing fatty acids, said bacteria being Enterobacteriaceae and comprising a genotype comprising: a) an overexpressed acyl ACP thioesterase (TE+); b) one or more of an overexpressed FabZ+, FadR+, or FabH+; c) one or more of a fadR, sucC, fabR, fadD, or ptsG; with the proviso that said bacteria does not have a genotype of fadD sucC TE+, wherein said bacteria produces at least 14% more fatty acid than having a control microorganism having fadD plus TE+.

2. The genetically engineered bacteria of claim 1, wherein the genotype of said bacteria comprises i) sucC FabZ+ TE+; ii) sucC fabR FabZ+ TE+; iv) fabR FadR+ TE+; or v) sucC fabR FadR+ TE+.

3. The genetically engineered bacteria of claim 1, wherein the genotype of said bacteria comprises sucC FabZ+ TE+.

4. The genetically engineered bacteria of claim 1, wherein the genotype of said bacteria comprises fadD sucC FabZ+ TE+.

5. The genetically engineered bacteria of claim 1, wherein the genotype of said bacteria comprises a genotype selected from the group consisting of: (1) fadD fabR acyl-ACP thioesterase+ (2) fadD fabR fabZ+ acyl-ACP thioesterase+ (3) fadD fadR acyl-ACP thioesterase+ (4) fadD fadR fabZ+ acyl-ACP thioesterase+ (5) fadD fadR fabR acyl-ACP thioesterase+ (6) fadD fabZ+ acyl-ACP thioesterase+ (7) fadD fadR+ acyl-ACP thioesterase+ (8) fadD sucC fabR acyl-ACP thioesterase+ (9) fadD sucC fabR fabZ+ acyl-ACP thioesterase+ (10) fadD sucC fadR acyl-ACP thioesterase+ (11) fadD sucC fadR fabZ+ acyl-ACP thioesterase+ (12) fadD sucC fabR fadR acyl-ACP thioesterase+ (13) fadD sucC fabZ+ acyl-ACP thioesterase+ (14) fadD sucC fadR+ acyl-ACP thioesterase+ (15) fabR acyl-ACP thioesterase+ (16) fabR fabZ+ acyl-ACP thioesterase+ (17) fadR acyl-ACP thioesterase+ (18) fadR fabZ+ acyl-ACP thioesterase+ (19) fadR fabR acyl-ACP thioesterase+ (21) fadR+ acyl-ACP thioesterase+ (22) sucC fabR acyl-ACP thioesterase+ (23) sucC fabR fabZ+ acyl-ACP thioesterase+ (24) sucC fadR acyl-ACP thioesterase+ (25) sucC fadR fabZ+ acyl-ACP thioesterase+ (26) fadD sucC fabR fadR acyl-ACP thioesterase+ (27) fadD sucC fabZ+ acyl-ACP thioesterase+ (28) fadD sucC fadR+ acyl-ACP thioesterase+ (29) fadD fabR fadR+ acyl-ACP thioesterase+ (30) fabR fadR+ acyl-ACP thioesterase+ (31) fadD sucC fabR fadR+ acyl-ACP thioesterase+ (32) sucC fabR fadR+ acyl-ACP thioesterase+ (33) fadD ptsG fabH+ acyl-ACP thioesterase+ (34) ptsG fabH+ acyl-ACP thioesterase+.

6. The genetically engineered bacteria of claim 1, wherein said bacteria produces at least 45% more fatty acid than having a control microorganism having fadD plus TE+.

7. The genetically engineered bacteria of claim 1, wherein said bacteria produces at least 65% more fatty acid than having a control microorganism having fadD plus TE+.

8. The genetically engineered bacteria of claim 1, wherein said bacteria produces at least 80% more fatty acid than having a control microorganism having fadD plus TE+.

9. A genetically engineered bacteria for producing increased amounts of fatty acids, said bacteria being Enterobacteriaceae and comprising a genotype selected from the group consisting of: (1) sucC fabZ+ acyl-ACP thioesterase+ (2) sucC fabR fabZ+ acyl-ACP thioesterase+ (4) fadR+ acyl-ACP thioesterase+ (5) sucC fabR acyl-ACP thioesterase+ (6) fabR acyl-ACP thioesterase (7) fadR fabZ+ acyl-ACP thioesterase+ (8) fabR fabZ+ acyl-ACP thioesterase+ (9) fadD sucC fabZ+ acyl-ACP thioesterase+ (10) fadD sucC fabR fabZ+ acyl-ACP thioesterase+ (11) fadD fabZ+ acyl-ACP thioesterase+ (12) fadD fadR+ acyl-ACP thioesterase+ (13) fadD sucC fabR acyl-ACP thioesterase+ (14) fadD fabR acyl-ACP thioesterase+ (15) fadD fadR fabZ+ acyl-ACP thioesterase+ (16) fadD fabR fabZ+ acyl-ACP thioesterase+ (17) fadD fabR fadR+ acyl-ACP thioesterase+ (18) fabR fadR+ acyl-ACP thioesterase+ (19) fadD sucC fabR fadR+ acyl-ACP thioesterase+ (20) sucC fabR fadR+ acyl-ACP thioesterase+ (21) fadD ptsG fabH+ acyl-ACP thioesterase+.

10. A genetically engineered Enterobacteriaceae comprising fadD sucC FabZ+ TE+.

11. A genetically engineered Enterobacteriaceae comprising fadD sucC fabR FabZ+ TE+.

12. A genetically engineered Enterobacteriaceae comprising fadD FabZ+ TE+.

13. A genetically engineered Enterobacteriaceae comprising sucC FabZ+ TE+.

14. A genetically engineered Enterobacteriaceae comprising sucC fabR FabZ+ TE+.

15. A method of producing fatty acids, comprising culturing a genetically engineered bacteria of any of claim 1-14 in a culture medium under conditions effective for the production of fatty acids; and harvesting said fatty acids from said bacteria and/or the culture medium.

16. The method of producing fatty acids of claim 15, comprising adding acetic acid to said culture medium and harvesting the fatty acids from said culture medium.

17. A method of producing fatty acids, comprising culturing a genetically engineered bacteria in a culture medium under conditions effective for the production of fatty acids; and harvesting said fatty acids from the microorganism and/or the culture medium, wherein the bacteria and the fatty acid profile are selected from the group consisting of those listed in Table 2.

18. The method of claim 17, wherein the bacteria and the fatty acid profile are selected from the group consisting of: TABLE-US-00008 Bacteria: Fatty Acid Profile: fadD fabR TE.sup.+ about 60% C16:1 fadD fadR TE.sup.+ about 60% C14 fadD fadR FabZ.sup.+ TE.sup.+ about 60% C14 fadD FabA.sup.+ TE.sup.+ about 90% C16

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Proposed metabolic map in which the introduction of additional appropriate pathways, the fatty acids can be converted to chemicals.

(2) FIG. 2. Simplified metabolic map showing the fatty acid synthesis pathway. The transcription factor FabR has shown to have negative effect on FabA and FabB; but the transcription factor FadR has the opposite effect on FabA and FabB (Fujita et al., 2007). Free fatty acids are formed in the presence of an acyl-ACP thioesterase, which breaks the fatty acid elongation cycle.

(3) FIG. 3. Cell viability of strain MLK163_18Z, in which at least 75% of the cells are still viable after 48 hours growth.

(4) FIG. 4. Graphic illustrating that over 70% of the cellular content of the mutant strains are fatty acids.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(5) FIG. 2 shows a simplified central aerobic metabolic pathway of Escherichia coli using glucose, for example, as a carbon source. Also included in FIG. 1 are the fatty acid biosynthesis pathways. Note that each fatty acid elongation cycle will increase the carbon chain length of the fatty acid by two. Free fatty acids can be produced by introducing an acyl-thioesterase gene. The presence of the thioesterase will break the fatty acid elongation cycle and release free fatty acids (Davis et al., 1993; Lu et al., 2008; Zhang et al., 2011).

(6) Also shown in FIG. 1 are the two transcription factors involved in the fatty acid biosynthesis and degradation pathways. The transcription factor FabR has shown to have negative effect on FabA and FabB; but the transcription factor FadR has the opposite effect on FabA and FabB (Fujita et al., 2007).

(7) Metabolically engineered strains were constructed based on the manipulation of the transcription factor(s) involved in the fatty acid biosynthesis and/or degradation pathways; a combination of these transcription factor(s) together with one or more of selected gene(s) in the fatty acid synthesis pathway, fatty acid degradation pathway or the central metabolic pathway.

Creating Strains

(8) The wild type E. coli strain was MG 1655 (F.sup. lambda.sup. ilvG.sup. rfb.sup. rph.sup. ATCC 47076). An fadD knockout was introduced to make ML103 (MG1655 (fadD) (Zhang 2011). The plasmid for overexpression was pTrc99a, a cloning vector from AMERSHAM PHARMACIA, but any suitable vector could be used.

(9) In this work, we used acyl ACP thioesterase from castor bean, added by plasmid per our prior work. See Zhang 2011 (pXZ18-pTrc99a carries an acyl-ACP thioesterase from Ricinus communis (castor bean) (XM002515518). However, any suitable enzyme can be used, and many are available in suitable expression plasmids already. Another vector, pXZ18R, is the same gene in the plasmid pXZ18 but with the addition of a fadR gene from E. coli.

(10) The strains studied in the present invention are created based on a fadD knock-out mutant strain ML103_18, where the 18 is a particular clone number. We used the fadD mutant strain as a base strain because it is often used in the literature and easily available. However, deleting this gene is completely optional, and it is not considered a critical component of the genetic background.

(11) The mutational set includes knockouts of one or more of FadR, SucC, FabR, FadD together with wild type or overexpressed acyl-ACP thioesterase together with overexpressed FabZ and FadR. The gene set should not include overexpressed FabA.

(12) TABLE-US-00003 Knockout Wild type or mutations Overexpression Overexpression BUT Excluding Zero, TE.sup.+ from any one or more of Optionally FabA.sup.+** one or more of species fabZ.sup.+, fadR.sup.+, fadD sucC fadR, sucC, or fabH.sup.+ acyl-ACP fabR, fadD, thioesterase+ ptsG bacteria with no further mutations **FabA is known to have negative effect when used with the full complement of mutations, but may be beneficial with certain subsets, and this is being tested.

(13) While our overexpression constructs were made by adding plasmid to a wild type background, this is not the only way to generate overexpression, and if desired the wild type gene can be completely replaced, other vectors could be used, and or the wild type gene could be upregulated. Further, while our gene sets included knockout mutations for simplicity and ease of interpreting results, knockout mutations can be replaced with reduced activity mutations.

Measuring Fatty Acid Production

(14) The metabolically engineered strains were studied in shake flasks. The strains were grown in 250 ml flasks, with 40 ml Luria-Bertani (LB) broth medium supplemented with 15 g/L of glucose, 1 mM IPTG, and appropriate amount of ampicillin. The cultures were grown in a rotary shaker at 250 rpm.

(15) Samples of the media were taken at 24 and 48 hrs after inoculation. The fatty acids were analyzed and quantified by GC/MS and GC/FID after extraction. Odd number saturated straight chain fatty acids, such as C13, C15 and/or C17, were used as the internal standard. The results shown in the table are the sum of all major free fatty acids in the sample. The data shown are means for triplicate experiments at 48 hrs.

(16) ML163 from WO2011116279 is very similar to MLK163_18 (bold font in Table 1) and has the same genetic construction of fadD, sucC and acyl-ACP thioesterase.sup.+, though the MLK variant has a kanamycin marker.

(17) ML103_18 (bold font) was chosen as the base or control strain in this work, although a bacteria wild type for the gene set in question would also be a suitable control, and the improvement in fatty acids levels would be even higher. Some constructs do not give a yield higher than ML163_18 (underlined in Table 1), a strain taught in earlier work. However, such strains may have other desirable properties, such as different fatty acid distribution and/or percentage excreted to the medium. Thus, such strains are included herein even though they may produce less fatty acid than strain ML163_18. Further, such strains when compared against wild type bacteria probably have some level of improved production.

(18) From the results shown below in Table 1, positive effects were observed in the strains shaded grey, and the best producers are indicated with an arrow.

(19) TABLE-US-00004 TABLE 1 Free fatty acid and yield (g of fatty acids produced/g of glucose used) fabA.sup.+ = overexpression of FabA by plasmid, plus wild type gene present fabZ.sup.+ = overexpression of FabZ by plasmid, plus wild type gene present; fadR.sup.+ = overexpression of FadR by plasmid, plus wild type gene present acyl-ACP thioesterase.sup.+ = overexpression of castor bean acyl ACP TE, plus wild type present. *percentage improvement based on ML103_18

(20) Inactivation of the transcription factor FabR improves fatty acid production and yield for both the parent strain and the sucC mutant strain (MLK211 vs ML103_18 and MLK212 vs MLK163_18).

(21) Overexpression of FabZ improves fatty acid production and yield for both the parent strain and the sucC mutant strain (ML103_18Z vs ML103_18 and MLK163_18Z vs MLK163_18). In fact, a combination of sucC inactivation and FabZ overexpression yield the best strain with a very high titer of 5.65 and a yield of 0.38 g/g (which is 81% improvement over the base strain ML103_18). The yield of 0.38 g/g is very close to the maximum theoretical value (The maximum theoretical yields of C14 and C16 straight chain fatty acids are 0.3629 and 0.3561 g/g, respectively).

(22) Overexpression of FadR improves fatty acid production and yield for the parent strain (ML103_18fadR vs ML103_18).

(23) Order of improvements: sucC fabZ.sup.+>sucCfabR fabZ.sup.+>fabZ.sup.+>fadR.sup.+>sucC>sucCfabR>fabR or fadR fabZ.sup.+>fabR fabZ.sup.+. By extracting only those with positive results in Table 1, we have the following Table 1.1, wherein the bolded strain MLK163-18 is similar to strain ML 163.

(24) TABLE-US-00005 TABLE 1.1 Free fatty acid and yield of positive strains Free FA % improve- Yield % improve- Strain name Relevant genotype (g/l) ment* (g/g) ment MLK163_18Z fadD sucC fabZ.sup.+ acyl-ACP 5.65 81 0.38 81 thioesterase.sup.+ MLK212_18Z fadD sucC fabR fabZ.sup.+ acyl- 5.15 65 0.34 62 ACP thioesterase.sup.+ ML103_18Z fadD fabZ.sup.+ acyl-ACP 4.61 48 0.31 48 thioesterase.sup.+ ML103_18fadR fadD fadR.sup.+ acyl-ACP 4.19 34 0.27 29 thioesterase.sup.+ MLK163.sub.18 fadD sucC acyl-ACP 3.96 27 0.27 29 thioesterase.sup.+ MLK212_18 fadD sucC fabR acyl-ACP 3.83 23 0.26 24 thioesterase.sup.+ MLK211_18 fadD fabR acyl-ACP 3.73 20 0.25 19 thioesterase.sup.+ MLK225_18Z fadD fadR fabZ.sup.+ acyl-ACP 3.71 19 0.25 19 thioesterase.sup.+ MLK211_18Z fadD fabR fabZ.sup.+ acyl-ACP 3.62 16 0.24 14 thioesterase.sup.+

(25) Additionally, some negative effects have been observed in the following strains:

(26) Overexpression of FabA significantly decreases fatty acid production and yield for both the parent strain and the sucC mutant strain (ML103_18A vs MLK103_18 and MLK163_18A vs ML163_18).

(27) Simultaneous deactivation of FadR and FabR also decreases fatty acid production and yield for the (MLK227_18 vs MLK103_18).

(28) Additional strains we have tested and gave very good results are MLK211(pXZ18R) and MLK212(pXZ18R)which are fadD fabR FadR.sup.+ acyl-ACP thioesterase.sup.+ and fadD sucC fabR FadR.sup.+ acyl-ACP thioesterase.sup.+. We are planning to test fadD fabR acyl-ACP thioesterase.sup.+ FadR.sup.+ FabZ.sup.+ and fadD sucC fabR acyl-ACP thioesterase.sup.+ FadR.sup.+ FabZ.sup.+, which we expect to give even better results.

(29) As a result, it is clear that random overexpression or deletion of transcription factors and/or with genes in the fatty acid synthesis and central metabolic pathway may not necessarily lead to positive results. Our data demonstrated that a selected single or selected combination of these manipulations is required to increase the yield and titer as shown in Table 2.

(30) Our invention also indicates that the long belief of the carboxylation of acetyl-CoA to malonyl-CoA is the limiting step of fatty acid biosynthesis may not necessarily be the case since we have constructed strains that can achieve high yield (close to maximum theoretical yield), high titer and high production rate of free fatty acids.

Distribution of Fatty Acids

(31) The genetic manipulations demonstrated herein also have significant effect on the distribution of the free fatty acids (Table 2). As such, this invention also will allow the manipulation or tailoring of the type of fatty acids to be produced. For example, while the engineered strain ML103_18A can produce more than 88% of C16, the other engineered strains MLK225_18 and MLK225_18Z can produce about 60% of C14, and MLK211_18 can produce about 60% of C16:1.

(32) This indicates that the exact genetic combination has a significant effect on the fatty acid composition, even one uses the same acyl-ACP thioesterase.

(33) TABLE-US-00006 TABLE 2 Fatty acid distribution (Percentage of total) Strain name Relevant genotype C14 C16:1 C16 C18:1 ML103_18 fadD acyl-ACP thioesterase.sup.+ 47.27 38.33 12.96 1.45 MLK163_18 fadD, sucC acyl-ACP thioesterase.sup.+ 37.30 31.74 28.06 2.91 MLK211_18 fadD fabR acyl-ACP thioesterase.sup.+ 19.14 59.12 17.50 4.24 MLK211_18A fadD fabR fabA.sup.+ acyl-ACP 16.28 15.45 64.40 3.88 thioesterase.sup.+ MLK211_18Z fadD fabR fabZ.sup.+ acyl-ACP 45.62 25.51 74.41 2.83 thioesterase.sup.+ MLK225_18 fadD fadR acyl-ACP thioesterase.sup.+ 59.63 23.36 14.02 ND MLK225_18Z fadD fadR fabZ.sup.+ acyl-ACP 58.56 3.05 38.40 ND thioesterase.sup.+ MLK227_18 fadD fadR fabR acyl-ACP 48.40 34.39 17.09 0.13 thioesterase.sup.+ ML103_18A fadD fabA.sup.+ acyl-ACP thioesterase.sup.+ 10.92 0.00 89.08 ND ML103_18Z fadD fabZ.sup.+ acyl-ACP thioesterase.sup.+ 57.91 8.16 33.92 ND ML103_18fadR fadD fadR.sup.+ acyl-ACP thioesterase.sup.+ 18.29 48.09 26.85 6.77 MLK212_18 fadD sucC fabR acyl-ACP 18.52 57.59 18.50 5.40 thioesterase.sup.+ MLK212_18A fadD sucC fabR fabA.sup.+ acyl-ACP 11.91 10.33 76.32 1.44 thioesterase.sup.+ MLK212_18Z fadD sucC fabR fabZ.sup.+ acyl-ACP 40.24 16.46 42.59 0.71 thioesterase.sup.+ MLK213_18 fadD sucC fadR acyl-ACP 55.95 27.05 16.31 0.69 thioesterase.sup.+ MLK213_18Z fadD sucC fadR fabZ.sup.+ acyl-ACP 44.58 3.51 43.74 ND thioesterase.sup.+ MLK228_18 fadD sucC fabR fadR acyl-ACP 38.26 38.13 22.78 0.83 thioesterase.sup.+ MLK163_18A fadD sucC fabA.sup.+ acyl-ACP 20.95 7.50 71.55 ND thioesterase.sup.+ MLK163_18Z fadD sucC fabZ.sup.+ acyl-ACP 53.25 10.55 36.20 ND thioesterase.sup.+ MLK163_18fadR fadD sucC fadR.sup.+ acyl-ACP 14.24 38.39 35.78 11.59 thioesterase.sup.+ ND: Not detected

(34) The super-producer strain MLK163_18Z (fadD sucC fabZ.sup.+ acyl-ACP thioesterase.sup.) was analyzed further by measuring the total cell dry weight. Samples were taken at 16, 24 36 and 48 hours after inoculation and tested for cell viability using propidium iodide staining of DNA and flow cytometry. The results are shown in FIG. 3.

(35) It was found that at least more than 75% of the cells remained viable after 48 hrs. The dry weight of the sample at 48 hours was also determined and used to estimate the fraction of free fatty acid inside the cells. As explained in FIG. 4, certain amounts of free fatty acids are released from the cell, whereas a greater amount of free fatty acids are still located within the cells. It was found that free fatty acid accounts for more than 70% of the cellular contents.

FabH Overexpression

(36) It has been reported that FabH is involved in the initiation of fatty acid biosynthesis, and we propose that overexpression of FabH may also contribute to the production of fatty acids. To study the impact of the initiation step in the fatty acids synthesis pathway, another engineered strain ML190_88-fabH (fadD ptsG FabH.sup.+) carrying a modified acyl-ACP thioesterase from C. palustris (Acc. No. Q39554) specific to shorter chain length and with FabH from E. coli (UNIPROTKB Acc. No. P0A6R0) overexpression was constructed and tested in Super Broth (SB) medium with 30 g/L of glucose. The strain ML190_88 fabH s about 19% improvement in fatty acids production over the control strain ML190_88, as shown in Table 3.

(37) TABLE-US-00007 TABLE 3 Free fatty acid (C8 straight chain fatty acid) Free FA % improve- Strain name Relevant genotype (C8) (g/l) ment* ML190_88 fadD ptsG acyl-ACP 1.08 thioesterase.sup.+ ML190_88_fabH fadD ptsG fabH.sup.+ 1.28 19 acyl-ACP thioesterase.sup.+ fabH.sup.+ = overexpression of FabH by plasmid, plus wild type enzyme present

(38) This result shows that with the same genetic background, overexpression of FabH improves fatty acid production. We will also plan to add FabH to the strains of Table 1.1, beginning with the best producers, such as fadD sucC FabZ.sup.+ TE.sup.+; fadD sucC fabR FabZ.sup. TE.sup.+; and fadD FabZ.sup.+ TE.sup.+. Although not yet available, it is predicted that the combination with further improve fatty acid production levels, although the effect on distribution of fats is not yet known.

(39) The present invention shows that manipulating repressor levels along with overexpression of certain fatty acid biosynthesis enzymes and overexpressed acyl ACP thioesterases can significantly increase fatty acid production, to level heretofore not thought possible. Further, the distribution of fatty acids can be manipulated by introducing different combinations of mutations. In addition, the present invention shows that overexpression of fabH can result in improvement of fatty acids production, at least in C-8 fatty acids.

(40) The following publications are incorporated by reference in their entirety for all purposes herein.

(41) Davies, H. M., et al., 1993. Fatty acid synthesis genes: Engineering the production of medium-chain fatty acids. p. 176-181. In: J. Janick and J. E. Simon (eds.), New crops. Wiley, N.Y.

(42) Fujita Y, et al., Regulation of fatty acid metabolism in bacteria. Mol Microbiol. November 2007; 66(4):829-39.

(43) Lu, X., et al., 2008. Overproduction of free fatty acids in E. coli: Implications for biodiesel production. Metabolic Engineering. 10: 333-339.

(44) Zhang X, et al. 2011. Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases Metabolic Engineering, Metab Eng. November 2011; 13(6):713-22.

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