Alpha omega bifunctional fatty acids

Abstract

The present disclosure describes an engineered microorganism for producing alpha omega bifunctional C6-16 fatty acids from renewable carbon sources.

Claims

1. An engineered bacterial cell comprising overexpressed enzymes to produce alpha, omega-bifunctionalized products, the overexpressed enzymes including: i) acyl-ACP thioesterase (TE); ii) -ketoacyl-ACP synthase III (KASIII) with substrate specificity for an omega functionalized-CoA primer; iii) a CoA synthase able to convert a primer precursor to said omega functionalized-CoA primer; and iv) optionally, reduced expression of any endogenous KASI, KASII or KASIII having a substrate preference for acetyl-CoA and propionyl-CoA, wherein said engineered bacterial cell comprises one of the following genotypes: a) TE+ and FabH+ and PrpE+; b) Rc TE+ and Sa FabH+ and Se PrpE+; c) Rc TE+ and Bs FabH+ and Se PrpE+; d) Ua TE+ and SaFabH+ and Se PrpE+; e) Ua TE+, Bs FabH+ and Se PrpE+; f) any of a-e) with added IL+ and GLYR+; g) any of a-e) with added Ec IL+ and At GLYR+; h) any of a-e) with added IL+and GLYR+and deleted endogenous icd and aceB, i) any of a-e) with added Ec IL+ and At GLYR+ and deleted endogenous icd and aceB; j) any of a-e) with added PYC+ and AAT+ and PAND+; k) any of a-e) with added PYC+ and AAT+ and PAND+ and PAL+; l) any of a-k) with added alcohol dehydrogenase (AlkJ) or AlkJ and aldehyde dehydrogenase (AlkH); and m) any of a-I) with added fadD; wherein Ec=E. coli, Re=Ricinus communis, Se=Salmonella enterica, At=Arabidopsis thaliana, Sa=Staphylococcus aureus, Bs=Bacillus subtilis, and Ua=Umbellularia californica, PrpE=propionyl-CoA synthetase, IL=isocitrate lyase, GLYR=glyoxylate reductase, PYC=pyruvate carboxylase, AAT=acetate-CoA transferase, PAND=aspartate 1-decarboxylase, PAL=bata-alanyl-CoA ammonia-lyase.

2. The bacteria cell of claim 1, wherein the omega functionalized-coA primers, the enzymes, and products are selected from: (i) primer: glycolate; enzymes: PrpE (propionyl-CoA synthetase), FabH (KASIII), and TE (acyl-ACP thioesterase); products; -hydroxy fatty acids (FA); (ii) primer: beta alanine; enzymes: PrpE, FabH and TE; products: -amino FA; or (iii) primer: acrylic acid; enzymes: PrpE, FabH and TE; products: -unsaturated FA.

3. The bacteria cell of claim 1, further comprising: a) overexpression of: i) a propionyl-CoA synthetase, ii) a glyoxylate reductase, and iii) an isocitrate lyase; and, b) deactivation of: i) endogenous malate synthase A, ii) endogenous isocitrate dehydrogenase, and iii) endogenous acyl-CoA synthetase.

4. The bacteria cell of claim 1, further comprising overexpression of a) pyruvate carboxylase (E.C. 6.4.1.1); b) acetate-CoA transferase (E.C. 2.8.3.8); and, c) aspartate 1-decarboxylase (E.C. 4.1.1.11).

5. The bacteria cell or claim 4, further comprising overexpressed beta-alanyl-CoA ammonia-lyase (EC 4.3.1.6).

6. The bacteria cell of claim 1, further including overexpression of a propionyl-CoA synthetase, overexpression of a glyoxylate reductase, overexpression of an isocitrate lyase, deactivation or deletion of endogenous malate synthase A, deactivation or deletion of endogenous isocitrate dehydrogenase, and deactivation or deletion of native acyl-CoA synthetase.

7. The bacteria cell of claim 1, wherein the KASIII is FabH from a Gram-positive bacteria.

8. The bacteria cell of claim 1, wherein the KASIII is FabH from Staphylococcus aureus S. pneumonia, Streptomyces glaucescens, A. acidocaldarius, B. vulgatus Legionella pneumophila, Aeromonas hydrophila, Bacteroides vulgatus, Brevibacterium linens, Capnocytophaga gingivalis, Thermus aquaticus, Bacillus licheniformis, Desulfovibrio vulgaris, Bacillus subtilis, S. Haliangium ochraceum, or Alicyclobacillus acidocaldarius.

9. The bacteria cell of claim 3, wherein the propionyl-CoA synthetase is from Salmonella enterica (Se prpE.sup.+).

10. The bacteria cell of claim 3, wherein the glyoxylate reductase is from Arabidopsis thaliana (At GLYR.sup.+).

11. The bacteria cell of claim 3, wherein the isocitrate lyase is from E. coli MG1655 (Ec IL.sup.+).

12. The bacteria cell of claim 1, comprising one of the following genotypes: a) TE.sup.+ and FabH.sup.+ and prpE.sup.+; b) Rc TE.sup.+ and Sa FabH.sup.+ and SE PrpE.sup.+; c) Rc TE.sup.+ and Bs FabH.sup.+ and SE PrpE.sup.+; d) Ua TE.sup.+ and Sa FabH.sup.+ and SE PrpE.sup.+; e) Ua TE.sup.+, Bs FabH.sup.+ and SE PrpE.sup.+; f) any of a-e) with add IL.sup.+ and GLYR.sup.+; g) any of a-e) with add EC IL.sup.+ and AT GLYR.sup.+; h) any of a-e) with add IL.sup.+ and GLYR.sup.+ and deleted endogenous icd and aceB; i) any of a-e) with add EC IL.sup.+ and AT GLYR.sup.+ and deleted endogenous icd and aceB; j) any of a-e) with add PYC.sup.+ and AAT.sup.+ and PAND.sup.+; k) any of a-e) with add PYC.sup.+ and AAT.sup.+ and PAND.sup.+ and PAL.sup.+; or l) any of a-k) with add fadD; wherein Ec=E, coli, Rc=Ricimus communis, Se=Salmonella enterica, At=arabidopsis thaliana, Sa=Staphylococcus aureus, Bs=Bacillus subtilis, and Ua=Umbellularia californica.

13. The engineered bacterial cell of claim 1, further comprising overexpressed alcohol dehydrogenase (AlkJ) or aldehyde dehydrogenase (AlkH) or both AlkJ and AlkH.

14. An engineered bacteria cell comprising overexpressed enzymes including: a) acyl-ACP thioesterase (TE); b) -ketoacyl-ACP synthase III (KASIII) with substrate specificity for an omega functionalized-CoA primer; c) a CoA synthase able to convert a primer precursor to said omega functionalized-CoA primer; d) optionally reduced expression of any endogenous KASI, KASII or KASIII having a substrate preference for acetyl-coA and propionyl-CoA; and, e) one of the following: i) overexpression of a propionyl-CoA synthetase, a glyoxylate reductase, an isocitrate lyase, and deactivation of endogenous malate synthase A, endogenous isocitrate dehydrogenase, and endogenous acyl-CoA synthetase, ii) overexpression of pyruvate carboxylase (E.C. 6.4.1.1), acetate-CoA transferase (E.C. 2.8.3.8); and aspartate 1-decarboxylase (E.C. 4.1.1.11), or iii) overexpression of pyruvate carboxylase (E.C. 6.4.1.1), acetate-CoA transferase (E.C. 2.8.3.8), aspartate 1-decarboxylase (E.C. 4.1.1.11) and beta-alanyl-CoA ammonia-lyase (EC 4.3.1.6).

15. A method of producing alpha-omega bi-functional C6-16 fatty acids, comprises the steps of: a) culturing the engineered bacterial cell of claim 1 in a medium under conditions suitable for growth and production of alpha-omega bifunctional C6-16 fatty acids; b) isolating said alpha-omega bifunctional C6-16 fatty acids or their derivatives from the bacterial cell or the medium or both, c) wherein said alpha-omega bifunctional C6-16 fatty acids are selected from omega-hydroxy fatty acids, alpha omega dicarboxylic fatty acids, omega aldehyde fatty acids, omega amino fatty acids, fatty acids with a double bond at the omega position, omega halogenated fatty acids, omega phenyl fatty acids, omega cyclic fatty acids and omega branched fatty acids.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Schematic diagram showing the genetically engineered biosynthetic pathway of hydroxy fatty acid and dicarboxylic fatty acid.

(2) FIG. 2. Schematic diagrams of the plasmid pWLAA with TE, GLYR and AceA overexpression. TE: acyl ACP thioresterase from Ricinus communis; GLYR: glyoxylate reductase from Arabidopsis thaliana; AceA: isocitrate lyase from Escherichia coli MG1655; pTrc-lacO, trc promoter without lacO binding site; pTrc, trc promoter; lacI: lac operon repressor; AmpR, ampicillin resistant gene; rrnBT 1,2, transcriptional terminator of rrnB.

(3) FIG. 3. Mass spectrum of the fragmentation patterns of derivatized 16-hydroxyhexadecanoic acid of sample.

(4) FIG. 4 shows the scheme of the disclosure. Strictly speaking, the donor involved in the condensation reaction is malonyl-ACP, but it is derived from acetyl-CoA, as shown here.

(5) FIG. 5 displays a partial listing of available KASIII sequences.

(6) FIG. 6 is a partial listing of embodiments, anyone or more of which can be combined with any other one or more.

DETAILED DESCRIPTION

(7) The specification in its entirety is to be treated as providing a variety of details that can be used interchangeably with other details, as the specification would be of inordinate length if one were to list every possible combination of genes/vectors/enzymes/hosts that can be made to enable FAS of omega functionalized fatty acids or derivatives thereof.

(8) The invention provides a novel method of producing alpha-omega bifunctional fatty acids of various carbon chain lengths using engineered microbial from renewable carbon sources. Various constructs have been engineered and C6-16 fatty acids were successfully produced from these constructs.

16-Hydroxyhexanoic Acid with Added Glycolate

(9) Omega-hydroxy fatty acid can be produced by providing a primer precursor to the engineered strains that can first activate this precursor by converting it to the active acyl-CoA form, and then incorporate the activated primer into the fatty acid synthesis cycle. Finally an acyl-ACP thioesterase will be used to release the free omega-hydroxy fatty acid.

(10) Glycolate was used as the priming precursor in this proof-of-concept experiment. Three engineered strains were studied. The strain ML103 (pWL1T, pBAD33) harbors a plasmid pWL1T carrying an acyl-ACP thioesterase from Ricinus communis under a constitutive promoter and a cloning vector pBAD33 as the control. The second strain ML103 (pWL1T, pBHE2) also harbors the same plasmid pWL1T carrying an acyl-ACP thioesterase from Ricinus communis and a plasmid pBHE2 carrying a -ketoacyl-ACP synthase III or (KASIII) from Staphylococcus aureus and a propionyl-CoA synthetase (to activate the glycolate) from Salmonella enterica in pBAD33. The other strain ML103 (pWL1T, pBHE3) is similar to the ML103 (pWL1T, pBHE2) but carrying a -ketoacyl-ACP synthase III from Bacillus subtilis.

(11) A seed culture was prepared by inoculating a single colony from a freshly grown plate in 5 mL of LB medium in an orbital shaker (New Brunswick Scientific, New Jersey, USA) operated overnight at 250 RPM and 37 C. A secondary preculture was prepared by inoculating 0.5 mL seed culture to a 250-mL flask containing 50 mL LB medium and incubating at 30 C. and 250 RPM for 9 h.

(12) The cells were harvested aseptically by centrifugation at 3,300g for 5 min, and resuspended in the appropriate volume of fresh fermentation medium which was calculated based on the inoculation size of 10%. LB medium was supplemented with 15 g/L of glucose and with or without the omega-functionalized primer, in this case glycolic acid (2.5 g/L or 5 g/L). The initial pH was set to be 7.5.

(13) All of the media were supplemented with 100 mg/L ampicillin and 34 mg/L chloramphenicol. The expression of the prpE and fabH on plasmid pBHE2 and pBHE3 was induced by 10 mM arabinose. The cells were then cultivated in an orbital shake operated at 250 RPM and 30 C. Samples were taken at 24, 48 and 72 h for hydroxy fatty acids, fatty acids and extracellular metabolite analysis. All experiments were carried out in triplicate.

(14) The ability of the engineered strain to incorporate the primer precursor-glycolateinto the fatty acid synthesis cycle to produce 16-hydroxyhexanoic acid was demonstrated in Table 1. The control strain did not produce any detectable quantity of hydroxyl fatty acid. Both strains, ML103 (pWL1T, pBHE2) and ML103 (pWL1T, pBHE3), produce significant quantities of omega hydroxy fatty acids (Table 1). The strain ML103 (pWL1T, pBHE2) produced more than 425 mg/L of omega hydroxy fatty acids with 5 g/L of glycolate addition (Table 1).

(15) The mass spectrum of the fragmentation patterns of derivatized 16-hydroxyhexadecanoic acid of sample is shown in FIG. 3, which is verified with that from the NIST/EPA/NIH Spectral Library.

(16) Note: the fadD mutant was for convenience only and is not essential, although it is beneficial. The protein FadD encodes a long-chain fatty acyl coenzyme A (acyl-CoA) ligase that is postulated to activate exogenous long-chain fatty acids by acyl-CoA ligation concomitant with transport across the cytoplasmic membrane. Thus, the mutant produces higher amounts of fatty acids because this null mutant reduces beta oxidation of fats.

(17) TABLE-US-00007 TABLE 1 Production of long chain length omega- hydroxy fatty acid using exogenous glycolate Glycolate Glucose - Relevant addition Time Cell density consumed hydroxyhexadecanoic Strains phenotype (g/L) (h) (OD.sub.600) (mM) acid (mg/L) ML103 fadD 2.5 24 9.27 0.15 47.2 1.78 ND (pWL1T, Rc TE.sup.+ 48 4.93 0.14 77.6 1.67 ND pBAD33) 72 4.22 0.13 86.5 1.11 ND ML103 fadD 5.0 24 10.3 0.11 45.1 0.76 ND (pWL1T, Rc TE.sup.+ 48 5.46 0.31 78.4 2.31 ND pBAD33) 72 3.97 0.14 85.3 0.40 ND ML103 fadD 2.5 24 4.81 0.09 31.6 1.79 171 2.07 (pWL1T, Rc TE.sup.+ 48 6.14 0.16 50.0 1.13 271 7.6 pBHE2) Sa fabH.sup.+ 72 8.81 0.36 61.4 0.34 288 10.6 Se prpE.sup.+ ML103 fadD 5.0 24 5.65 0.11 26.4 0.41 191 4.46 (pWL1T, Rc TE.sup.+ 48 7.09 0.36 46.8 1.03 386 5.82 pBHE2) Sa fabH.sup.+ 72 10.3 0.24 64.5 0.67 428 6.06 Se prpE.sup.+ ML103 fadD 2.5 24 6.59 0.12 39.6 0.60 158 3.06 (pWL1T, Rc TE.sup.+ 48 8.44 0.09 55.5 0.58 257 0.88 pBHE3) Bs fabH.sup.+ 72 9.94 0.10 66.0 0.17 279 4.23 Se prpE.sup.+ ML103 fadD 5.0 24 7.30 0.38 34.4 1.04 179 2.75 (pWL1T, Rc TE.sup.+ 48 9.45 0.40 54.2 0.72 309 1.43 pBHE3) Bs fabH.sup.+ 72 10.5 0.17 71.6 0.79 332 2.44 Se prpE.sup.+ ND: not detected. Rc_TE.sup.+: overexpression of acyl-ACP thioesterase from Ricinus communis under a constitutive promoter system in a modified pTrc99a cloning vector Sa fabH.sup.+: overexpression of a -ketoacyl-ACP synthase III from Staphylococcus aureus in pBAD33 Se prpE.sup.+: overexpression of a propionyl-CoA synthetase from Salmonella enterica Bs fabH.sup.+: overexpression of a -ketoacyl-ACP synthase III from Bacillus subtilis in pBAD33

Omega-Hydroxytetradecanoic Acid from Glycolate

(18) Glycolate was used as the primer precursor in this experiment, which changed the TE to make a shorter omega functionalized fat. Two engineered strains were studied. The strain ML103 (pXZM12, pBHE2) also harbors the plasmid pXZM12 carrying an acyl-ACP thioesterase from Umbellularia californica and a plasmid pBHE2 carrying a -ketoacyl-ACP synthase III from Staphylococcus aureus and a propionyl-CoA synthetase from Salmonella enterica in pBAD33. The other strain ML103 (pXZM12, pBHE3) is similar to the ML103 (pXZM12, pBHE2) but carrying a -ketoacyl-ACP synthase III from bacillus subtilis.

(19) The seed culture and secondary preculture were prepared as described. The cells were harvested and inoculated at 10% as described in LB, pH 7.5, with 15 g/L of glucose and with or without glycolic acid (2.5 g/L or 5 g/L). All of the media were supplemented with 100 mg/L ampicillin and 34 mg/L chloramphenicol. The expression of TE from U. Californica (pXZM12) was induced by the addition of isopropyl--D-thiogalactopyranoside (IPTG) to the final concentration of 200 M. The expression of the prpE and fabH on plasmid pBHE2 and pBHE3 was induced by 10 mM arabinose.

(20) The cells were then cultivated in an orbital shaker operated at 250 RPM and 30 C. Samples were taken at 24, 48 and 72 h for hydroxy fatty acids, fatty acids and extracellular metabolite analysis. All experiments were carried out in triplicate.

(21) The ability of the engineered strain to incorporate the primer precursorglycolateinto the fatty acid synthesis cycle to produce co-hydroxyhexanoic acid was demonstrated in Table 2. Both strains, ML103 (pXZM12, pBHE2) and ML103 (pXZM12, pBHE3), produce significant quantities of omega hydroxy fatty acids (Table 2). The strain ML103 (pXZM12, pBHE2) produced more than 240 mg/L of omega hydroxy fatty acids with 5 g/L of glycolate addition (Table 2).

(22) TABLE-US-00008 TABLE 2 Production of medium chain length (C14) omega- hydroxy fatty acid using exogenous glycolate Glycolate Glucose - Relevant Addition Time Cell density consumed hydroxytetradecanoic Strains phenotype (g/L) (h) (OD.sub.600) (mM) acid (mg/L) ML103 fadD 2.5 24 5.22 0.21 35.8 1.42 72.4 2.54 (pXZM12, Ua_TE.sup.+ 48 7.36 0.14 55.6 1.21 161 9.88 pBHE2) Sa fabH.sup.+ 72 9.11 0.25 66.2 0.68 196 7.45 Se prpE.sup.+ ML103 fadD 5.0 24 5.35 0.16 28.4 0.55 106 3.36 (pXZM12, Ua_TE.sup.+ 48 7.68 0.22 48.2 1.21 189 8.90 pBHE2) Sa fabH.sup.+ 72 9.72 0.31 65.1 0.88 248 11.7 Se prpE.sup.+ ML103 fadD 2.5 24 6.15 0.11 36.2 0.69 68.5 4.2.sup. (pXZM12, Ua_TE.sup.+ 48 8.23 0.07 58.9 0.42 112 8.65 pBHE3) Bs fabH.sup.+ 72 9.96 0.22 70.1 0.21 156 7.93 Se prpE.sup.+ ML103 fadD 5.0 24 6.84 0.25 26.3 1.56 90.4 4.36 (pXZM12, Ua_TE.sup.+ 48 8.85 0.32 51.8 0.65 172 7.82 pBHE3) Bs fabH.sup.+ 72 10.6 0.16 67.4 0.88 216 9.91 Se prpE.sup.+ Ua_TE.sup.+: overexpression of acyl-ACP thioesterase from Umbellularia californica under a constitutive promoter system in a modified pTrc99a cloning vector Sa fabH.sup.+: overexpression of a -ketoacyl-ACP synthase III from Staphylococcus aureus in pBAD33 Se prpE.sup.+: overexpression of a propionyl-CoA synthetase from Salmonella enterica Bs fabH.sup.+: overexpression of a -ketoacyl-ACP synthase III from bacillus subtilis in pBAD33

Engineered Strains Producing Glycolate

(23) In the above proof of concept experiments, glycolate was provided to the cells for use as a primer precursor. However, strains can be built to provide their own primers and/or precursors, and this experiment demonstrates proof of concept for microbes engineered to make glycolate.

(24) Several strains were designed, constructed and tested for their ability to produce glycolate in vivo from renewable carbon source such as glucose. The design is based on first producing glyoxylate from glucose using the glyoxylate bypass pathway and then converting glyoxylate to glycolate using the enzyme glyoxylate reductase.

(25) Several metabolic engineering approaches were used to further divert the carbon flux to glyoxylate; these include the overexpression of isocitrate lyase, and inactivation of both malate synthase and isocitrate dehydrogenase.

(26) The control strain ML013(pWL1T) did not produce any detectable glycolate. However, the strain ML103 (pWLA), carrying the glyoxylate reductase from A. thaliana is capable of producing 186 mg/L of glycolate (Table 3). Glycolate production further increased to 304 mg/L with the overexpression of both glyoxylate reductase and isocitrate lyase.

(27) Using the malate synthase mutant strain, DW101, as the host strain further increases the glycolate production. The strain DW101 (pWLA) with overexpression of glyoxylate reductase from A. thaliana produced 633 mg/L of glycolate while the strain DW101 (pWLAA) with overexpression of glyoxylate reductase and isocitrate lyase produced as high as 939 mg/L of glycolate.

(28) The malate synthase and isocitric dehydrogenase double mutant strain, DW102, produces even higher levels of glycolate. The DW102 (pWLA) and DW102 (pWLAA) produced 819 mg/L and 1204 mg/L of glycolate, respectively. This set of experiments demonstrated that glycolate can be produced at high levels with the engineered strains.

(29) TABLE-US-00009 TABLE 3 Production of glycolate by engineered strains Glucose Relevant Time Cell density consumed Acetate glycolate Strains phenotype (h) (OD.sub.600) (mM) (mM) (mg/L) ML103 fadD 24 7.76 0.30 43.2 0.84 12.7 0.34 ND (pWL1T) Rc_TE.sup.+ 48 5.41 0.17 74.8 0.57 24.0 1.74 ND 72 5.08 0.12 85.4 0.33 8.60 0.30 ND ML103 fadD 24 8.21 0.11 40.8 2.26 14.6 0.43 ND (pWLA) Rc_TE.sup.+ 48 6.00 0.12 69.5 1.80 23.2 0.46 ND At GLYR.sup.+ 72 4.47 0.15 80.1 0.85 12.4 0.03 186 7.39 ML103 fadD 24 5.68 0.25 31.5 1.52 15.5 0.62 ND (pWLAA) Rc_TE.sup.+ 48 6.15 0.10 58.4 0.52 26.8 0.98 121 4.49 At GLYR.sup.+ 72 5.01 0.07 69.7 0.63 14.5 0.30 304 7.90 Ec IL.sup.+ DW101 fadD 24 4.34 0.13 27.9 0.44 14.4 0.36 ND (pWLA) aceB 48 5.43 0.31 54.5 0.68 45.2 0.10 212 8.21 Rc_TE.sup.+ 72 3.40 0.13 67.4 0.29 57.0 0.81 633 8.85 At GLYR.sup.+ DW101 fadD 24 4.18 0.06 30.2 1.00 17.9 0.55 385 6.12 (pWLAA) aceB 48 4.65 0.04 51.5 0.94 42.9 0.48 605 17.0 Rc_TE.sup.+ 72 3.14 0.15 67.5 0.97 56.0 0.53 939 10.1 At GLYR.sup.+ Ec IL.sup.+ DW102 fadD 24 4.56 0.22 25.8 0.94 20.9 0.54 324 14.2 (pWLA) aceB 48 4.82 0.11 46.7 0.63 46.0 0.60 556 13.4 icd 72 3.59 0.13 61.9 0.38 60.6 0.74 819 9.45 Rc_TE.sup.+ At GLYR.sup.+ DW102 fadD 24 3.54 0.11 21.9 0.48 18.2 0.24 318 7.34 (pWLAA) aceB 48 4.10 0.22 40.3 0.46 46.0 0.67 1012 26.4 icd 72 4.36 0.14 55.1 0.71 62.6 0.66 1206 38.5 Rc_TE.sup.+ At GLYR.sup.+ Ec IL.sup.+ ND: not detected. Rc_TE.sup.+: overexpression of acyl-ACP thioesterase from Ricinus communis under a constitutive promoter system in a modified pTrc99a cloning vector At GLYR.sup.+: overexpression of a glyoxylate reductase from Arabidopsis thaliana Ec IL.sup.+: overexpression of an isocitrate lyase from E. coli MG1655 aceB: deactivation or deletion of malate synthase A, aceB icd: deactivation or deletion of isocitrate dehydrogenase

De Novo Hydroxy Fatty Acid from Glucose

(30) The above experiments proved that we can design strains to make omega hydroxy fats of varying length with added primer precursor. We also showed that we could add a pathway to make the precursor in vivo. These next experiments provide proof-of-concept that both concepts function together, to make omega-hydroxy fats de novo from a carbon source, such as glucose.

(31) In this experiment as an example, the de novo production of hydroxy fatty acid, specifically w-hydroxyhexadecanoic (c16) acid, from glucose was demonstrated using the engineered strains.

(32) Three engineered strains were studied. The control strain DW102 (pTrc99A, pBAD33) harbors two cloning vectors pTrc99a and pBAD33.

(33) The second strain DW102 (pWLAA, pBHE2) harbors two plasmids; plasmid pWLAA carries an acyl-ACP thioesterase from Ricinus communis, a glyoxylate reductase from Arabidopsis thaliana and an isocitrate lyase from E. coli MG1655 while plasmid pBHE2 carries a -ketoacyl-ACP synthase III from Staphylococcus aureus and a propionyl-CoA synthetase from Salmonella enterica.

(34) The third strain DW102 (pWLAA, pBHE3) also harbors two plasmids which include the same plasmid pWLAA and plasmid pBHE3 carrying a -ketoacyl-ACP synthase III from bacillus subtilis and a propionyl-CoA synthetase from Salmonella enterica.

(35) The experiments were as described above, and the expression of the prpE and fabH on plasmid pBHE2 and pBHE3 was induced by 10 mM arabinose, and the expression of the TE was induced by IPTG. Isocitrate lyase was induced with IPTG.

(36) The control strain, DW102 (pTrc99A, pBAD33) did not show any detectable co-hydroxyhexadecanoic acid. Both DW102 (pWLAA, pBHE2) and DW102 (pWLAA, pBHE3) produced more than 300 mg/L of hydroxyhexadecanoic (C16) acid in 72 h (Table 4). These results clearly demonstrate the ability of the engineered strain to produce hydroxyl fatty acids from glucose.

(37) TABLE-US-00010 TABLE 4 De novo hydroxy fatty acid production from glucose Cell Glucose - Relevant Time density consumed Acetate hydroxyhexadecanoic Strains phenotype (h) (OD.sub.600) (mM) (mM) acid (mg/L) DW102 fadD 24 4.37 0.25 28.7 0.57 17.9 0.73 ND (pTrc99A, 48 5.35 0.27 43.4 0.20 41.3 0.80 ND pBAD33) 72 7.52 0.24 54.2 1.63 60.7 0.50 ND DW102 fadD 24 4.11 0.19 26.4 0.65 14.6 0.43 188 4.73 (pWLAA, aceB 48 5.68 0.06 32.6 1.27 36.3 0.48 296 6.86 pBHE2) icd 72 6.47 0.15 39.0 0.93 43.7 1.10 342 13.6 Rc_TE.sup.+ At GLYR.sup.+ Sa fabH.sup.+ Se prpE.sup.+ DW102 fadD 24 3.80 0.15 24.05 0.83 16.8 0.79 170 2.09 (pWLAA, aceB 48 4.48 0.26 30.42 0.95 34.9 1.88 288 7.16 pBHE3) icd 72 5.25 0.07 35.70 0.15 46.7 0.57 329 12.0 Rc_TE.sup.+ At GLYR.sup.+ Ec IL.sup.+ Bs fabH.sup.+ Se prpE.sup.+ Rc_TE.sup.+: overexpression of acyl-ACP thioesterase from Ricinus communis under a constitutive promoter system in a modified pTrc99a cloning vector At GLYR.sup.+: overexpression of a glyoxylate reductase from Arabidopsis thaliana Ec IL.sup.+: overexpression of an isocitrate lyase from E. coli MG1655 Sa fabH.sup.+: overexpression of a -ketoacyl-ACP synthase III from Staphylococcus aureus in pBAD33 Se prpE.sup.+: overexpression of a propionyl-CoA synthetase from Salmonella enterica Bs fabH.sup.+: overexpression of a -ketoacyl-ACP synthase III from bacillus subtilis in pBAD33 aceB: deactivation or deletion of malate synthase A, aceB icd: deactivation or deletion of isocitrate dehydrogenase

Amino Fats with Beta-Alanine Added

(38) Beta-alanine is used as the priming molecule in this experiment to produce amino fatty acids. Three engineered strains will be studied. The strain ML103(pWL1T, pBAD33) harbors a plasmid pWL1T carrying an acyl-ACP thioesterase from Ricinus communis under a constitutive promoter and a cloning vector pBAD33 as the control. The second strain ML103 (pWL1T, pBHE2) also harbors the same plasmid pWL1T carrying an acyl-ACP thioesterase from Ricinus communis and a plasmid pBHE2 carrying a -ketoacyl-ACP synthase III from Staphylococcus aureus and a propionyl-CoA synthetase from Salmonella enterica in pBAD33. The other strain ML103 (pWL1T, pBHE3) is similar to the ML103 (pWL1T, pBHE2), but carrying a -ketoacyl-ACP synthase III from bacillus subtilis.

(39) The seed culture is prepared by inoculating a single colony from a freshly grown plate in 5 mL of LB medium in an orbital shaker (New Brunswick Scientific, New Jersey, USA) is operated overnight at 250 RPM and 37 C. The secondary preculture is prepared by inoculating 0.5 mL seed culture to a 250-mL flask containing 50 mL LB medium and incubating at 30 C. and 250 RPM for 9 h.

(40) The cells are harvested aseptically by centrifugation at 3,300g for 5 min, and resuspended in the appropriate volume of fresh fermentation medium which is calculated based on the inoculation size of 10%. LB medium is supplemented with 15 g/L of glucose and with or without beta-alanine acid (2.5 g/L or 5 g/L) as the primer precursor. The initial pH is set to be 7.5.

(41) All of the media are supplemented with 100 mg/L ampicillin and 34 mg/L chloramphenicol. The expression of the prpE and fabH on plasmid pBHE2 and pBHE3 is induced by 10 mM arabinose.

(42) The cells are then cultivated in an orbital shake is operated at 250 RPM and 30 C. Samples are taken at 24, 48 and 72 h for amino fatty acids, fatty acids and extracellular metabolite analysis. All experiments are carried out in triplicate.

Amino Fats with Beta-Alanine In Vivo

(43) Beta-alanine produced in vivo is used as the priming molecule in this experiment. Beta alanine overproducing strains, named KSBA100, with overexpression of pyruvate carboxylase (Pyc), aspartate aminotransferase (AAT) and aspartate decarboxylase (PAND) from various sources are constructed.

(44) Three engineered strains will be studied in this experiment to generate amino fatty acids. The strain KSBA100 (pWL1T, pBAD33) harbors the genes encoded for Pyc, AAT, PAND and TE from Ricinus communis and a cloning vector pBAD33 as the control. The second strain KSBA100 (pWL1T, pBHE2) also harbors the same set of genes encoded for Pyc, AAT, PAND and TE from Ricinus communis and a plasmid pBHE2 carrying a -ketoacyl-ACP synthase III from Staphylococcus aureus and a propionyl-CoA synthetase from Salmonella enterica in pBAD33. The other strain KSBA100 (pWL1T, pBHE3) is similar to the KSBA100 (pWL1T, pBHE2) but carrying a -ketoacyl-ACP synthase III from Bacillus subtilis.

(45) The experiments were as described above, except a pathway is added to synthesize beta-alanine in vivo from glucose instead of externally added beta-alanine, and thus no beta alanine need be added to the culture medium.

Omega-Unsaturated Fatty Acids with Acrylic Acid

(46) Externally added acrylic acid is used as the priming molecule in this experiment to produce omega unsaturated fatty acids. Three engineered strains will be studied. The strain ML103 (pWL1T, pBAD33) harbors a plasmid pWL1T carrying an acyl-ACP thioesterase from Ricinus communis under a constitutive promoter and a cloning vector pBAD33 as the control. The second strain ML103 (pWL1T, pBHE2) also harbors the same plasmid pWL1T carrying an acyl-ACP thioesterase from Ricinus communis and a plasmid pBHE2 carrying a -ketoacyl-ACP synthase III from Staphylococcus aureus and a propionyl-CoA synthetase from Salmonella enterica in pBAD33. The other strain ML103 (pWL1T, pBHE3) is similar to the ML 103 (pWL1T, pBHE2) but carrying a -ketoacyl-ACP synthase III from bacillus subtilis.

(47) The experiments were as described above, except the priming precursor, acrylic acid, has an unsaturated bond, and is added to the medium instead of glycolate.

Omega-Unsaturated Fats with Propenoyl-CoA In Vivo

(48) Beta-alanine produced in vivo is used as the priming molecule in this experiment to produce omega unsaturated fatty acids. Beta alanine overproducing strains, named KSBA100, with overexpression pyruvate carboxylase (Pyc), aspartate aminotransferase (AAT) and aspartate decarboxylase (PAND) from various sources constructed above will be used. In addition, beta-alanyl-CoA ammonia-lyase (PAL) will be overexpressed to convert beta-alanyl-CoA to propenoyl-CoA:

(49) ##STR00018##

(50) This strain is named KSPC100.

(51) Three engineered strains will be studied. The strain KSPC100 (pWL1T, pBAD33) harbors the genes encoded for Pyc, AAT, PAND, TE from Ricinus communis and a cloning vector pBAD33 as the control. The second strain KSPC100 (pWL1T, pBHE2) also harbors the same set of genes encoded for Pyc, AAT, PAND, TE from Ricinus communis but a plasmid pBHE2 carrying a -ketoacyl-ACP synthase III from Staphylococcus aureus and a propionyl-CoA synthetase from Salmonella enterica in pBAD33. The other strain KSPC100 (pWL1T, pBHE3) is similar to the KSPC100 (pWL1T, pBHE2) but carrying a -ketoacyl-ACP synthase III from bacillus subtilis.

(52) The experiments were as described above, except a pathway is added to synthesize acrylic acid in vivo from glucose, instead of externally added acrylic acid

Alpha, Omega Dicarboxylic Acid

(53) Engineered strains with the ability to produce omega-hydroxy fatty acids are further engineered to overexpress alcohol dehydrogenase (AlkJ) and aldehyde dehydrogenase (AlkH) for the conversion of hydroxyl fatty acids to dicarboxylic acids. Hence, this strain has the relevant genotype of fadD, aceB, icd, Rc_TE.sup.+, At GLYR.sup.+, Sa fabH.sup.+, Se prpE.sup.+, alkJ.sup.+ and alkH.sup.+.

(54) The experiments were as described above, except an additional pathway is added to convert the hydroxyl end to carboxylic acid, or to convert the hydroxyl to aldehyde if just the AlkJ is added. Although these experiments are not yet complete, this conversion has already been shown to work in other engineered strains.

Alpha, Omega Dicarboxylic Acids by Co-Culture

(55) Engineered strains with the ability to produce omega-hydroxy fatty acids are co-cultured with strain ML103(pAlkJH). The plasmid pALkJH carries the genes encoded for alcohol dehydrogenase (alkJ) and aldehyde dehydrogenase (alkH) for the conversion of hydroxy fatty acids to dicarboxylic acids. The use of alkJ alone will produce aldehydes, while the use of both will produce the dicarboxylates.

(56) The seed culture is prepared by inoculating a single colony of these two strains (ML103 (pWLAA, pBHE3) and ML103(pAlkJH)) from a freshly grown plate in 5 mL of LB medium in an orbital shaker (New Brunswick Scientific, New Jersey, USA) is operated overnight at 250 RPM and 37 C. The secondary preculture is prepared by inoculating 0.5 mL seed culture to a 250-mL flask containing 50 mL LB medium and incubating at 30 C. and 250 RPM for 9 h.

(57) The two cultures are harvested aseptically by centrifugation at 3,300g for 5 min, and the cells are resuspended in the appropriate volume of fresh fermentation medium, which is calculated based on the inoculation size of 10%. LB medium is supplemented with 15 g/L of glucose. Optionally, glycolate (2.5 g/L or 5 g/L) will be added depending on the strains being used. The initial pH is set to be 7.5.

(58) All of the media are supplemented with 100 mg/L ampicillin and 34 mg/L chloramphenicol. The expression of the prpE and fabH on plasmid pBHE2 and pBHE3 is induced by 10 mM arabinose.

(59) The cells are then cultivated in an orbital shake is operated at 250 RPM and 30 C. Samples are taken at 24, 48 and 72 h for dicarboxlic acids, fatty acids and extracellular metabolite analysis. All experiments are carried out in triplicate.

(60) Although these experiments are not yet complete, they are expected to work since we have demonstrated it before by using externally added octanoic acid.

(61) The above experiments are repeated in Bacillus subtilis. The same genes can be used, especially since Bacillus has no significant codon bias. A protease-deficient strain like WB800N is preferably used for greater stability of heterologous protein. The E. coli-B. subtilis shuttle vector pMTLBS72 exhibiting full structural stability can be used to move the genes easily to a more suitable vector for Bacillus. Alternatively, two vectors pHT01 and pHT43 allow high-level expression of recombinant proteins within the cytoplasm. As yet another alternative, plasmids using the theta-mode of replication such as those derived from the natural plasmids pAM3 and pBS72 can be used. Several other suitable expression systems are available. Since the FAS enzymes are ubiquitous, the invention is predicted to function in Bacillus.

(62) The above experiments are repeated in yeast. The same genes can be used, but it may be preferred to accommodate codon bias. Several yeast E. coli shuttle vectors are available for ease of the experiments. Since the FAS enzymes are ubiquitous, the invention is predicted to function in yeast, especially since yeast are already available with exogenous functional TE genes and various modified FAS pathways have also been made to run in yeast.

(63) The following references are incorporated by reference in their entirety for all purposes. Choi, K. H., R. J. Heath, and C. O. Rock. 2000. -Ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J. Bacteriol. 182:365-370. He, X., and K. A. Reynolds. 2002. Purification, characterization, and identification of novel inhibitors of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus. Antimicrob. Agents Chemother. 46:1310-1318. WO2000075343 Engineering B-Ketoacyl ACP Synthase For Novel Substrate Specificity US20160090576 Materials and methods for characterizing and using kasiii for production of bi-functional fatty acids. Qiu, X, et al., Crystal structure and substrate specificity of the b-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus, Protein Science (2005), 14:2087-2094 (2005).