Production of fatty acids esters

Description

BACKGROUND OF THE INVENTION
Discussion of the Background

Fatty acid esters may be used for several purposes commercially. For example, biodiesel, an alternative fuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odour which makes them useful as fragrances or flavouring agents. Fatty acid esters may also be used as solvents for lacquers, paints, varnishes and the like. Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. Further, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals. Accordingly, fatty acid esters are very useful in this day and age.

Fatty acid esters may be extracted from petroleum. However, this method is energy consuming and costly. Also, it is an inefficient process because frequently the long chain hydrocarbons in crude petroleum are cracked to produce smaller monomers. These monomers are then used as the raw material to manufacture the more complex specialty chemicals. This process of cracking gasoline or petroleum is bad for the environment. Also, since the costs for these starting materials will be linked to the price of petroleum, with the expected increase in petroleum prices in the future, cost of making these fatty acid esters may also increase relative to the increase in the petroleum prices.

Due to the inherent challenges posed by petroleum, there is a need for a renewable petroleum source which does not need to be explored, extracted, transported over long distances, or substantially refined like petroleum. There is also a need for a renewable petroleum source that can be produced economically and that does not cause the environmental damage as that produced by the petroleum industry and the burning of petroleum based fuels.

Fatty acid esters may be found in several biofuels. However, the yield of the fatty acid esters from these biofuels and/or plant based fuels is low. Thus, a need exists to develop an alternate biological source of fatty acid esters. One option is to recombinantly engineer a microbial species for efficient production of fatty acid esters.

Fatty acid esters are known to be the product of a condensation reaction between an acyl-CoA molecule and an alcohol of any chain length sometimes in the presence of wax ester synthases. For example, a fatty acid ester can be the condensation product of methanol, ethanol, propanol, butanol, isobutanol, 2-methylbutanol, 3-methylbutanol, or pentanol with an acyl-CoA molecule. In some instances, fatty acid esters such as fatty acid methyl esters (“FAME”) or fatty acid ethyl esters (“FAEE”) can be produced by supplying the alcohol used in the reaction (e.g., methanol or ethanol) to the culture media. Similarly, wax esters can be produced by supplying fatty alcohols.

Most fatty acid esters have useful functions as mentioned above. One of these esters, methyl laurate, CH₃(CH₂)₁₀COOCH₃a water-insoluble, clear, colourless ester, has several uses in the commercial industry including the pharmaceutical and cosmetic industry.

However, the current methods used to make fatty acid esters are inefficient as they produce a large amount of by-products that result in a waste of resources. Also, the currently available methods do not allow for selecting specific fatty acid esters. There is thus a need for more energy efficient and specific production of fatty acid esters including methyl laurate.

SUMMARY OF THE INVENTION

The present invention attempts to solve the problems above by providing at least one method of producing fatty acid esters from genetically engineered microorganisms. In particular, the fatty acid esters are produced by culturing a microorganism that is genetically engineered to produce a fatty acid and express at least one wax ester synthase, in the presence of exogenous alcohol, such as exogenous ethanol, exogenous methanol or the like.

In one embodiment, the present invention relates to a microbial cell for producing at least one fatty acid ester, wherein the cell is genetically modified to comprise

- (i) at least one first genetic mutation that enables the cell to produce at least one fatty acid and/or acyl coenzyme A (CoA) thereof by increased enzymatic activity in the cell relative to the wild type cell of malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway, wherein the fatty acid comprises at least 5 carbon atoms; and
- (ii) a second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell and the wax ester synthase has sequence identity of at least 50% to a polypeptide of SEQ ID NO: 1-8 and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the fatty acid ester.

In another embodiment, the present invention relates to a method for producing methyl laurate, the method comprising:

contacting lauric acid and/or lauroyl coenzyme A with an isolated wax ester synthase that has sequence identity of at least 50% to a polypeptide of SEQ ID NOs: 1-8 and combinations thereof.

The present invention also related to the above method, which is carried out within a microbial cell which is genetically modified to comprise

- (i) at least one first genetic mutation that enables the cell to produce at least one fatty acid and/or acyl coenzyme A (CoA) thereof by increased enzymatic activity in the cell relative to the wild type cell of malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway, wherein the fatty acid comprises at least 5 carbon atoms; and
- (ii) a second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell and the wax ester synthase has sequence identity of at least 50% to a polypeptide of SEQ ID NO: 1-8 and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the fatty acid ester.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing free fatty acid (FFA) production in shake flask cultures after 68 hours for four FFA production strains.

FIG. 2 is a graph showing the lauric acid production profile for BXF_169 evaluated in 50 ml shake flask, OD₆₀₀=2.

FIG. 3 is a graph showing the mRNA expression relative to cysG for various pathway genes in BXF_169 measured at 8, 24 and 48 hrs.

FIG. 4 is a graph showing the enzyme activity for various elongation steps measured in lysate for BXF_169 at 8 and 24 hours. EnCR activity is encoded by the Ter gene. KAS activities are encoded by the NphT7 and FabH genes.

FIG. 5 is a graph showing intermediate product formation and utilization specific to the C4→C6 elongation steps of the lauroyl-CoA production pathway (data collected but not shown for the C6→C12 intermediates) from in vitro pathway reconstitution with FadB, FadB+Crt and FadB+Crt+Hbd.

FIG. 6 is a graph showing product formation measured for 20 soluble WES candidates. Purified candidate enzymes were incubated individually with methanol and C6- to C14-CoA substrates to assess product formation.

FIG. 7 is a graph showing FAME production at 24 hours for nine thiolase-based strains expressing different wax ester synthase candidates evaluated with the 2 mL small-scale protocol.

FIG. 8 is a graph showing the production of methyl laurate and other FAMEs (C6-C14) in 1 ml, 24 hour strain screening assay by 50 strains engineered with various versions of the synthase pathway for methyl laurate production.

FIG. 9 is a graph showing FAME product distribution exhibited by strain BXE_022 after 40 hrs in 1 L fermentation under various test conditions.

FIG. 10 is a graph showing FAME and FFA concentration distribution for strains BXE_058 and BXE_062 at approximately 45 hours of production; methyl myristate concentration was not include in the analysis as 50 g/L was added as a second phase.

FIG. 11 is an illustration of the metabolic pathways of a cell related to genetic modifications for increasing flux through the intermediate malonyl-CoA

FIG. 12 is a graph showing the evaluation of intermediate product accumulation specific to the C4→C6 elongation steps of the lauroyl-CoA production pathway (data collected but not shown for the C6→C12 intermediates.)

FIG. 13 is a graph showing specific enzyme activities for three FabH mutant enzymes with C6-C10-CoA substrates compared to wild-type FabH.

FIG. 14 is a graph showing FAME production in standard 1 mL method after 20 hours of production. (Note: BXE_198 is the control comparison for BXE_271-273 and BXE_233 is the control for BXE_243-244).

FIG. 15 is a graph showing FAME production in standard 1 mL method after 20 hours of production with alternative WES enzyme candidates.

FIG. 16 is a graph showing in vitro product formation from methanol and C12-CoA after incubation for 5 minutes with purified Mhyd variants as measured by GC-MS.

FIG. 17 is a graph showing FAME production in standard 1 mL method after 20 hours of production with Maqu mutants (SEQ ID NOs:7 and 8).

FIG. 18 is a graph showing WES enzyme activities for BXE_062 and BXE_233 cell lysates as measured by product formation over time by GC-MS. The target activity for WES in cell lysates is 0.02 U/mg.

FIG. 19 is a graph showing average production profile for methyl laurate and total FAME for 1 L fermentations with BXE_276 in the FM14 process.

DETAILED DESCRIPTION OF THE INVENTION

The ranges described below include all values and subvalues between the lower and upper limit of the range.

According to one aspect, the present invention provides a microbial cell for producing at least one fatty acid ester, wherein the cell is genetically modified to comprise:

- at least one first genetic mutation that enables the cell to produce at least one fatty acid and/or acyl coenzyme A thereof, wherein the fatty acid comprises at least 5 carbon atoms; and
- at least one second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell and the wax ester synthase has sequence identity of at least 50% to a polypeptide of SEQ ID NOs: 1-8 and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the fatty acid ester.

In particular, the cell may be capable of producing the fatty acid and/or acyl coenzyme A thereof by means of increased enzymatic activity in the cell relative to the wild type cell of the malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway.

The microbial cells according to any aspect of the present invention may be prokaryotes or eukaryotes. These can be mammalian cells (such as, for example, cells from man), plant cells or microorganisms such as yeasts, fungi or bacteria, wherein microorganisms in particular bacteria and yeasts may be used.

Suitable bacteria, yeasts or fungi are in particular those bacteria, yeasts or fungi that are deposited in the Deutsche Sammlung von Mikroorganismen and Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH (DSMZ), Brunswick, Germany, as bacterial, yeast or fungal strains. Bacteria suitable according to the invention belong to the genera that are listed in the Deutsche Sammlung von Mikroorganismen and Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH(DSMZ) Germany.

Yeasts suitable according to the invention belong to the genera that are listed in the Deutsche Sammlung von Mikroorganismen and Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH (DSMZ), Brunswick, Germany.

Fungi suitable according to the invention belong to the genera that are listed in the Deutsche Sammlung von Mikroorganismen and Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH (DSMZ), Brunswick, Germany.

In particular, the cells may be selected from the genera Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium and Cupriavidus. More in particular, the cells may be selected from the group consisting of Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Burkholderia andropogonis, B. brasilensis, B. caledonica, B. caribensis, B. caryophylli, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururiensis, B. phenazinium, B. phymatum, B. phytofirmans, B. plantarii, B. sacchari, B. singaporensis, B. sordidicola, B. terricola, B. tropica, B. tuberum, B. ubonensis, B. unamae, B. xenovorans, B. anthina, B. pyrrocinia, B. thailandensis, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Escherichia coli, Hansenula polymorpha, Kluveromyces lactis, Methylobacterium extorquens, Paracoccus versutus, Pseudomonas argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. fragi, P. lundensis, P. taetrolens, P. antarctica, P. azotoformans, ‘P. blatchfordae’, P. brassicacearum, P. brenneri, P. cedrina, P. corrugata, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. cremoricolorata, P. fulva, P. monteilii, P. mosselii, P. parafulva, P. putida, P. balearica, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, ‘P. helianthi’, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. psychrophila, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. thermotolerans, P. aeruginosa, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, Ralstonia eutropha, Rhodospirillum rubrum, Rhodobacter sphaeroides, Saccharomyces cerevisiae, Yarrowia lipolytica and Zymomonas mobile. More in particular, the cell may be a bacterial cell selected from the group consisting of Pseudomonas, Corynebacterium, Bacillus and Escherichia. Even more in particular, the cells may be selected from the group consisting of Pseudomonas putida and Escherichia coli.

The genetically modified cell may be genetically different from the wild type cell. The genetic difference between the genetically modified cell according to any aspect of the present invention and the wild type cell may be in the presence of a complete gene, amino acid, nucleotide etc. in the genetically modified cell that may be absent in the wild type cell. In one example, the genetically modified cell according to any aspect of the present invention may comprise enzymes that enable the cell to produce at least one fatty acid and/or acyl coenzyme A thereof; and convert the fatty acid and/or acyl coenzyme A thereof to the fatty acid ester. The wild type cell relative to the genetically modified cell of the present invention may have none or no detectable activity of the enzymes that enable the genetically modified cell to produce at least one fatty acid and/or acyl coenzyme A thereof; and the enzymes that enable genetically modified cell to convert the fatty acid and/or acyl coenzyme A thereof to the respective fatty acid ester.

The phrase “wild type” as used herein in conjunction with a cell may denote a cell with a genome make-up that is in a form as seen naturally in the wild. The term may be applicable for both the whole cell and for individual genes. The term “wild type” therefore does not include such cells or such genes where the gene sequences have been altered at least partially by man using recombinant methods.

A skilled person would be able to use any method known in the art to genetically modify a cell. According to any aspect of the present invention, the genetically modified cell may be genetically modified so that in a defined time interval, within 2 hours, in particular within 8 hours or 24 hours, it forms at least twice, especially at least 10 times, at least 100 times, at least 1000 times or at least 10000 times more fatty acid and/or acyl coenzyme A thereof and the respective fatty acid ester than the wild-type cell. The increase in product formation can be determined for example by cultivating the cell according to any aspect of the present invention and the wild-type cell each separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specified time interval in a suitable nutrient medium and then determining the amount of target product (fatty acid, acyl coenzyme A thereof and the respective fatty acid ester) in the nutrient medium.

In particular, the cell comprises at least one first genetic mutation that enables the cell to produce at least one fatty acid and/or acyl coenzyme A thereof. In particular, the first genetic mutation may enable the cell to produce at least one fatty acid and/or acyl coenzyme A thereof by means of a malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway. More in particular, there is an increase in enzymatic activity in the malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway in the cell relative to the wild type cell.

The cell may be genetically modified for increased enzymatic activity in the microorganism's malonyl-CoA dependent, malonyl-ACP independent, fatty acyl-CoA metabolic pathway (“MDMIFAA”) This pathway is also referred to herein as malonyl-CoA dependent, but malonyl-ACP independent, fatty acyl-CoA metabolic pathway. Such increase in the cell's malonyl-CoA dependent, malonyl-ACP independent fatty acyl-CoA metabolic pathway can be achieved by an increased activity or expression of a gene or a pathway comprising an acetoacetyl-CoA synthase, a ketoacyl-CoA synthase (or elongase), an enoyl-CoA reductase, a ketoacyl-CoA reductase and/or a 3-hydroxyacyl-CoA dehydratase in combination with a decrease in expression or activity of acetoacetyl-CoA thiolase. Alternatively, increased activity in the microorganism's malonyl-CoA dependent, malonyl-ACP independent fatty acyl-CoA metabolic pathway can be achieved by an increased expression of a gene or a pathway comprising an acetoacetyl-CoA synthase, a ketoacyl-CoA thiolase, a enoyl-CoA reductase, a ketoacyl-CoA reductase and/or a 3-hydroxyacyl-CoA dehydratase in combination with a decrease in expression or activity of acetoacetyl-CoA thiolase.

A list of non-limiting genetic modifications to enzymes or enzymatic activities that may lead a cell to produce a fatty acid and/or acyl coenzyme A thereof and that may be considered as the first genetic mutation according to any aspect of the present invention are provided below in Table 1 and explained in US20140051136.

In particular, fatty acid biosynthetic pathways in the cells of the present invention use precursors acetyl-CoA and malonyl-CoA. The enzymes that may be involved are provided in FIG. 11.

In one example, nucleic acid sequences that encode temperature-sensitive forms of these polypeptides may be introduced in place of the native enzymes, and when such genetically modified microorganisms are cultured at elevated temperatures (at which these thermolabile polypeptides become inactivated, partially or completely, due to alterations in protein structure or complete denaturation), there is observed an increase in a chemical product. For example, in E. coli, these temperature-sensitive mutant genes could include FabIts(S241F), FabBts(A329V) or FabDts(W257Q) amongst others. In most of these examples, the genetic modifications may increase malonyl-CoA utilization so that there is a reduced conversion of malonyl-CoA to fatty acids via the native pathway, overall biomass, and proportionally greater conversion of carbon source to a chemical product including a fatty acid or fatty acid derived product via a malonyl-CoA dependent and malonyl-ACP independent route. Also, additional genetic modifications, such as to increase malonyl-CoA production, may be made for some examples.

In another example, the enzyme, enoyl-acyl carrier protein reductase (EC No. 1.3.1.9, also referred to as enoyl-ACP reductase) is a key enzyme for fatty acid biosynthesis from malonyl-CoA. In Escherichia coli this enzyme, FabI, is encoded by the gene FabI (Richard J. Heath et al., 1995).

In one example, the expression levels of a pyruvate oxidase gene (Chang et al., 1983, Abdel-Ahmid et al., 2001) can be reduced or functionally deleted in the cell according to any aspect of the present invention. The pyruvate oxidase gene may encode an enzyme of, for example, EC 1.2.3.3. In particular, the pyruvate oxidase gene may be a PoxB gene.

In one example, the expression levels of a lactate dehydrogenase gene (Mat-Jan et al., Bunch et al., 1997) can be reduced or functionally deleted. In some examples, the lactate dehydrogenase gene encodes an enzyme of, for example, EC 1.1.1.27. The lactate dehydrogenase gene may be an NAD-linked fermentative D-lactate dehydrogenase gene. In particular, the lactate dehydrogenase gene is an IdhA gene.

In one example, the first genetic mutation may be in at least one feedback resistant enzyme of the cell that results in increased expression of the feedback resistant enzyme. In particular, the enzyme may be pantothenate kinase, pyruvate dehydrogenase or the like. In E. coli, these feedback resistant mutant genes could include CoaA(R106A) and/or lpd(E354K).

In a further example, the increase in the cell's malonyl-CoA dependent, but malonyl-ACP independent fatty acyl-CoA metabolic pathway may occur through reduction in the acetoacetyl-CoA thiolase activity and/or trigger factor activity and/or in the activity of a molecular chaperone involved in cell division. In one example, the cell may comprise a genetic mutation in Ttig gene.

In one example, the first genetic mutation in the cell may result in increased enzymatic activity in the NADPH-dependent transhydrogenase pathway relative to the wild type cell. This result may occur by introduction of a heterologous nucleic acid sequence coding for a polypeptide encoding nucleotide transhydrogenase activity.

In another example, the first genetic mutation in the cell may result in decreased expression of fatty acyl-CoA synthetase and/or ligase activity via any method known in the art.

In yet another example, the first genetic mutation in the cell may result in overexpression of an enzyme having acetyl-CoA carboxylase activity.

In one example, the cell may have increased intracellular bicarbonate levels brought about by introduction of a heterologous nucleic acid sequence coding for a polypeptide having cyanase and/or carbonic anhydrase activity.

More in particular, the first genetic mutation according to any aspect of the cell may result in increased and/or decreased levels of fatty acyl-CoA thioesterase activity. This result may increase chain length specificity of a desired fatty acid product by increasing levels of chain length specific fatty acyl-CoA thioesterase activity and decreasing the activity of fatty acyl-CoA thioesterase activity on undesired fatty acid chain lengths. In one example, the increased chain length specificity of fatty acid or fatty acid derived product may occur by increasing levels of chain length specific ketoacyl-CoA thiolase, enoyl-CoA reductase, ketoacyl-CoA reductase or 3-hydroxyacyl-CoA dehydratase activities either individually or in combination.

The first genetic mutation in the cell according to any aspect of the present invention may result in an increase or decrease in expression of only one enzyme selected from the list of enzymes mentioned above or an increase or decrease in expression of a combination of enzymes mentioned above.

In another example, the first genetic mutation in the cell may be in at least one enzyme selected from the group consisting of acetoacetyl-CoA synthase, ketoacyl-CoA synthase (or elongase), enoyl-CoA reductase, ketoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and acetoacetyl-CoA thiolase. More in particular, the first genetic mutation in the cell may result in an increase in expression of acetoacetyl-CoA synthase, ketoacyl-CoA synthase (or elongase), enoyl-CoA reductase, ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydratase in combination optionally with a decrease in expression or activity of acetoacetyl-CoA thiolase. In particular, the enoyl-CoA reductase and/or ketoacyl-CoA reductase may either utilize the cofactor NADH and/or NADPH. In particular, the genetic modification in the cell according to any aspect of the present invention may comprise any of the enzymes listed in Table 1 in combination with the following enzymes acetoacetyl-CoA synthase, ketoacyl-CoA synthase (or elongase), enoyl-CoA reductase, ketoacyl-CoA reductase and/or 3-hydroxyacyl-CoA dehydratase and acetoacetyl-CoA thiolase wherein the expression or activity of enzymes acetoacetyl-CoA synthase, ketoacyl-CoA synthase (or elongase), enoyl-CoA reductase, ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydratase is increased and the activity of acetoacetyl-CoA thiolase is decreased.

In yet another example, malonyl-CoA dependent, malonyl-ACP independent fatty acyl-CoA metabolic pathway in the cell according to any aspect of the present invention can be achieved by an increased expression of a gene or a pathway comprising acetoacetyl-CoA synthase, ketoacyl-CoA thiolase, enoyl-CoA reductase, ketoacyl-CoA reductase and/or 3-hydroxyacyl-CoA dehydratase in combination with a decrease in expression or activity of acetoacetyl-CoA thiolase.

In particular, the first genetic modification in the cell according to any aspect of the present invention may comprise any of the enzymes listed in Table 1 in combination with the following enzymes acetoacetyl-CoA synthase, ketoacyl-CoA thiolase, enoyl-CoA reductase, ketoacyl-CoA reductase and/or 3-hydroxyacyl-CoA dehydratase in combination with a decrease in expression or activity of acetoacetyl-CoA thiolase.

In one example, the cell according to any aspect of the present invention may comprise a first genetic modification in any of the enzymes listed in Table 1 in combination with the following enzymes acetyl-CoA carboxylase, malonyl-CoA:ACP transacylase (FabD), β-ketoacyl-ACP synthase III, β-ketoacyl-ACP synthase I (FabB), β-ketoacyl-ACP synthase II (FabF), 3-oxoacyl-ACP-synthase I and enoyl ACP reductase.

More in particular, the first genetic mutation may result in an increase in the expression of at least one enzyme selected from the group consisting of acetyl-CoA carboxylase, malonyl-CoA:ACP transacylase (FabD), β-ketoacyl-ACP synthase III, β-ketoacyl-ACP synthase I (FabB), β-ketoacyl-ACP synthase II (FabF), 3-oxoacyl-ACP-synthase I and enoyl ACP reductase relative to the wild type cell. In particular, the first genetic mutation may result in an increase in the expression of more than one enzyme in the cell according to any aspect of the present invention that enables the cell to produce a fatty acid and/or acyl coenzyme A thereof by means of increased enzymatic activity in the cell relative to the wild type cell of malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway.

In one example, there may be an increase in expression of β-ketoacyl-ACP synthase and 3-oxoacyl-ACP-synthase in the cell according to any aspect of the present invention. In another example, there may be an increase in expression of β-ketoacyl-ACP synthase and Malonyl-CoA-ACP transacylase in the cell according to any aspect of the present invention. In yet another example, there may be an increase in expression of β-ketoacyl-ACP synthase and enoyl ACP reductase in the cell according to any aspect of the present invention. In one example, there may be an increase in expression of β-ketoacyl-ACP synthase, Malonyl-CoA-ACP transacylase and enoyl ACP reductase in the cell according to any aspect of the present invention. In all examples, there may be an increase in the expression of enoyl ACP reductase and/or acyl-CoA thioesterase.

The phrase “increased activity of an enzyme”, as used herein is to be understood as increased intracellular activity. Basically, an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences that code for the enzyme, using a strong promoter or employing a gene or allele that code for a corresponding enzyme with increased activity and optionally by combining these measures. Genetically modified cells used according to any aspect of the present invention are for example produced by transformation, transduction, conjugation or a combination of these methods with a vector that contains the desired gene, an allele of this gene or parts thereof and a vector that makes expression of the gene possible. Heterologous expression is in particular achieved by integration of the gene or of the alleles in the chromosome of the cell or an extra-chromosomally replicating vector.

Accordingly, the cells and methods of the present invention may comprise providing a genetically modified microorganism that comprises both a production pathway to a fatty acid or fatty acid derived product, and a modified polynucleotide that encodes an enzyme of the malonyl-ACP dependent fatty acid synthase system that exhibits reduced activity, so that utilization of malonyl-CoA shifts toward the production pathway compared with a comparable (control) microorganism lacking such modifications. The methods involve producing the chemical product using a population of such genetically modified microorganism in a vessel, provided with a nutrient media. Other genetic modifications described herein, to other enzymes, such as acetyl-CoA carboxylase and/or NADPH-dependent transhydrogenase, may be present in some such examples. Providing additional copies of polynucleotides that encode polypeptides exhibiting these enzymatic activities is shown to increase a fatty acid or fatty acid derived product production. Other ways to increase these respective enzymatic activities is known in the art and may be applied to various examples of the present invention.

TABLE 1

Examples of genetic modifications in cells of microorganisms for production of

fatty acids and/or acyl coenzyme A thereof

Genetic Modifications

E.C. CLASSIFICATION
GENE NAME

ENZYME FUNCTION
No.
IN E. COLI
COMMENTS

Glucose transporter
N/A
galP
Increase function

Pyruvate dehydrogenase E1p
1.2.4.1
aceE
Increase function

lipoate acetyltransferase/
2.3.1.12
aceF
Increase function

dihydrolipoamide acetyltransferase

Pyruvate dehydrogenase
1.8.1.4
lpd
Increase function or

E3 (lipoamide dehydrogenase)

alter such as by

mutation to increase

resistance to NADH

inhibition.

Lactate dehydrogenase
1.1.1.28
ldhA
Decrease function,

including by mutation

Pyruvate formate lyase
2.3.1.—
pflB
Decrease function,

(B “inactive”)

including by mutation

Pyruvate oxidase
1.2.2.2
poxB
Decrease function,

including by mutation

Phosphate acetyltransferase
2.3.1.8
Pta
Decrease function,

including by mutation

acetate kinase
2.7.2.15 2.7.2.1
ackA
Decrease function,

including by mutation

methylglyoxal synthase
4.2.3.3
mgsA
Decrease function,

including by mutation

Heat stable, histidyl
N/A
ptsH
Decrease function,

phosphorylatable protein (of PTS)

(HPr)
including by mutation

Phosphoryl transfer protein
N/A
ptsI
Decrease function,

(of PTS)

including by mutation

Polypeptide chain (of PTS)
N/A
Crr
Decrease function,

including by mutation

3-oxoacyl-ACP synthase I
2.3.1.179
fabF
Decrease function,

3-oxoacyl-ACP synthase II
2.3.1.41

including by mutation

monomer

β-ketoacyl-ACP synthase I,
2.3.1.41
fabB
Decrease function,

3-oxoacyl-ACP-synthase I
2.3.1.—

including by mutation

Malonyl-CoA-ACP
2.3.1.39
fabD
Decrease function,

transacylase

including by mutation

enoyl acyl carrier protein
1.3.1.9.
fabI
Decrease function,

reductase
1.3.1.10

including by mutation

β-ketoacyl-acyl carrier
2.3.1.180
fabH
Decrease function,

protein synthase III

including by mutation

Carboxyl transferase
6.4.1.2
accA
Increase function

subunit α subunit

Biotin carboxyl carrier
6.4.1.2
accB
Increase function

protein

Biotin carboxylase subunit
6.3.4.14
accC
Increase function

Carboxyl transferase
6.4.1.2
accD
Increase function

subunit β subunit

long chain fatty acyl
3.1.2.2.
tesA
Increase function as

thioesterase I
3.1.1.5

well as alter by

mutation to express in

cytoplasm or deletion

acyl-CoA synthase
2.3.1.86
fadD
Decrease via deletion

or mutation

acetate CoA-transferase
2.8.3.8
atoD
Decrease via deletion

or mutation

acetate CoA-transferase
2.8.3.8
atoA
Decrease via deletion

or mutation

Transporter
N/A
atoB
Decrease via deletion

or mutation

acetyl-CoA acetyltransferase
2.3.1.9
atoB
Decrease via deletion

or mutation

pantothenate kinase
2.7.1.33
coaA
Increase via expression

or feedback resisant

mutation

lactose repressor
N/A
lacI
Decrease via deletion

or mutation

γ-glutamyl-γ-
1.2.1.—
puuC
Decrease via deletion

aminobutymidehyrte

or mutation

dehydrogenase

malate synthase A
2.3.3.9
aceB
Decrease via deletion

or mutation

isocitrate lyase
4.1.3.1
aceA
Decrease via deletion

or mutation

isocitrate dehydrogenase
3.1.3.—
aceK
Decrease via deletion

phosphatase/isocitrate
2.7.11.5.

or mutation

dehydrogenase kinase

pyruvate formate-lyase
1.2.1.10 1.1.1.1
adhE
Decrease via deletion

deactivate

or mutation

aldehyde dehydrogenase A,
1.2.1.21 1.2.1.22
aldA
Decrease via deletion

NAD-linked

or mutation

acetaldehyde
1.2.1.4
aldB
Decrease via deletion

dehydrogenase

or mutation

Lambda phage DE3 lysogen
N/A
λDE3
Increase

T7 mRNA polymerase
N/A
T7pol
Increase

trigger factor
5.2.1.8
tig
Decrease via deletion

or mutation

3-ketoacyl-CoA thiolase
2.3.1.16
fadA
Increase

dodecenoyl-CoA δ-isomerase,
5.3.3.8 1.1.1.35
fadB
Increase

enoyl-CoA hydratase, 3-
5.1.2.3 4.2.1.17

hydroxybutyryl-CoA epimerase,

3-hydroxyacyl-CoA dehydrogenase

Sucrose permease
N/A
cscB
Increase

Invertase
3.2.1.26
cscA
Increase

fructokinase
2.7.1.4
cscK
Increase

carbonic anhydrase
4.2.1.1
cynT
Increase

carbonic anhydrase
4.2.1.1
can
Increase

pyridine nucleotide
1.6.1.2
pntAB
Increase

transhydrogenase

pyridine nucleotide
1.6.1.1
udhA
Increase

transhydrogenase

acyl-CoA thioesterase
3.1.2.20 3.1.2.2
yciA
Increase and or

decrease

thioesterase II
3.1.2.20 3.1.2.2
tesB
Increase and or

decrease

thioesterase III
3.1.2.—
fadM
Increase and or

decrease

hydroxyphenylacetyl-CoA
N/A
paaI
Increase and or

thioesterase

decrease

esterase/thioesterase
3.1.2.28
ybgC
Increase and or

decrease

proofreading thioesterase in

entH
Increase and or

enterobactin biosynthesis

decrease

acetoacetyl-CoA synthase
2.3.1.194
npth07
Increase

3-ketoacyl-CoA synthase/elongase
2.3.1
Elo1
Increase

3-ketoacyl-CoA synthase/elongase
2.3.1
Elo2
Increase

3-Hydroxybutyryl-CoA dehydrogenase
1.1.1.157
hbd
Increase

3-oxoacyl-CoA reductase
1.1.1.100
fabG
Increase

enoyl-CoA hydratase
4.2.1.17
crt
Increase

enoyl-CoA hydratase
4.2.1.17
ech2
Increase

Trans-2-enoyl-reductase
1.3.1.9
ter
Increase

thioesterase
3.1.2.20
paaI
Decrease

E.C. No = “Enzyme Commission number”

Also, without being limiting, a first step in some multi-phase methods of making a fatty acid may be exemplified by providing into a vessel, such as a culture or bioreactor vessel, a nutrient media, such as a minimal media as known to those skilled in the art, and an inoculum of a genetically modified microorganism so as to provide a population of such microorganism, such as a bacterium, and more particularly a member of the family Enterobacteriaceae, such as E. coli, where the genetically modified microorganism comprises a metabolic pathway that converts malonyl-CoA to a fatty acid. This inoculum is cultured in the vessel so that the cell density increases to a cell density suitable for reaching a production level of a fatty acid or fatty acid derived product that meets overall productivity metrics taking into consideration the next step of the method. In various alternative embodiments, a population of these genetically modified microorganisms may be cultured to a first cell density in a first, preparatory vessel, and then transferred to the noted vessel so as to provide the selected cell density. Numerous multi-vessel culturing strategies are known to those skilled in the art. Any such examples provide the selected cell density according to the first noted step of the method.

Also without being limiting, a subsequent step may be exemplified by two approaches, which also may be practiced in combination in various examples. A first approach provides a genetic modification to the genetically modified microorganism such that its enoyl-ACP reductase enzymatic activity may be controlled. As one example, a genetic modification may be made to substitute a temperature-sensitive mutant enoyl-ACP reductase (e.g., fabI^TSin E. coli) for the native enoyl-ACP reductase. The former may exhibit reduced enzymatic activity at temperatures above 30° C. but normal enzymatic activity at 30° C., so that elevating the culture temperature to, for example to 34° C., 35° C., 36° C., 37° C. or even 42° C., reduces enzymatic activity of enoyl-ACP reductase. In such case, more malonyl-CoA is converted to a fatty acid than at 30° C., where conversion of malonyl-CoA to fatty acids is not impeded by a less effective enoyl-ACP reductase.

Other genetic modifications that may be useful in the production of fatty acids may be included in the cell. For example, the ability to utilize sucrose may be provided, and this would expand the range of feed stocks that can be utilized to produce a fatty acid or fatty acid derived product or other chemical products. Common laboratory and industrial strains of E. coli, such as the strains described herein, are not capable of utilizing sucrose as the sole carbon source. Since sucrose, and sucrose-containing feed stocks such as molasses, are abundant and often used as feed stocks for the production by microbial fermentation, adding appropriate genetic modifications to permit uptake and use of sucrose may be practiced in strains having other features as provided herein. Various sucrose uptake and metabolism systems are known in the art (for example, U.S. Pat. No. 6,960,455).

Also, genetic modifications may be provided to add functionality for breakdown of more complex carbon sources, such as cellulosic biomass or products thereof, for uptake, and/or for utilization of such carbon sources. For example, numerous cellulases and cellulase-based cellulose degradation systems have been studied and characterized (Beguin, P and Aubert, J-P, 1994; Ohima, K. et al., 1997.)

In some examples, genetic modifications increase the pool and availability of the cofactor NADPH, and/or, consequently, the NADPH/NADP⁺ ratio may also be provided. For example, in E. coli, this may be done by increasing activity, such as by genetic modification, of one or more of the following genes: pgi (in a mutated form), pntAB, overexpressed, gapA:gapN substitution/replacement, and disrupting or modifying a soluble transhydrogenase such as sthA, and/or genetic modifications of one or more of zwf, gnd, and edd.

Any such genetic modifications may be provided to species not having such functionality, or having a less than desired level of such functionality. More generally, and depending on the particular metabolic pathways of a microorganism selected for genetic modification, any subgroup of genetic modifications may be made to decrease cellular production of fermentation product(s) selected from the group consisting of acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene, isobutylene, ethyl acetate, vinyl acetate, other acetates, 1,4-butanediol, 2,3-butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate, 2-OH-isobutyrate, 3-OH-butyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate, caprolactam, adipic acid, propanol, isopropanol, fusel alcohols, and 1,2-propanediol, 1,3-propanediol, formate, fumaric acid, propionic acid, succinic acid, valeric acid, and maleic acid. Gene deletions may be made as disclosed generally herein, and other approaches may also be used to achieve a desired decreased cellular production of selected fermentation products.

The first genetic mutation according to any aspect of the present invention may result in the formation of at least one fatty acid and/or acyl coenzyme A (CoA) thereof, wherein the fatty acid comprises at least 5 carbon atoms. In particular, the fatty acid may be of any chain length from 5 to greater than 18 carbons. The fatty acid may be selected from the group consisting of: pentanoic acid, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, and oleic acid. In particular, these fatty acids may be produced from a fatty acyl-CoA intermediate via the activity of a fatty acyl-CoA thioesterase. Alternatively, these fatty acids may be produced from a fatty acyl-CoA intermediate via concerted activities of a fatty acyl-CoA phosphotransferase first producing a fatty acyl-phosphate and then the action of a fatty acid kinase operating to produce a fatty acid from the fatty acyl-phosphate.

According to any aspect of the present invention the cell according to any aspect of the present invention may be combined with a carbon source to be able to produce the fatty acid. In particular, the carbon source provided to the cell may have a ratio of carbon-14 to carbon-12 of about 1.0×10⁻¹⁴or greater. The carbon source may be selected from the group consisting of glucose, sucrose, fructose, dextrose, lactose, xylose, pentose, polyol, hexose, other cellulosic sugars or a combination thereof. In one example, the carbon source may be glycerol. In another example, the carbon source may be synthesis gas. Synthesis gas can for example be produced as a by-product of coal gasification. Accordingly, the microorganism according to any aspect of the present invention may be capable of converting a substance which is a waste product into a valuable resource.

In another example, synthesis gas may be a by-product of gasification of widely available, low-cost agricultural raw materials for use with the mixed culture of the present invention to produce substituted and unsubstituted organic compounds. There are numerous examples of raw materials that can be converted into synthesis gas, as almost all forms of vegetation can be used for this purpose. In particular, raw materials are selected from the group consisting of perennial grasses such as miscanthus, corn residues, processing waste such as sawdust and the like.

In general, synthesis gas may be obtained in a gasification apparatus of dried biomass, mainly through pyrolysis, partial oxidation and steam reforming, wherein the primary products of the synthesis gas are CO, H₂and CO₂. Usually, a portion of the synthesis gas obtained from the gasification process is first processed in order to optimize product yields, and to avoid formation of tar. Cracking of the undesired tar and CO in the synthesis gas may be carried out using lime and/or dolomite. These processes are described in detail in for example, Reed, 1981.

In particular, the cell culture may comprise an inhibitor of fatty acid synthase the cell may be genetically modified for reduced enzymatic activity in the cell's fatty acid synthase pathway. This may allow better control for producing the specific desired fatty acid.

The cell according to any aspect of the present invention may comprise at least one second genetic mutation that may increase the activity of at least one wax ester synthase in the cell relative to the wild type cell. The wax ester synthase may comprise sequence identity of at least 50% to a polypeptide of SEQ ID NOs: 1-23 and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the respective fatty acid ester. In particular, the wax ester synthase may comprise sequence identity of at least 50% to a polypeptide of SEQ ID NOs: 1-8 and combinations thereof. More in particular, the wax ester synthase used according to any aspect of the present invention may comprise sequence identity of at least 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% to a polypeptide of any one of sequences of SEQ ID NOs: 1-8 and combinations thereof. These sequences are only reference amino acid sequences. In particular, the sequence of the wax ester synthase used according to any aspect of the present invention may comprise amino acids other than those essential for the function, for example the catalytic activity of a protein, or the fold or structure of a molecule are deleted, substituted or replaced by insertions or essential amino acids are replaced in a conservative manner to the effect that the biological activity of the reference sequence or a molecule derived therefrom is preserved. The state of the art comprises algorithms that may be used to align two given amino acid sequences and to calculate the degree of identity, see Arthur Lesk (2008), and Katoh et al., 2005. In particular, the wax ester synthase sequences used according to any aspect of the present invention may comprise the amino acids that provide the function to the protein. More in particular, the wax ester synthase sequences may comprise deletions, insertions or substitutions in amino acid sequences as well as fusions that still retain the function of the wax ester synthase capable of catalyzing the conversion of fatty acid and/or acyl coenzyme A thereof to the respective fatty acid ester. In one example, a mutant of any one of the sequences of SEQ ID NO:1-8 may be used in any aspect of the present invention. A ‘mutant’ used herein indicates a mutant derived from any one of the sequences of SEQ ID NO:1-8 that is capable of maintaining the function of a wax ester synthase of converting a fatty acid and/or acyl coenzyme A thereof to the respective fatty acid ester. Such a mutant has an amino acid sequence subjected to deletion, substitution, insertion, or addition of at least one amino acid. The mutant of the present invention can be adequately produced with the use of any methods known to persons skilled in the art.

TABLE 2

Sequence of wax ester synthases

Accession No.

SEQ ID NO:
(NCBI)
Organism
Sequence

1
522136843

Singularimonas

MESPRTPMHVGGLMTFRLPPD

(Svar)

variicoloris

APPDFLRQLFARLRAQMPSTEP

FNLRLARTPWSALAPAWEPAP

DIDIDYHVRHSALPYPGGEREL

GVLVSRLHSHPLDLRRPPWEIT

LIEGLENDRFAFFLKVHHSALD

GMGALKLVRRWLSADALQRD

MPALWALPAQPREARDAPHGH

AVEQGVEALRTQLRASGELLST

LRRMARRRDNPEGGILSALSTP

RTLLNVPITPQRRLATQLFELSR

IKAVSAATQSTVNDVALALIAG

AVRRYLLELDALPHEPLVASVP

VGLPRADGKPGNAVAGFVVPL

ETQADDPLDCLHVVRAVTQRT

KDQLRGMSPEALAQFTMLGLS

PLILGQMARVLSHLPPIFNFVVS

NVVASKELLYLEGAELEAMYPI

SVLFDGYALNVTLVGYHDRLS

LGFTGCRDALPSLQRLAVYSAE

ALEELERAAGLVPHAAGAAEH

APARRTRRRGAH

2
YP_436128.1

Hahella

MTPLSPVDQIFLWLEKRQQPM

(Hche)

chejuensis

HVGGLHIFSFPDDADAKYMTEL

KCTC 2396
AQQLRAYATPQAPFNRRLRQR

WGRYYWDTDAQFDLEHHFRH

EALPKPGRIRELLAHVSAEHSN

LMDRERPMWECHLIEGIRGRRF

AVYYKAHHCMLDGVAAMRM

CVKSYSFDPTATEMPPIWAISK

DVTPARETQAPAAGDLVHSLS

QLVEGAGRQLATVPTLIRELGK

NLLKARDDSDAGLIFRAPPSILN

QRITGSRRFAAQSYALERFKAI

GKAFQATVNDVVLAVCGSALR

NYLLSRQALPDQPLIAMAPMSI

RQDDSDSGNQIAMILANLGTHI

ADPVRRLELTQASARESKERFR

QMTPEEAVNYTALTLAPSGLNL

LTGLAPKWQAFNVVISNVPGPN

KPLYWNGARLEGMYPVSIPVD

YAALNITLVSYRDQLEFGFTAC

RRTLPSMQRLLDYIEQGIAELE

KAAGV

3
480024154

Acinetobacter

MRPLHPIDFIFLSLEKRQQPMH

(Ajun)

junii NIPH 182
VGGLFLFEIPENASPTFVHDLVQ

DIRQSKSIPVPPFNNQLNGLFW

GEDPEFDIDHHFRHIALPNPGRI

RELLVYISQQHSSLIDRAKPLW

TCDIIEGIEGNRFAMYFKIHHA

MVDGVAGMRLIEKSLSKDPNE

KHVVPLWCVEGKRTKRLKAPK

PPSVSKIKGIMDGIKSQLEVTPK

VMQELSQTIFKEIGKNPDYVST

FQAPPSILNQRVSSSRRFAAQSF

ELDRFRNIAKSLGVTINDVVLA

VCAGALREYLISHESLPKKPLIA

MVPASLRTDDSDVSNRITMILA

NLATHIEDPIERLQIIRRSVQNSK

QRFSRMTANEILNYSALVYGPA

GLNIVSGMLPKRQAFNLVISNV

PGPREPLYWNGAKLDALYPASI

VMDGQALNITMTSYLDKLEVG

LIACRNALPKMQNLLTHLEDEI

QRFESAILSLPKQAAEG

4
449424446

Amycolatopsis

MPFMPVTDSMFLLVETREHPM

(Aazu)

azurea DSM
HVGGLQLFKKPEDAGPDYLRD

43854
LRRKLLDSDNMRDVFRRRPAR

PVNTAGHVAWATDNDLELDY

HFRHSALPQPGRIRELLELTGR

WHSTLLDRHRPLWEIHLVEGL

QDGRFAIYSKIHHALMDGVSAL

RHLQGTLSDDPTDLDCPPPWGR

RPKPDGGRNGKASPSVLSTFGK

TVNQLAGIAPAAMKVAREAFQ

EHTLTLPAQAPKTMLNVPIGGA

RRFAAQSWSLDRVRKVATAAG

VSRNDVVLAMCSGALRDYLIE

QNSLPDAPLTAMVPVSLRRKDS

GDAAGNNIGALLCNLATHLTD

PAARLATINASMRNGKKLFSEL

TPLQTLLLSGINVAQLGVSPIPG

FVNNTKPPFNLVISNVPGPRKQ

MYWNGASLDGIYPASVLLDGQ

ALNITLTSNGDNLDFGVTGCRR

SVPHLQRILTHLDTALAELEHA

VSVGRS

5
479966651

Acinetobacter

MRPLHPIDFIFLSLEKRQQPMH

(Acip)

sp. CIP 56.2
VGGLFLFELPENASPTFVHDLV

NEIRQSKSIPVPPFNNQLNGLFW

GEDSEFDLDHHFRHIALPNPGRI

RELLVYISQQHSSLIDRAKPLW

TCDIIEGIEGNRFAMYFKIHHA

MVDGVAGMRLIEKSLSQDPNE

KHVVPLWCVEGKRTKRLKAPK

PPTVSKIKGVMEGIKSQLEVAP

KVMQELSQTIFKEMGKNPDYV

STFQAPPSILNQRVSSSRRFAAQ

SFELGRFRRIAKSLGVTLNDVIL

AVCSGALREYLISHNSLPKKPLI

AMVPASLRTDDSDVSNRITMIL

ANLATHIEDPIERLEVIRRSVQN

SKQRFSRMTANEILNYSAVVYG

PAGLNIASGMLPKRQAFNLVIS

NVPGPREPLYWNGAKLDALYP

ASIVMDGQALNITMTSYLDKLE

VGLIACRNALPKMQNLLTHLEE

EIQRFEQAIQDLPQKVAN

6
ABO21021

Marinobacter

MKRLGTLDASWLAVESEDTPM

hydrocarbono-
HVGTLQIFSLPEGAPETFLRDM

clasticus ATCC
VTRMKEAGDVAPPWGYKLAW

49840
SGFLGRVIAPAWKVDKDIDLDY

HVRHSALPRPGGERELGILVSR

LHSNPLDFSRPLWECHVIEGLE

NNRFALYTKMHHSMIDGISGV

RLMQRVLTTDPERCNMPPPWT

VRPHQRRGAKTDKEASVPAAV

SQAMDALKLQADMAPRLWQA

GNRLVHSVRHPEDGLTAPFTGP

VSVLNHRVTAQRRFATQHYQL

DRLKNLAHASGGSLNDIVLYLC

GTALRRFLAEQNNLPDTPLTAG

IPVNIRPADDEGTGTQISFMIAS

LATDEADPLNRLQQIKTSTRRA

KEHLQKLPKSALTQYTMLLMS

PYILQLMSGLGGRMRPVFNVTI

SNVPGPEGTLYYEGARLEAMY

PVSLIAHGGALNITCLSYAGSLN

FGFTGCRDTLPSMQKLAVYTG

EALDELESLILPPKKRARTRK

7
YP_957462

Marinobacter

MGSSHHHHHHSSGLVPRGSHM

(Maqu

aquaeolei VT8
TPLNPTDQLFLWLEKRQQPMH

T373M,

T373M, Q420R
VGLQLFSFPEGAPDDYVAQLA

Q420R)

DQLRQKTEVTAPFNQRLSYRLG

QPVWVEDEHLDLEHHFRFEAL

PTPGRIRELLSFVSAEHSHLMDR

ERPMWEVHLIEGLKDRQFALY

TKVHHSLVDGVSAMRMATRM

LSENPDEHGMPPIWDLPCLSRD

RGESDGHSLWRSVTHLLGLSG

RQLGTIPTVAKELLKTINQARK

DPAYDSIFHAPRCMLNQKITGS

RRFAAQSWCLKRIRAVCEAYG

TTVNDVVTAMCAAALRTYLM

NQDALPEKPLVAFVPVSLRRDD

SSGGNQVGVILASLHTDVQEAG

ERLLKIHHGMEEAKQRYRHMS

PEEIVNYTALTLAPAAFHLLTG

LAPKWQMFNVVISNVPGPSRPL

YWNGAKLEGMYP

VSIDMDRLALNMTLTSYNDRV

EFGLIGCRRTLPSLQRMLDYLE

QGLAELELNAGL

8
YP_957462

Marinobacter

MGSSHHHHHSSGLVPRGSHMT

(Maqu

aquaeolei VT8
PLNPTDQLFLWLEKRQQPMHV

E72K)

E72K (no final
GGLQLFSFPEGAPDDYVAQLA

H in His-tag)
DQLRQKTEVTAPFNQRLSYRLG

QPVWVKDEHLDLEHHFRFEAL

PTPGRIRELLSFVSAEHSHLMDR

ERPMWEVHLIEGLKDRQFALY

TKVHHSLVDGVSAMRMATRM

LSENPDEHGMPPIWDLPCLSRD

RGESDGHSLWRSVTHLLGLSG

RQLGTIPTVAKELLKTINQARK

DPAYDSIFHAPRCMLNQKITGS

RRFAAQSWCLKRIRAVCEAYG

TTVNDVVTAMCAAALRTYLM

NQDALPEKPLVAFVPVSLRRDD

SSGGNQVGVILASLHTDVQEAG

ERLLKIHHGMEEAKQRYRHMS

PEEIVNYTALTLAPAAFHLLTG

LAPKWQTFNVVISNVPGPSRPL

YWNGAKLEGMYPVSIDMDRL

ALNMTLTSYNDQVEFGLIGCRR

TLPSLQRMLDYLEQGLAELELN

AGL

23 (Maqu)
YP_957462

Marinobacter

MTPLNPTDQLFLWLEKRQQPM

aquaeolei VT8
HVGGLQLFSFPEGAPDDYVAQ

LADQLRQKTEVTAPFNQRLSYR

LGQPVWVEDEHLDLEHHFRI-th

ALPTPGRIRELLSFVSAEHSHLM

DRERPMWEVHLIEGLKDRQFA

LYTKVHHSLVDGVSAMRMAT

RMLSENPDEHGMPPIWDLPCLS

RDRGESDGHSLWRSVTHLLGL

SGRQLGTIPTVAKELLKTINQA

RKDPAYDSIFHAPRCMLNQKIT

GSRRFAAQSWCLKRIRAVCEA

YGTTVNDVVTAMCAAALRTYL

MNQDALPEKPLVAFVPVSLRR

DDSSGGNQVGVILASLHTDVQE

AGERLLKIHHGMEEAKQRYRH

MSPEEIVNYTALTLAPAAFHLL

TGLAPKWQTFNVVISNVPGPSR

PLYWNGAKLEGMYPVSIDMDR

LALNMTLTSYNDQVEFGLIGCR

RTLPSLQRMLDYLEQGLAELEL

NAGL

Throughout this application, any data base code, unless specified to the contrary, refers to a sequence available from the NCBI data bases, more specifically the version online on 12 Jun. 2014, and comprises, if such sequence is a nucleotide sequence, the polypeptide sequence obtained by translating the former.

The cell according to any aspect of the present invention may comprise a third genetic mutation that reduces the fatty acid degradation capacity of the cell relative to the wild type cell.

Degradation of fatty acids is accomplished by a sequence of enzymatically catalyzed reactions. First of all, fatty acids are taken up and translocated across the cell membrane via a transport/acyl-activation mechanism involving at least one outer membrane protein and one inner membrane-associated protein which has fatty acid-CoA ligase activity, referred to in the case of E. coli as FadL and FadD/FadK, respectively. Inside the cell, the fatty acid to be degraded is subjected to enzymes catalyzing other reactions of the β-oxidation pathway. The first intracellular step involves the conversion of acyl-CoA to enoyl-CoA through acyl-CoA dehydrogenase, the latter referred to as FadE in the case of E. coli. The activity of an acyl-CoA dehydrogenase may be assayed as described in the state of art, for example by monitoring the concentration of NADH spectrophotometrically at 340 nm in 100 mM MOPS, pH 7.4, 0.2 mM Enoyl-CoA, 0.4 mM NAD⁺. The resulting enoyl-CoA is converted to 3-ketoacyl-CoA via 3-hydroxyacyl-CoA through hydration and oxidation, catalyzed by enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, referred to as FadB and FadJ in E. coli. Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase activity, more specifically formation of the product NADH may be assayed spectrophotometrically as described in the state of the art, for example as outlined for FadE. Finally, 3-ketoacyl-CoA thiolase, FadA and FadI in E. coli, catalyzes the cleavage of 3-ketoacyl-CoA, to give acetyl-CoA and the input acyl-CoA shortened by two carbon atoms. The activity of ketoacyl-CoA thiolase may be assayed as described in the state of the art, for example in Antonenkov, V., D. et al. (1997) Substrate specificities of 3-oxoacyl-CoA thiolase and sterol carrier protein 2/3-oxoacyl-coa thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates. In one example, the term “a cell having a reduced fatty acid degradation capacity”, as used herein, refers to a cell having a reduced capability of taking up and/or degrading fatty acids. The fatty acid degradation capacity of a cell may be reduced in various ways. In another example, the cell has, compared to its wild type, a reduced activity of an enzyme involved in the β-oxidation pathway. In a further example, the term “enzyme involved in the β-oxidation pathway”, as used herein, refers to an enzyme that interacts directly with a fatty acid or a derivative thereof formed as part of the degradation of said fatty acid via the β-oxidation pathway the sequence of reactions effecting the conversion of a fatty acid to acetyl-CoA and the CoA ester of the shortened fatty acid, for example by recognizing the fatty acid or derivative thereof as a substrate, and converts it to a metabolite formed as a part of the β-oxidation pathway. More in particular, the reduced fatty acid degradation capacity in the cell according to any aspect of the present invention may be the third genetic mutation which results in a decrease in the expression of at least one enzyme selected from the group consisting of acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase relative to the wild type cell.

For example, the acyl-CoA dehydrogenase is an enzyme involved in the β-oxidation pathway as it interacts with fatty acid-CoA and converts fatty acid-CoA ester to enoyl-CoA, which is a metabolite formed as part of the β-oxidation. In a further example, the term “enzyme involved in the β-oxidation pathway”, as used herein, comprises any polypeptide from the group comprising acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-keto-acyl-CoA thiolase. Subsequently, the acyl-CoA synthetase may catalyze the conversion a fatty acid to the CoA ester of a fatty acid, i.e. a molecule, wherein the functional group —OH of the carboxy group is replaced with —S-CoA, for introducing said fatty acid into the β-oxidation pathway. For example, the polypeptides FadD and FadK in E. coli (access code: BAA15609.1) are acyl-CoA dehydrogenases. In an example, the term “acyl-CoA dehydrogenase”, as used herein, is a polypeptide capable of catalyzing the conversion of an acyl-CoA to enoyl-CoA, as part of the β-oxidation pathway. For example, the polypeptide FadE in E. coli (access code: BAA77891.2) is an acyl-CoA dehydrogenase. In one example, the term “2,4-dienoyl-CoA reductase”, as used herein, is a polypeptide capable of catalyzing the conversion of the 2,4-dienoyl CoA from an unsaturated fatty acid into enoyl-CoA, as part of the β-oxidation pathway. For example, the polypeptide FadH in E. coli is a 2,4-dienoyl-CoA reductase. In an example, the term “enoyl-CoA hydratase”, as used herein, also referred to as 3-hydroxyacyl-CoA dehydrogenase, refers to a polypeptide capable of catalyzing the conversion of enoyl-CoA to 3-ketoacyl-CoA through hydration and oxidation, as part of the β-oxidation pathway. For example, the polypeptides FadB and FadJ in E. coli (access code: BAE77457.1) are enoyl-CoA hydratases. In one example, the term “ketoacyl-CoA thiolase”, as used herein, refers to a polypeptide capable of catalyzing the conversion of cleaving 3-ketoacyl-CoA, resulting in an acyl-CoA shortened by two carbon atoms and acetyl-CoA, for example as the final step of the β-oxidation pathway. For example, the polypeptides FadA and FadI in E. coli (access code: AP009048.1) are ketoacyl-CoA thiolases.

In particular, the cell according to any aspect of the present invention may comprise genetic mutations that result in an increase in the expression of β-ketoacyl-ACP synthase III. This may be the first mutation in the cell according to any aspect of the present invention. Any β-ketoacyl-ACP synthase III (fabH) known in the art may be used in the method according to any aspect of the present invention. In particular, the fabH may be selected from Table 3a.

TABLE 3a

Possible sources of FabH that may be used to produce

fatty acids such as lauric acid.

FabH
Accession No.

Shewanella sp. MR-4
gi|113969844

Shewanella frigidimarina NCIMB 400
gi|114563637

Shewanella sp. ANA-3
gi|117920011

Shewanella amazonensis SB2B
gi|119774647

Shewanella sp. W3-18-1
gi|120598458

Shewanella baltica OS185
gi|153001200

Gordonia bronchialis DSM 43247
gi|262201496

Gordonia neofelifaecis NRRL B-
gi|326383808

59395

putative Shewanella sp. HN-41
gi|336312046

Rheinheimera sp. A13L
gi|336314652

Gordonia araii NBRC 100433
gi|359421305

Gordonia polyisoprenivorans NBRC
gi|359767552

16320

Gordonia effusa NBRC 100432
gi|359774344

Alishewanella jeotgali KCTC 22429
gi|375108677

Gordonia otitidis NBRC 100426
gi|377561073

Gordonia sputi NBRC 100414
gi|377565709

Gordonia terrae NBRC 100016
gi|377571475

Gordonia polyisoprenivorans VH2
gi|378716896

Alishewanella agri BL06
gi|393761603

Gordonia sp. KTR9
gi|404214055

Shewanella oneidensis MR-1
gi|414562081

Gordonia hirsuta DSM 44140
gi|441517717

Gordonia sihwensis NBRC 108236
gi|441522685

Gordonia soli NBRC 108243
gi|444431726

Gordonia malaquae

gi|495656093

Gordonia sp. NB4-1Y
gi|464805365

Thalassolituus oleivorans MIL-1
gi|473830078

Colwellia psychrerythraea 34H
gi|71278947

Shewanella denitrificans OS217
gi|91793871

Rheinheimera nanhaiensis E407-8
gi383934006

Acinetobacter sp. CIP 53.82
480152603

Hahella chejuensis KCTC 2396
83645428

Acinetobacter sp. SH024
293608659

Acinetobacter sp. NBRC 10098
359430113

Acinetobacter sp. ADP1
50085221

Acinetobacter ursingii DSM 16037 =
406040759

CIP 107286

Acinetobacter bohemicus ANC 3994
479867614

Candidatus Accumulibacter phosphatis

257092603

clade IIA str. UW-1

blood disease bacterium R229
344167953

Ralstonia solanacearum CMR15
410684104

Marinobacter sp. BSs20148
399545195

Marinobacter algicola DG893
149375225

Ralstonia sp. 5_7_47FAA
309779507

Rubrivivax gelatinosus IL144
383757692

Oceanobacter sp. RED65
94501061

gamma proteobacterium HTCC5015
254447852

Ilumatobacter coccineus YM16-304
470180366

marine gamma proteobacterium
254480565

HTCC2148

Marinobacter aquaeolei VT8
120554511

Alcanivorax sp. DG881
254427265

Hydrocarboniphaga effusa AP103
392950783

Curvibacter putative symbiont of Hydra
260220470

magnipapillata

gamma proteobacterium HdN1
304312991

marine gamma proteobacterium
119503170

HTCC2080

gamma proteobacterium IMCC3088
329897271

gamma proteobacterium NOR5-3
254514195

Acinetobacter radioresistens SK82
255318218

Acinetobacter sp. NIPH 899
479953276

Acinetobacter schindleri CIP 107287
480002578

Acinetobacter towneri DSM 14962 =
480029713

CIP 107472

Acinetobacter junii CIP 107470
480007780

Acinetobacter sp. CIP 56.2
479964140

Acinetobacter baumannii AYE
169796586

baumannii MDR-ZJ06

Acinetobacter gerneri DSM 14967 =
479991204

CIP 107464

Acinetobacter bouvetii DSM 14964 =
480043238

CIP 107468

Acinetobacter sp. ANC 3789
479932652

Acinetobacter lwoffii SH145
262375396

Acinetobacter soli NIPH 2899
480019083

Acinetobacter baumannii WC-323
425744072

Acinetobacter calcoaceticus NIPH 13
479856262

Acinetobacter johnsonii SH046
262369694

Acinetobacter haemolyticus CIP 64.3
480080132

Acinetobacter sp. CIP 102529
479942708

Acinetobacter sp. CIP-A165
479879420

Acinetobacter guillouiae CIP 63.46
479909393

Ralstonia solanacearum FQY_4
469776065

Ralstonia solanacearum UW551
83747353

The β-ketoacyl-ACP synthase III (FabH) may comprise sequence identity of at least 50% to a polypeptide selected from the group consisting of SEQ ID NOs: 24-27 and combinations thereof or to a functional fragment of any of the polypeptides. In particular, the FabH may comprise sequence identity of at least 50% to a polypeptide of SEQ ID NOs: 24-27 and combinations thereof. More in particular, the FabH used according to any aspect of the present invention may comprise sequence identity of at least 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% to a polypeptide of any one of sequences of SEQ ID NOs: 24-27 and combinations thereof. More in particular, the cell according to any aspect of the present invention may have a first mutation that comprises a combination of sequences of FabH. For example, the cell according to any aspect of the present invention may be genetically modified to comprise a polypeptide with sequence identity of at least 50, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% to a polypeptide of comprising SEQ ID NOs: 24 and 27, SEQ ID NOs: 25 and 27, SEQ ID NOs: 26 and 27 and the like.

These sequences are only reference amino acid sequences. In particular, the sequence of the FabH used according to any aspect of the present invention may comprise amino acids other than those essential for the function, for example the catalytic activity of a protein, or the fold or structure of a molecule are deleted, substituted or replaced by insertions or essential amino acids are replaced in a conservative manner to the effect that the biological activity of the reference sequence or a molecule derived therefrom is preserved.

TABLE 3b

Sequences of FabH, crt, Hbd that may be used in the cells according

to any aspect of the present invention

SEQ ID
Accession No.

NO:
(NCBI)/
Organism
Sequence

24
WP_014577218.1|

Marinobacter

MIKAVISGTGLYTPPATISNDE

adhaerens

LVEAFNQYVELFNAENADAI

HP15
ASGDVTPLQPSSSSFIEKASGI

KRRHVIDKDGILDPNRMKPYI

PDRSNEEPSVQCDMAVTACR

EALEQAGKSAEDVDAVIVAC

SNLQRAYPAVSIEVQEALGID

GFAYDMNVACSSATFGLQAA

VNSVENGSARAVLVVSPEICS

GHLNFRDRDSHFIFGDACTAI

LVEREEDTREGQGFEILGTSL

KTKFSNNIRNNFGFLNRADES

GVGKPDKLFVQQGRKVFKEV

SPLVAETIQKQLQSLSLAPDD

LRRMWLHQANLNMNQLIAR

KVLGRDATEEEAPVILDEYAN

TSSAGSIIAFHKNKDDLVSGD

LGVICSFGAGYSIGSVVVRRR

25
ZP_10350240

Alishewanella

MQQVVISGSGLFTPQHRISNE

agri BL06
ELVQSYNQYVDQFNEEHAAA

IAAGEIEALEYSSTEFIEKASGI

KARHVLYKDGILDPKIMHPVF

RKRGEDELPEMVEMAVQAA

TQALAQANKTAADIDLIICAA

SNMQRPYPALSVELQQALGA

GGYAFDMNVACSSATFAISN

AVNAIRGGTAKVVLVVNPEF

ASPQVDYRSRDSHFIFGDVCT

ATIIEAESSCSSQQAFRILGMR

LKTTFSNNIRCDIGYTEHCFTE

QDPKAPFFKQQGRKVFKELLP

IVADVIQDEMAAQNLAPDDL

KRLWLHQANINMNIFAAKKI

LGRDPLPEEAPLVLDTYANTA

SAGSIIAFHKYQQGLVSGDKA

ILCSFGAGYSVGCVVLEKC

26
ENU26638

Acinetobacter

MGIRITGTGLFHPTESISNEEL

sp. NIPH 236
VESLNAYVEQFNQENAEQIA

AGEIEALRGSSPEFIEKASGIQ

RRYVVEKSGILDPKRLRPRLQ

ERSNDELSLQAEWGVIAAKQ

AMENAGVTAEDIDVVILACS

NMQRAYPAVAIEIQSALGIQG

YAYDMNVACSAATFGLKQA

YDAVKCGARRVLLLNVEITS

GHLDYRTRDAHFIFGDVATA

SIIEETETKSGYEILDIHLFTQF

SNNIRNNFGFLNRSEDAVVDD

KLFRQDGRKVFKEVCPLVAKI

ITAQLEKLELTPEQVKRFWLH

QANANMNELILKLVVGKEAD

LERAPIILDEFANTSSAGVIIA

MHRTGEQVNNGEYAVISSFG

AGYSVGSIIVQKHIA

27
YP_006031367

Ralstonia

MHDVVISGTGLWVAPEVITN

solanacearum

EELVASFNAYARHYNEANAT

Po82
AIAAGTLAAVAESSVEFIEKA

SGIRQRYVIDKAGVLDPARM

RPRLAPRGDDALSLQAEIGVA

AAREALAAAGRDAGDIDMLI

CSAANMQRPYPAMGIEIQNA

LGADGYAFDMNVACSSATFG

LEQAINAVRTGSARVALMVN

PEITSGHLAWKDRDCHFIFGD

VCTAVVVERADDARAPDQW

QVLGTRMATRFSNSIRNNAGF

LSRSEDRDPDDRDQLFRQEGR

KVFKEVCPMAAEHIAGHLQS

LGHAPADVRRFWLHQANLG

MNQLIGKRLLGRDASADEAP

VILDEFANTASAGSIIAFHRHR

ADLQPGDLGLICSFGAGYSIG

SVAVRKR

28
AAA95967

Clostridium

MELNNVILEKEGKVAVVTIN

acetobutylicum

RPKALNALNSDTLKEMDYVI

ATCC 824
GEIENDSEVLAVILTGAGEKS

FVAGADISEMKEMNTIEGRKF

GILGNKVFRRLELLEKPVIAA

VNGFALGGGCEIAMSCDIRIA

SSNARFGQPEVGLGITPGFGG

TQRLSRLVGMGMAKQLIFTA

QNIKADEALRIGLVNKVVEPS

ELMNTAKEIANKIVSNAPVAV

KLSKQAINRGMQCDIDTALAF

ESEAFGECFSTEDQKDAMTAF

IEKRKIEGFKNR

29
NP_349314

Clostridium

MKKVCVIGAGTMGSGIAQAF

acetobutylicum

AAKGFENVLRDIKDEFVDRG

ATCC 824
LDFINKNLSKLVKKGKIEEAT

KVEILTRISGTVDLNMAADCD

LVIEAAVERMDIKKQIFADLD

NICKPETILASNTSSLSITEVAS

ATKRPDKVIGMHFFNPAPVM

KLVEVIRGIATSQETFDAVKE

TSIAIGKDPVEVAEAPGFVVN

RILIPMINEAVGILAEGIASVE

DIDKAMKLGANHPMGPLELG

DFIGLDICLAIMDVLYSETGDS

KYRPHTLLKKYVRAGWLGR

KSGKGFYDYSK

As used herein, the term “fatty ester” means an ester. In particular, a fatty ester is any ester made from a fatty acid to produce a fatty acid ester. In one example, a fatty ester contains an A side (i.e., the carbon chain attached to the carboxylate oxygen) and a B side (i.e., the carbon chain comprising the parent carboxylate). In a particular, when the fatty ester is derived from the fatty acid biosynthetic pathway, the A side is contributed by an alcohol, and the B side is contributed by a fatty acid. Any alcohol can be used to form the A side of the fatty esters. For example, the alcohol can be derived from the fatty acid biosynthetic pathway. Alternatively, the alcohol can be produced through non-fatty acid biosynthetic pathways. In one example, the alcohol can be provided exogenously. For example, the alcohol can be supplied in the fermentation broth in instances where the fatty ester is produced by an organism that can also produce the fatty acid. Alternatively, a carboxylic acid, such as a fatty acid or acetic acid, can be supplied exogenously in instances where the fatty ester is produced by an organism that can also produce alcohol.

The carbon chains comprising the A side or B side can be of any length. In one example, the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. The B side of the ester is at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. The A side and/or the B side can be straight or branched chain. The branched chains may have one or more points of branching. The branched chains may also include cyclic branches and/or the A side and/or B side can be saturated or unsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation.

In one example, the fatty ester according to any aspect of the present invention may be produced biosynthetically. In particular, the fatty acid may be “activated” to produce at least one compound selected from the group consisting of acyl Coenzyme A (acyl-CoA), and acyl phosphate. More in particular, the fatty ester may be activated to Acyl-CoA, a direct product of fatty acid biosynthesis or degradation. Acyl-CoA may also be synthesized from a free fatty acid, a CoA, or an adenosine nucleotide triphosphate (ATP). An example of an enzyme which produces acyl-CoA is acyl-CoA synthase. Acyl-CoA may then be transferred to a recipient nucleophile such as alcohols, thiols, phosphates and the like.

The cell according to any aspect of the present invention may be further genetically modified to increase the expression of 3-hydroxyacyl coenzyme A dehydratase (3HCDh) and/or keto acyl-CoA reductase (KCR) relative to the wild type cell. This is increase in expression may improve the activity of fadB. In particular, the 3HCDh may crotonase/enoyl-CoA hydratase (Crt) and/or the KCR may be hydroxybutyric dehydrogenase (Hbd). More in particular, the Crt may have sequence identity of at least 50, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% identity to a polypeptide of SEQ ID NO:28 and/or the Hbd has sequence identity of at least 50, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% identity to a polypeptide of SEQ ID NO:29.

In particular, the fatty acid ester may be produced in the presence of at least one exogenous alcohol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, hexadecanol and the like.

More in particular, the fatty acid may be 12 carbons in length. The fatty acid may be lauric acid, the acyl coenzyme A thereof may be lauroyl coenzyme A and the fatty acid ester may be methyl laurate. According to another aspect of the present invention there is provided a method for producing methyl laurate, the method comprising contacting lauric acid and/or lauroyl coenzyme A with an isolated wax ester synthase that has sequence identity of at least 50% to a polypeptide of SEQ ID NOs: 1-8 and combinations thereof. More in particular, the wax ester synthase used according to any aspect of the present invention may comprise sequence identity of at least 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% to a polypeptide of any one of sequences of SEQ ID NOs: 1-8 and combinations thereof. These sequences are only reference amino acid sequences. In particular, the sequence of the wax ester synthase used according to any aspect of the present invention may comprise amino acids other than those essential for the function, for example the catalytic activity of a protein, or the fold or structure of a molecule are deleted, substituted or replaced by insertions or essential amino acids are replaced in a conservative manner to the effect that the biological activity of the reference sequence or a molecule derived therefrom is preserved.

In particular, the method according to any aspect of the present invention is carried out within the cell according to any aspect of the present invention.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

EXAMPLES
Example 1

Optimization of C12 Fatty Acyl-CoA Production in E. coli

Production of Fatty Acids Via Malonyl-CoA and Acetyl-CoA in a Shake Flask Experiment

TABLE 4a

List of microorganism strains that were used to produce fatty acids in the

subsequent examples. The method of production and the sequences of

the strains are provided in Table 3.2 of WO2014026162A1

(OPX Biotechnologies Inc., USA).

Strain

designation
Host
Plasmid
SEQ ID NOs.

BXF_0012
BX_864
1)pBMT-3_ccdAB
30

BXF_0013
BX_864
1)pBMT-3_ccdAB_P_T7-
31

′tesA

BXF_0014
BX_864
1)pBMT-3_ccdAB_P_T7-
32

nphT7-hbd-crt-ter

BXF_0015
BX_864
1)pBMT-3_ccdAB_P_T7-
33

′tesA_PT7-nph_T7-hbd-crt-

ter

BXF_0018
BX_864
pBMT-3_ccdAB_PT7-
32

nphT7-hbd-crt-ter

BXF_0020
BX_860
1)pBMT-3_ccdAB_PT7-
33

′tesA_PT7-nphT7-hbd-crt-

ter

BXF_0021
BX_876
1)pBMT-3_ccdAB_PT7-
33

′tesA_PT7-nphT7-hbd-crt-

ter

BXF_0022
BX_874
1)pBMT-3_ccdAB
30

BXF_0023
BX_874
1)pBMT-3_ccdAB_PT7-
31

′tesA

BXF_0024
BX_874
1)pBMT-3_ccdAB_PT7-
33

′tesA_PT7-nphT7-hbd-crt-

ter

BXF_0025
BX_875
1)pBMT-3_ccdAB
30

BXF_0026
BX_875
1)pBMT-3_ccdAB_PT7-
31

′tesA

BXF_0027
BX_875
1)pBMT-3_ccdAB_PT7-
33

′tesA_PT7-nphT7-hbd-crt-

ter

BXF_0028
BX_878
1)pBMT-3ccdAB-T7-
33

′tesA-

PT7_nphT7_hbd_crt_ter

BXF_0028
BX_878
1)pBMT-3_ccdAB_PT7-
33

′tesA_PT7-nphT7-hbd-crt-

ter

BXF_0029
BX_879
1)pBMT-3_ccdAB_PT7-
33

′tesA_PT7-nphT7-hbd-crt-

ter

BXF_0030
BX_881
1)pBMT-3_ccdAB_PT7-
33

′tesA_PT7-nphT7-hbd-crt-

ter

BXF_0031
BX_864
1)pBMT-3_ccdAB_PT7-
33

′tesA_PT7-nphT7-hbd-crt-

ter

2)pET-28b(empty vector)

BXF_0033
BX_878
1)pBMT-3_ccdAB_PT7-
32

nphT7-hbd-crt-ter

BXF_0034
BX_879
2)pBMT-3_ccdAB_PT7-
32

nphT7-hbd-crt-ter

A base strain was constructed by chromosomal integration of Hbd and Crt into the BX_1018 parent strain. The following set of strains were transformed and evaluated in small scale for both FAME production and metabolite accumulation.

TABLE 4b

list of strains used for testing hbd and crt presence in FAME production

Strain
Parent(s)
Plasmid 1
Plasmid 2

BXE_062
BX_1018
pET-PpstsIH-Aagr
pACYC-PpstsIH-nphT7(SV)-ter-

PpstsIH-fadB-Maqu

BXE_207
BX_1018
pET-PpstsIH-Aagr
pACYC-PpstsIH-nphT7(SV)-ter-

PpstsIH-fadB-PphoE-Maqu

BXE_229
BX_1018
pET-PpstsIH-Aagr
pACYC-PpstsIH-nphT7(SV)-ter-

ΔyibD:PyibD-hbd-crt

PpstsIH-fadB-PphoE-Maqu

BXE_230
BX_1018
pET-PpstsIH-Aagr
pACYC-PpstsIH-nphT7(SV)-ter-

ΔyibD:PyibD-hbd-crt

PpstsIH-fadB-Maqu

To better understand the impact of Hbd/Crt expression, metabolite accumulation was monitored in cell lysate following incubation with malonyl-CoA and acetyl-CoA substrates. The results of this assay, as presented in FIG. 12, showed increased accumulation of C4-C6-CoA intermediates consistent with the expected activity on keto- and hydroxy-C4-CoAs.

Engineering β-Keto Acyl-CoA Synthases (FabH)

A screening approach was employed for β-keto acyl-CoA synthase homologs to identify candidates for lauric acid production based on demonstrated activity on C4- to C10 acyl-CoA substrates. Greater than 70 homologs have been synthesized, expressed, purified and evaluated for activity in vitro.

Synthase candidates identified to have significant activity on C4- to C10-CoA substrates were incorporated into production hosts and evaluated for FFA production in shake flask. FIG. 1 shows FFA production profiles at 68 hours for several of the engineered strains (Table 4c) exhibiting significant lauric acid production. As shown, both lauric acid and total FFA production profiles are modulated by the individual synthase candidate(s) incorporated. For example, strain BXF_198, which contains the Aagr fabH construct, shows the highest specificity for lauric acid, whereas co-expression of Rsol fabH corresponds to the highest titer in total FFA. All four synthase combinations shown in FIG. 1 have been selected for Example 3 focused on production of methyl laurate.

TABLE 4c

Bacterial strains with specific plasmids used in the screening phase

Strain
Host
Plasmid 1
Plasmid 2

BXF_166
BX_978
1009_pACYC-PpstsIH-
1007_pET28b-

nphT7(I147S,F217V)-ter-
ΔlacI-PpstsIH-Madh

TT_PpstsIH-fadB
fabH-Aagr fab H

BXF_169
BX_985
1009_pACYC-PpstsIH-
1008_pET28b-

nphT7(I147S,F217V)-ter-
ΔlacI-PpstsIH-Anip

TT_PpstsIH-fadB
fabH-Aagr fab H

BXF_185
BX_985
1009_pACYC-PpstsIH-
1045_pET28b-

nphT7(I147S,F217V)-ter-
ΔlacI-PpstsIH-Rsol

TT_PpstsIH-fadB
fabH-Aagr fab H

BXF_198
BX_985
1009_pACYC-PpstsIH-
1123_pET28b-

nphT7(I147S,F217V)-ter-
ΔlacI-PpstsIH-Aagr

TT_PpstsIH-fadB
fab H

Baseline FFA Analysis

A more comprehensive baseline analysis was completed with a subset of FFA production strains with various synthase combinations as described above. Data for strain BXF_169 is presented; similar trends were observed among all strains that were evaluated in the more comprehensive tests. The data presented in FIG. 2 shows a time course of lauric acid production for strain BXF_169. In the shake flask test conditions, the strain exhibited a stable initial production rate for 10-20 hours. After the 20 hours, the rate decreased significantly and product titer levels off.

For all strains analyzed, samples were taken at each time point and analyzed for transcript, expression, and activity for key enzymes in the fatty acid production pathway. FIGS. 3 and 4 show the relative mRNA expression of the pathway genes and the corresponding enzyme activity over time for BXF_169. Transcription levels were good, but also differed between the five genes despite being driven from the same promoter. Both transcript and enzyme activity showed a decreasing trend over time, with enzyme activities dropping for several reactions at the 24 hour time point. Similar trends were observed for all other FFA production strains evaluated.

In addition to reduced mRNA and enzyme activity, the 24-hour time point was also characterized by increased insoluble protein accumulation and decreased glucose consumption for all strains (data not shown). As fatty acids including lauric acid are known to accumulate inside the cells in the absence of specific transporters, it was hypothesized that the reduction in productivity at 24 hours is due to intracellular FFA toxicity.

In Vitro Analysis of the Fatty Acid Synthesis Pathway

An assay was developed by reconstructing the fatty acyl-CoA pathway in vitro with purified enzymes. The in vitro reconstruction simplified identification of rate-limiting steps, which were characterized by metabolite accumulation under reaction conditions. There were several benefits of in vitro pathway reconstitution including isolation of pathway flux from competitive enzymes (e.g. thioesterases), substrate limitations (e.g. NADH pools), and balanced expression of multiple pathway enzymes. The equilibrium of the pathway was evaluated by quantifying all 20 pathway intermediates from malonyl-CoA to lauroyl-CoA in the presence of varying amounts of enzymes or substrates (acyl-CoA intermediates).

FIG. 5 shows the accumulation of pathway intermediates for the C4 to C6 elongation cycle observed while varying the enzymes for the keto acyl-CoA reductase (KCR) and 3-hydroxyacyl-CoA dehydratase (3HCDh) reactions. As shown, when the pathway was reconstructed with FadB, there was significant accumulation of 3-hydroxybutyryl-CoA, suggesting insufficient 3HCDh activity to drive the reaction forward. The observed accumulation may be due to the preference for this enzyme to catalyze the reverse reaction, which may also be reducing overall forward pathway flux in vivo. However, when FadB is supplemented with Hbd and/or Crt (alternative KCR and 3HCDh enzymes that have significant activity in the forward direction with C4 intermediates), a reduction in 3-hydroxybutyryl-CoA accumulation was observed. These data suggested that the supplemental activity was sufficient to drive the pathway forward to butyryl-CoA and that integration of Hbd and Crt into production hosts may enhance production of longer chain fatty acids.

Synthase Mutant Evaluations

FabH variants were isolated from a 96-well plate-based screen developed to detect beneficial mutations with improved activity on C10-CoA. Following the initial screen of >1000 mutants, positive variants were sequenced and activity on C6-C10-CoA was evaluated. As shown in FIG. 13, three of the purified variants identified were confirmed to have increased activity on C6-C10-CoA substrates.

Following positive confirmation, FabH mutants were evaluated in vivo. The following strains were constructed by incorporating the FabH mutations into BXE_198 and BXE_233.

Strains were evaluated in the standard 1 mL screening protocol for FAME production in 20 hours (FIG. 14). As shown below, increased methyl laurate observed with the E37K (strain BXE_271) and D196G/K342E (strains BXE_273 and BXE_243) variants compared with the control strains (strains BXE_198 and BXE_233). However, as with the hbd/crt module, it is expected that these mutations may have a greater impact in production strains with improved WES activity.

It has been consistently demonstrated with current production strains that the required expression of Aagr FabH to achieve target activities in lysate requires a significant fraction of the total protein pool. Furthermore, even with a high level of expression, the FabH activity on C10-CoA is often times at or slightly below the target activity.

TABLE 4d

The following strains were constructed by incorporating the FabH mutations into

BXE_198 and BXE_233.

Strain
Parent(s)
Plasmid 1
Plasmid 2

BXE_198
BX_1018
pET-PpstsIH-Aagr-TS Maqu
pACYC_PpstsIH nphT7(SV)-ter-

PpstsIH-fadB

BXE_233
BX_1018
pET_PpstsIH-Aagr-PtpiA-
pACYC-PpstsIH-nphT7(SV)-ter-

accDA_PrpiA-accB-accC
PpstsIH-fadB-PphoE-Mhyd

BXE_271
BX_1018
pET_PpstsIH-Aagr(E37K)-
pACYC_PpstsIH nphT7(SV)-ter-

PTS-Maqu
PpstsIH-fadB

BXE_272
BX_1018
pET_PpstsIH-Aagr(E51G,
pACYC_PpstsIH nphT7(SV)-ter-

S357G)-PTS-Maqu
PpstsIH-fadB

BXE_273
BX_1018
pET_PpstsIH-Aagr(D196G,
pACYC_PpstsIH nphT7(SV)-ter-

E342E)-PTS-Maqu
PpstsIH-fadB

BXE_243
BX_1018
pET_PpstsIH-Aagr(D196G,
pACYC_PpstsIH nphT7(SV)-ter-

K342E)-Ptpi-accDA_PrpiA-
PpstsIH-fadB-PphoE-Mhyd

accB-accC

BXE_244
BX_1018
pET_PpstsIH-Aagr(D196G,
pACYC_PpstsIH nphT7(SV)-ter-

Q219R)-PtpiA-accDA_PrpiA-
PpstsIH-fadB-PphoE-Mhyd

accB-accC

Example 2

Optimization of Wax Ester Synthase (WES) Activity for Methyl Laurate Production

WES candidates were expressed, purified, and evaluated for solubility and FAME production in vitro. Based on the results of the initial screen, 21 WES candidates were chosen for further evaluation including substrate specificity and in vivo FAME production. In vitro assays were completed by measuring product formation following the addition of individual CoA substrates. As summarized in FIG. 6, a range of activities was observed on the various substrates and a number of candidates were identified based on high overall activity (Svar, Maqu) or specificity for substrates ≥C12-CoA (M360.2, Acip). The sequences of the WES used are provided in Table 1 above.

As an orthogonal method for testing in vivo activity, nine of the WES candidates (Table 4f) shown to be active on lauroyl-CoA in the presence of methanol were expressed in a fatty acid production host.

TABLE 4f

Nine WES candidates shown to be active on lauroyl-CoA in the presence

of methanol were expressed in a fatty acid production host

Strain
Host
Plasmid 1
Plasmid 2

BXE_003
BX_926
583_pET28b-ΔlacI-
1005_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-TT_PpstsIH-

Abor

BXE_013
BX_926
583_pET28b-ΔlacI-
1071_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-PpstsIH-

Gpro

BXE_016
BX_926
583_pET28b-ΔlacI-
1076_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-PpstsIH-

Requ

BXE_017
BX_926
583_pET28b-ΔlacI-
1077_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-PpstsIH-

LMED

BXE_018
BX_926
583_pET28b-ΔlacI-
1078_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-PpstsIH-

Aazu

BXE_019
BX_926
583_pET28b-ΔlacI-
1079_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-PpstsIH-

Msme

BXE_020
BX_926
583_pET28b-ΔlacI-
1080_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-PpstsIH-

Ajun

BXE_021
BX_926
583_pET28b-ΔlacI-
1081_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-PpstsIH-

M360.2

BXE_022
BX_926
583_pET28b-ΔlacI-
1082_pACYC-PpstsIH-

PpstsIH-fadBA
nphT7-ter-PpstsIH-

ACIP

Small-scale evaluations were completed for each strain cultured at OD₆₀₀=2.0 in a 20 mL capped glass test tube with a working volume of 2 mL. Samples were taken at 24 hours by removing 1 mL of the culture for growth and glucose measurement and extracting the remaining broth with MTBE prior to analyzing for FAME production by GC-MS. As shown in FIG. 7, methyl laurate titers ranging from 0.04-0.5 g/L were observed, indicating modest in vivo WES activity.

Specificity of product formation observed in vivo was significantly different than what would be predicted based upon the in vitro assays. This discrepancy may have been due to the production profile of the thiolase FFA strains, which produced nearly equivalent titers from C6 to C16 FFA. Due to the limitations of screening the wax ester synthase candidates in the thiolase strain, subsequent in vivo characterization was performed in synthase-based strains, which produced C12 FFA and FAME products at higher specificity, rate and titer.

WES High-Throughput Screening

A 96-well plate, Nile red-based assay has been developed for high-throughput quantification of FAME production by fluorescence. Mutant libraries were constructed with Maqu WES. The top 20 mutants identified in the screen have been isolated, sequenced, cloned into production hosts and evaluated for FAME and FFA production in the 1 mL method. While comprehensive data analysis for the 1 mL confirmations is pending, initial results showed significantly improved FAME production with at least two WES constructs identified by this method. As demonstrated in FIG. 17, both mutations resulted in improved methyl laurate production when compared with the control strain, while maintaining a high degree of specificity.

Similarly, mutants of Mhyd were made and the methyl laurate production determined. The results are shown in FIG. 16.

Example 3

Construction and Evaluation of Methyl Laurate Producing Strains

Engineering of Strains for Methyl Laurate Production

Numerous methyl laurate production strains have been constructed, incorporating key pathway modules developed in Examples 1 and 2 above and building upon the malonyl-CoA production technology as described in WO2014026162A1 (OPX Biotechnologies Inc., USA). Small-scale evaluations were completed for each strain cultured at OD₆₀₀=2.0 in a 20 mL capped glass test tube with a working volume of 1 mL. Samples were taken at 24 hours by extracting the entire culture with MTBE and analyzing for FAME production by GC-MS. This 1 mL protocol was instituted in an effort to reduce volatile loss of FAME products seen during shake flask evaluations.

TABLE 5

methyl laurate production strains used in Example 3

Strain
Base strain
Plasmid 1
Plasmid 2

BXE_233
BX_1018
pET_PpstsIH-Aagr-PtpiA-
pACYC-PpstsIH-nphT7(SV)-ter-

accDA-PrpiA-accB-accC
PpstsIH-fadB-PphoE-Mhyd

BXE_275
BX_1018
pET_PpstsIH-Aagr-PtpiA-
pACYC-PpstsIH-nphT7(SV)-ter-

accDA-PrpiA-accB-accC
PpstsIH-fadB-PphoE-Aazu

BXE_276
BX_1018
pET_PpstsIH-Aagr-PtpiA-
pACYC-PpstsIH-nphT7(SV)-ter-

accDA-PrpiA-accB-accC
PpstsIH-fadB-PphoE-Hche

BXE_279
BX_1018
pET_PpstsIH-Aagr-PtpiA-
pACYC-PpstsIH-nphT7(SV)-ter-

accDA-PrpiA-accB-accC
PpstsIH-fadB-PphoE-Maqu

In FIG. 8, 24 hour FAME production data is shown for 50 of the >100 methyl laurate producing strains screened in the 1 ml assay since the May SCM. In this strain set, titers >1 g/L have been obtained in 20 hours of production with biomass levels ˜1 gDCW/L. Furthermore, specificities between 70-98% on a FAME basis have been observed.

FIG. 15 shows the 1 mL screening results for strains expressing four WES candidates shown in Table 5. These results show that varying the WES leads to significant changes in methyl laurate production. Furthermore, Hche showed high levels of production. FIG. 18 also shows that while the majority of the activities (FabH, FadB, Ter) were not significantly different between the two hosts, BXE_233 and BXE_062, the WES activity was nearly three-fold higher in BXE_233 when compared with BXE_062. The significant differences in methyl laurate production seen with differing WES candidates, and with different expression constructs for the same WES enzyme, support the hypotheses that WES activity remains limiting in current production strains.

Example 4

Fermentation Method Development and Optimization for Methyl Laurate Production

Initial Development of a 1 L Bioprocess for Methyl Laurate Production

Development of a 1 L bioprocess for methyl laurate was carried out to support evaluation of production strains under pH-controlled conditions and at biomass concentrations more representative of commercial production.

Several process parameters (Table 6) were explored, including pH setpoint, methanol feeding, temperature profile and incorporation of a second phase for increased FAME recovery.

A subset of the data generated is shown in FIG. 9. Overall, the methanol feeding and control strategy did not appear to have a significant impact on methyl laurate production or overall fermentation performance. The inclusion of a second phase appeared to be a slight, positive effect on FAME recovery. The incorporation of the 35° C. to 37° C. temperature shift profile showed a positive effect on production, Based on these results, the following baseline process as shown in Table 7 was established and utilized for the subsequent 1 L fermentations and evaluations of methyl laurate producing strains engineered in Example 3.

TABLE 6

Several process parameters used in Example 4

Production

Methanol Feed
2^nd
Temperature
Production

Condition
Profile
Phase
Profile
pH
Medium

Control
1% (v/v) MeOH
N/A
37° C.
7.4
FM12 + 9 mM

added at TS; bolus

phosphate

additions every 8-12

hours to

maintain 1%

MeC14
1% (v/v) MeOH
50 g/L
37° C.
7.4
FM12 + 9 mM

FAME
added at TS; bolus
C14

phosphate

additions every 8-12
FAME

hours to

maintain 1%

35 C. to
1% (v/v) MeOH
N/A
35° C. →37° C.
7.4
FM12 + 9 mM

37 C.
added at TS; bolus

phosphate

additions every 8-12

hours to

maintain 1%

MeOH
0.5% (v/v) MeOH
N/A
37° C.
7.4
FM12 + 9 mM

Feed
added at TS; bolus

phosphate

additions every 8-12

hours to

maintain 0.5%

pH 7
1% (v/v) MeOH
N/A
37° C.
7.4
FM12 + 9 mM

added at TS; bolus

phosphate

additions every 8-12

hours to

maintain 1%

TABLE 7

Baseline process used in fermentation of methyl laurate producing strains

engineered in Example 3

Production

Methanol Feed

Temperature
Production

Condition
Profile
2^ndPhase
Profile
pH
Medium

Baseline
1% (v/v) MeOH
50 g/L C14
35° C. →37° C.
7
FM12 +

Process
added at TS;
FAME

9 mM

bolus additions at

phosphate

every 8-12 hours

to maintain 1%

BXE_276 strain was tested as mentioned above. The average production time course for the triplicate BXE_276 was run. Fairly constant methyl laurate production rates were observed over 36.5 hours with an average titer of 3 g/L methyl laurate. Interestingly, this strain produced a significant amount of methyl decanoate (3-5 g/L), resulting in significantly higher total FAME production than observed with previous strains (FIG. 19). BXE_276 showed higher specificity for production of methyl decanoate than methyl laurate in 1 L, which is in contrast to the higher specificity demonstrated in 1 mL for methyl laurate production (69%).

REFERENCES

Heath et al., Prog. Lipid Res. 40(6):467-97 (2001)

McCue et al., Nucleic Acids Res., 29(3):774-82 (2001)

Zhang et al., J. Biol. Chem. 277 (18):15558-65 (2002)

Chang et al., J. Bacteriol. 154(2):756-62 (1983)

Abdel-Ahmid et al., Microbiol. 147(6):2001

Mat-Jan et al., J. Bacteriol. 171(1):342-8

Bunch et al., Microbiol. 143(1):187-95 (1997)

Enoyl-Acyl Carrier Protein (fabI) Plays a Determinant Role in Completing Cycles of Fatty Acid Elongation in Escherichia coli,” Richard J. Heath and Charles 0. Rock, J. Biol. Chem. 270:44, pp. 26538-26543 (1995),

Beguin, P and Aubert, J-P (1994) FEMS Microbial. Rev. 13: 25-58

Ohima, K. et al. (1997) Biotechnol. Genet. Eng. Rev. 14: 365414.

Antonenkov, V., D. et al. Van Veldhoven, P., P., Waelkens, E., and Mannaerts, G. P. J. Biol. Chem. 1997, 272:26023-26031.

Reed, 1981

Arthur Lesk (2008), Introduction to bioinformatics, 3^rdedition,

Thompson et al., Nucleic Acids Research 22, 4637-4680, 1994,

Katoh et al., Genome Information, 16(1), 22-33, 2005.

U.S. provisional patent application No. 62/044,621 filed Sep. 2, 2015, is incorporated herein by reference.

Numerous modifications and variations on the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

1. A microbial cell for producing at least one fatty acid ester, wherein the cell comprises: (i) a first genetic mutation that enables the cell to produce at least one fatty acyl coenzyme A (CoA) intermediate by an increase in the expression of at least one enzyme selected from the group consisting of acetoacetyl-CoA synthase, ketoacyl-CoA synthase (or elongase), ketoacyl-CoA thiolase, enoyl-CoA reductase, ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydratase relative to a wild type cell; and (ii) a second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell, wherein the wax ester synthase has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO: 1, at least 80% to a polypeptide set forth in SEQ ID NO: 2, at least 85% to a polypeptide as set forth in SEQ ID NO: 3, at least 85% to a polypeptide as set forth in SEQ ID NO: 4, at least 90% to a polypeptide as set forth in SEQ ID NO: 5, at least 95% to a polypeptide as set forth in SEQ ID NO: 7, or at least 98% to a polypeptide as set forth in SEQ ID NO: 8, and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of the fatty acyl coenzyme A intermediate to a fatty acid ester.
2. The cell according to claim 1, wherein the at least one fatty acyl coenzyme A (CoA) intermediate comprises a C4 to C10 acyl-CoA substrate.
3. The cell according to claim 1, further comprising a genetic mutation that results in a decrease in the expression of acetoacetyl-CoA thiolase relative to the wild type cell.
4. The cell according to claim 1, wherein the cell comprises a third genetic mutation that reduces the fatty acid degradation capacity of the cell relative to the wild type cell.
5. The cell according to claim 4, wherein the third genetic mutation results in a decrease in the expression of at least one enzyme selected from the group consisting of acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase, each relative to the wild type cell.
6. The cell according to claim 1, wherein the cell is genetically modified to increase the expression of β-ketoacyl-ACP synthase III (fabH) relative to the wild type cell.
7. The cell according to claim 6, wherein the β-ketoacyl-ACP synthase III (fabH) has sequence identity of at least 85% to a polypeptide selected from the group consisting of SEQ ID NOs: 24-27 and combinations thereof.
8. The cell according to claim 1, wherein the cell is genetically modified to increase the expression of 3-hydroxyacyl coenzyme A dehydratase (3HCDh) and/or keto acyl-CoA reductase (KCR) relative to the wild type cell.
9. The cell according to claim 8, wherein the 3HCDh is crotonase/enoyl-CoA hydratase (Crt) and the KCR is hydroxybutyric dehydrogenase (Hbd).
10. The cell according to claim 9, wherein the Crt has sequence identity of at least 85% to a polypeptide as set forth in SEQ ID NO:28 and/or the Hbd has sequence identity of at least 85% to a polypeptide of SEQ ID NO:29.
11. The cell according to claim 1, wherein the fatty acid ester is a fatty acid methyl ester (FAME) and/or a fatty acid ethyl ester (FAEE).
12. The cell according to claim 1, wherein the fatty acid ester is produced in the presence of at least one exogenous alcohol selected from the group consisting of methanol, ethanol, propanol, butanol, and pentanol.
13. The cell according to claim 1, wherein the cell is a yeast cell, a fungi cell, or a bacteria cell.
14. The cell according to claim 13, wherein the cell is a selected from the group consisting of Pseudomonas putida, Escherichia coli, and Saccharomyces cerevisiae.
15. A method for producing fatty acid ester, the method comprising: contacting at least one fatty acyl coenzyme A (CoA) intermediate with an isolated wax ester synthase that has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO: 1, at least 85% to a polypeptide as set forth in SEQ ID NO: 3, at least 85% to a polypeptide as set forth in SEQ ID NO: 4, at least 90% to a polypeptide as set forth in SEQ ID NO: 5, at least 95% to a polypeptide as set forth in SEQ ID NO: 7, or at least 98% to a polypeptide as set forth in SEQ ID NO: 8.
16. A method for producing at least one fatty acid ester, the method comprising culturing a microbial cell which comprises: (i) a first genetic mutation that enables the cell to produce at least one fatty acyl coenzyme A (CoA) intermediate by an increase in the expression of at least one enzyme selected from the group consisting of acetoacetyl-CoA synthase, ketoacyl-CoA synthase (or elongase), ketoacyl-CoA thiolase, enoyl-CoA reductase, ketoacyl-CoA reductase and 3-hydroxyacyl-CoA dehydratase relative to a wild type cell; and (ii) a second genetic mutation that increases the activity of at least one wax ester synthase in the cell relative to the wild type cell, wherein the wax ester synthase has sequence identity of at least 80% to a polypeptide set forth in SEQ ID NO: 1, at least 80% to a polypeptide set forth in SEQ ID NO: 2, at least 85% to a polypeptide as set forth in SEQ ID NO: 3, at least 85% to a polypeptide as set forth in SEQ ID NO: 4, at least 90% to a polypeptide as set forth in SEQ ID NO: 5, at least 95% to a polypeptide as set forth in SEQ ID NO: 7, or at least 98% to a polypeptide as set forth in SEQ ID NO: 8, and combinations thereof or to a functional fragment of any of the polypeptides for catalyzing the conversion of the fatty acyl coenzyme A intermediate to a fatty acid ester.
17. The cell according to claim 16, wherein the fatty acid ester is a fatty acid methyl ester (FAME) and/or a fatty acid ethyl ester (FAEE).
18. The cell according to claim 16, wherein the fatty acid ester is produced in the presence of at least one exogenous alcohol selected from the group consisting of methanol, ethanol, propanol, butanol, and pentanol.

FIELD OF THE INVENTION

This application is a continuation of U.S. application Ser. No. 14/843,525, filed Sep. 2, 2015, and entitled PRODUCTION OF FATTY ACIDS ESTERS, which claims the benefit of U.S. Provisional Application No. 62/044,621, filed Sep. 2, 2014, and entitled PRODUCTION OF FATTY ACIDS ESTERS, each of which is hereby incorporated by reference in its entirety. The present invention relates to a biotechnological method and cell for producing at least one fatty acid ester from a sugar.

US Referenced Citations (256)

Number	Name	Date	Kind
2408889	Short et al.	Oct 1946	A
2464768	Griffin et al.	Mar 1949	A
2469701	Redmon et al.	May 1949	A
2798053	Brown et al.	Jul 1957	A
3687885	Murray et al.	Aug 1972	A
3872037	MacLeod	Mar 1975	A
3875101	MacLeod	Apr 1975	A
3891591	Chang et al.	Jun 1975	A
3904685	Shahidi	Sep 1975	A
3915921	Schlatzer	Oct 1975	A
4029577	Godlewski et al.	Jun 1977	A
4268641	Koenig et al.	May 1981	A
4301266	Muenster et al.	Nov 1981	A
4431547	Dubin et al.	Feb 1984	A
4666983	Tsubakimoto et al.	May 1987	A
4685915	Hasse et al.	Aug 1987	A
4708997	Stanley et al.	Nov 1987	A
4734478	Tsubakimoto et al.	Mar 1988	A
4857610	Pauen et al.	Aug 1989	A
4952505	Cho	Aug 1990	A
4985518	Alexander et al.	Jan 1991	A
5009653	Osborn	Apr 1991	A
5093472	Bresciani et al.	Mar 1992	A
5135677	Yamaguchi et al.	Aug 1992	A
5145906	Chambers et al.	Sep 1992	A
5180798	Nakamura et al.	Jan 1993	A
5252474	MacNeil et al.	Oct 1993	A
5274073	Gruber et al.	Dec 1993	A
5331059	Engelhardt et al.	Jul 1994	A
5342899	Graham et al.	Aug 1994	A
5350799	Woodrum et al.	Sep 1994	A
5426199	Lundquist	Jun 1995	A
5470928	Harwood et al.	Nov 1995	A
5487989	Fowler et al.	Jan 1996	A
5510307	Narayanan et al.	Apr 1996	A
5510526	Baniel et al.	Apr 1996	A
5558656	Bergman	Sep 1996	A
5616496	Draths et al.	Apr 1997	A
5723639	Datta et al.	Mar 1998	A
5817870	Haas et al.	Oct 1998	A
5827255	Crainic	Oct 1998	A
5876983	Suzuki et al.	Mar 1999	A
6004773	Yoshihara et al.	Dec 1999	A
6013494	Picataggio et al.	Jan 2000	A
6087140	Cameron et al.	Jul 2000	A
6117658	Dennis et al.	Sep 2000	A
6143538	Somerville et al.	Nov 2000	A
6284495	Sato et al.	Sep 2001	B1
6297319	Nagasuna et al.	Oct 2001	B1
6306636	Haselkorn et al.	Oct 2001	B1
6355412	Stewart et al.	Mar 2002	B1
6472188	Lee et al.	Oct 2002	B1
6489508	Van Gansbeghe et al.	Dec 2002	B1
6509156	Stewart et al.	Jan 2003	B1
6534679	Eyal et al.	Mar 2003	B2
6586229	Ben-Bassat et al.	Jul 2003	B1
6593116	Huisman et al.	Jul 2003	B1
6623944	Rieping	Sep 2003	B2
6709919	Tu et al.	Mar 2004	B2
6723799	Higley et al.	Apr 2004	B2
6852517	Suthers et al.	Feb 2005	B1
6960455	Akhverdian et al.	Nov 2005	B2
7090998	Ishikawa et al.	Aug 2006	B2
7118896	Kalscheuer et al.	Oct 2006	B2
7141154	Lin et al.	Nov 2006	B2
7153663	Payne et al.	Dec 2006	B2
7166743	Whitehouse et al.	Jan 2007	B2
7186541	Buckel et al.	Mar 2007	B2
7186856	Meng et al.	Mar 2007	B2
7223567	Sanchez et al.	May 2007	B2
7279598	Meng et al.	Oct 2007	B2
7285406	Payne et al.	Oct 2007	B2
7309597	Gokarn et al.	Dec 2007	B2
7326557	San et al.	Feb 2008	B2
7358071	Payne et al.	Apr 2008	B2
7393676	Buckel et al.	Jul 2008	B2
7524660	Caimi et al.	Apr 2009	B2
7538247	Craciun et al.	May 2009	B2
7638316	Buckel et al.	Dec 2009	B2
7678869	Matyjaszewski et al.	Mar 2010	B2
7687661	Lilga et al.	Mar 2010	B2
7803620	Zirkle et al.	Sep 2010	B2
7826975	Maranas et al.	Nov 2010	B2
7833761	Terashita et al.	Nov 2010	B2
7846688	Gill et al.	Dec 2010	B2
7943362	Frost et al.	May 2011	B2
7987056	Gill et al.	Jul 2011	B2
8048624	Lynch et al.	Nov 2011	B1
8076111	Fukui et al.	Dec 2011	B2
8097439	Alibhai et al.	Jan 2012	B2
8110093	Friedman et al.	Feb 2012	B2
8110670	Valle et al.	Feb 2012	B2
8183028	Schirmer et al.	May 2012	B2
8268599	Schirmer et al.	Sep 2012	B2
8283143	Valle et al.	Oct 2012	B2
8313934	Bhatia et al.	Nov 2012	B2
8323924	Schirmer et al.	Dec 2012	B2
8372610	Haliburton et al.	Feb 2013	B2
8377666	Niu et al.	Feb 2013	B2
8467975	Ryan et al.	Jun 2013	B2
8530221	Hu et al.	Sep 2013	B2
8535916	Del et al.	Sep 2013	B2
8597922	Rude et al.	Dec 2013	B2
8652816	Lynch et al.	Feb 2014	B2
8658404	Rude et al.	Feb 2014	B2
8753840	Vermaas et al.	Jun 2014	B2
8809027	Mercogliano et al.	Aug 2014	B1
8835137	Cross et al.	Sep 2014	B2
8859259	Rude	Oct 2014	B2
8883464	Gill et al.	Nov 2014	B2
9388419	Gill et al.	Jul 2016	B2
9428778	Gill et al.	Aug 2016	B2
9447438	Louie et al.	Sep 2016	B2
9587231	Trinh et al.	Mar 2017	B2
20020081684	Grobler et al.	Jun 2002	A1
20020164729	Skraly et al.	Nov 2002	A1
20030004375	Mizrahi et al.	Jan 2003	A1
20030087381	Gokarn et al.	May 2003	A1
20030101486	Facciotti et al.	May 2003	A1
20030158441	Zhong et al.	Aug 2003	A1
20030159175	Ghulam et al.	Aug 2003	A1
20030191146	Kabbash et al.	Oct 2003	A1
20030211131	Martin et al.	Nov 2003	A1
20030233675	Cao et al.	Dec 2003	A1
20030235892	Katz et al.	Dec 2003	A1
20040009466	Maranas et al.	Jan 2004	A1
20040076982	Gokarn et al.	Apr 2004	A1
20040077090	Short	Apr 2004	A1
20040152159	Causey et al.	Aug 2004	A1
20040152174	Cervin et al.	Aug 2004	A1
20040209337	Frost et al.	Oct 2004	A1
20040210087	Meng et al.	Oct 2004	A1
20040214294	Rieping et al.	Oct 2004	A1
20050003481	Gabriel et al.	Jan 2005	A1
20050054060	Chateau et al.	Mar 2005	A1
20050196758	Rock et al.	Sep 2005	A1
20050221457	Tsobanakis et al.	Oct 2005	A1
20050221466	Liao et al.	Oct 2005	A1
20050222458	Craciun et al.	Oct 2005	A1
20050233031	Hughes et al.	Oct 2005	A1
20050239179	Skraly et al.	Oct 2005	A1
20050272135	Datta et al.	Dec 2005	A1
20050283029	Meng et al.	Dec 2005	A1
20060014977	Miller et al.	Jan 2006	A1
20060068468	Knopf et al.	Mar 2006	A1
20060084098	Gill et al.	Apr 2006	A1
20060166342	Taoka et al.	Jul 2006	A1
20070010708	Ness et al.	Jan 2007	A1
20070027342	Meng et al.	Feb 2007	A1
20070031918	Dunson et al.	Feb 2007	A1
20070087403	Bestel-Corre et al.	Apr 2007	A1
20070092957	Donaldson et al.	Apr 2007	A1
20070107080	Liao et al.	May 2007	A1
20070141574	Keasling et al.	Jun 2007	A1
20070148749	Yasuda et al.	Jun 2007	A1
20070184524	Gokarn et al.	Aug 2007	A1
20070219390	Zacher et al.	Sep 2007	A1
20070245431	Metz et al.	Oct 2007	A1
20070270494	Metz et al.	Nov 2007	A1
20070281343	Arslanian	Dec 2007	A9
20080076167	Gokarn et al.	Mar 2008	A1
20080124785	Liao et al.	May 2008	A1
20080182308	Donaldson et al.	Jul 2008	A1
20080193989	Verser et al.	Aug 2008	A1
20080199926	Burgard et al.	Aug 2008	A1
20080274523	Renninger et al.	Nov 2008	A1
20090017514	Datta et al.	Jan 2009	A1
20090023006	Bub et al.	Jan 2009	A1
20090031453	Jessen et al.	Jan 2009	A1
20090053783	Gokarn et al.	Feb 2009	A1
20090076297	Bogan et al.	Mar 2009	A1
20090082286	Huang et al.	Mar 2009	A1
20090111151	Julien et al.	Apr 2009	A1
20090148914	Causey et al.	Jun 2009	A1
20090203097	Flint et al.	Aug 2009	A1
20090234146	Cooney et al.	Sep 2009	A1
20090246141	Hirai et al.	Oct 2009	A1
20090291480	Jessen et al.	Nov 2009	A1
20090298144	Tsobanakis et al.	Dec 2009	A1
20090305369	Donaldson et al.	Dec 2009	A1
20090325248	Marx et al.	Dec 2009	A1
20100021978	Burk et al.	Jan 2010	A1
20100028962	Hu et al.	Feb 2010	A1
20100037329	Frommer et al.	Feb 2010	A1
20100064381	Zou et al.	Mar 2010	A1
20100068773	Eggeling et al.	Mar 2010	A1
20100099910	Meng et al.	Apr 2010	A1
20100113822	Craciun et al.	May 2010	A1
20100151536	Baynes et al.	Jun 2010	A1
20100170148	Steen et al.	Jul 2010	A1
20100186117	Fabijanski et al.	Jul 2010	A1
20100210017	Gill et al.	Aug 2010	A1
20100257777	Sanchez-Riera et al.	Oct 2010	A1
20100257778	Gaertner et al.	Oct 2010	A1
20100261239	Soucaille et al.	Oct 2010	A1
20100274033	Sanchez-Riera et al.	Oct 2010	A1
20100285549	Muramatsu et al.	Nov 2010	A1
20100291644	Marx et al.	Nov 2010	A1
20110020883	Roessler et al.	Jan 2011	A1
20110038364	Monsieux et al.	Feb 2011	A1
20110072714	Gaertner et al.	Mar 2011	A1
20110089016	Winkelaar et al.	Apr 2011	A1
20110124063	Lynch et al.	May 2011	A1
20110125118	Lynch et al.	May 2011	A1
20110144377	Eliot et al.	Jun 2011	A1
20110159558	Grady et al.	Jun 2011	A1
20110162259	Gaertner et al.	Jul 2011	A1
20110171702	Reinecke et al.	Jul 2011	A1
20110183382	Schmalisch et al.	Jul 2011	A1
20110183388	Sabirova et al.	Jul 2011	A1
20110183391	Frost et al.	Jul 2011	A1
20110190513	Lynch et al.	Aug 2011	A1
20110214979	Chen et al.	Sep 2011	A1
20110244575	Lipscomb et al.	Oct 2011	A1
20110275851	Orjuela et al.	Nov 2011	A1
20110281314	Lynch et al.	Nov 2011	A1
20120041232	Lynch et al.	Feb 2012	A1
20120058530	Zhang et al.	Mar 2012	A1
20120116108	Basu et al.	May 2012	A1
20120129231	Wang et al.	May 2012	A1
20120135481	Jessen et al.	May 2012	A1
20120240289	Feussner et al.	Sep 2012	A1
20120244586	Gokarn et al.	Sep 2012	A1
20120244588	Park et al.	Sep 2012	A1
20120264902	Lipscomb et al.	Oct 2012	A1
20120329110	Kim et al.	Dec 2012	A1
20130071893	Lynch et al.	Mar 2013	A1
20130078684	Holtzapple et al.	Mar 2013	A1
20130078686	Holtzapple et al.	Mar 2013	A1
20130122541	Lynch et al.	May 2013	A1
20130122562	Aldor et al.	May 2013	A1
20130189787	Lynch et al.	Jul 2013	A1
20130316413	Gonzalez et al.	Nov 2013	A1
20130345470	Tengler et al.	Dec 2013	A1
20140051136	Liao et al.	Feb 2014	A1
20140121118	Warner	May 2014	A1
20140135526	Lynch et al.	May 2014	A1
20140215904	Pandey et al.	Aug 2014	A1
20140242648	Ochiai et al.	Aug 2014	A1
20140309451	Tengler et al.	Oct 2014	A1
20140330032	Trahan et al.	Nov 2014	A1
20150044746	Meerman et al.	Feb 2015	A1
20150056651	Gill et al.	Feb 2015	A1
20150056669	Louie et al.	Feb 2015	A1
20150056684	Gill et al.	Feb 2015	A1
20150057455	Harkrader et al.	Feb 2015	A1
20150064754	Louie et al.	Mar 2015	A1
20150072384	Mercogliano et al.	Mar 2015	A1
20150072399	Lipscomb et al.	Mar 2015	A1
20150119601	Louie et al.	Apr 2015	A1
20150299679	Shumaker et al.	Oct 2015	A1
20160060663	Grammann et al.	Mar 2016	A1
20160090576	Garg et al.	Mar 2016	A1
20160257975	Lynch et al.	Sep 2016	A1
20160340700	Liao et al.	Nov 2016	A1
20160362710	Lee et al.	Dec 2016	A9

Foreign Referenced Citations (113)

Number	Date	Country
2520795	Oct 2004	CA
2591599	Jul 2006	CA
2654133	Dec 2007	CA
101573451	Nov 2009	CN
101679924	Mar 2010	CN
102008002309	Dec 2009	DE
1124789	Sep 2004	EP
1036190	May 2005	EP
1305439	Jun 2006	EP
1124979	Aug 2006	EP
1731604	Dec 2006	EP
1105514	Feb 2008	EP
1778840	Jun 2008	EP
1975236	Oct 2008	EP
1654212	Jul 2009	EP
2133420	Dec 2009	EP
1706457	Feb 2012	EP
3103867	Dec 2016	EP
2473755	Sep 2011	GB
2007096348	Oct 2007	KR
20120108538	Oct 2012	KR
9821339	May 1998	WO
9855442	Dec 1998	WO
9914343	Mar 1999	WO
0039287	Jul 2000	WO
0056693	Sep 2000	WO
0061740	Oct 2000	WO
0116346	Mar 2001	WO
0138284	May 2001	WO
0208428	Jan 2002	WO
0234784	May 2002	WO
0242418	May 2002	WO
02090312	Nov 2002	WO
03040690	May 2003	WO
03062173	Jul 2003	WO
03082795	Oct 2003	WO
2004018621	Mar 2004	WO
2004033646	Apr 2004	WO
2005003074	Jan 2005	WO
2005047498	May 2005	WO
2005105770	Nov 2005	WO
2005118719	Dec 2005	WO
2006034156	Mar 2006	WO
2006052871	May 2006	WO
2006052914	May 2006	WO
2006121755	Nov 2006	WO
2007012078	Jan 2007	WO
2007030830	Mar 2007	WO
2007042494	Apr 2007	WO
2007047680	Apr 2007	WO
2007093848	Aug 2007	WO
2007106903	Sep 2007	WO
2007130745	Nov 2007	WO
2007136762	Nov 2007	WO
2008021765	Feb 2008	WO
2008023039	Feb 2008	WO
2008027742	Mar 2008	WO
2008028002	Mar 2008	WO
2008072920	Jun 2008	WO
2008089102	Jul 2008	WO
2008091627	Jul 2008	WO
2008145737	Dec 2008	WO
2008149951	Dec 2008	WO
2009006430	Jan 2009	WO
2009031737	Mar 2009	WO
2009036095	Mar 2009	WO
2009062190	May 2009	WO
2009089457	Jul 2009	WO
2009094485	Jul 2009	WO
2009111513	Sep 2009	WO
2009111672	Sep 2009	WO
2009121066	Oct 2009	WO
2009143401	Nov 2009	WO
2009151342	Dec 2009	WO
2010006076	Jan 2010	WO
2010011874	Jan 2010	WO
2010017230	Feb 2010	WO
2010031083	Mar 2010	WO
2010105095	Sep 2010	WO
2011002892	Jan 2011	WO
2011008565	Jan 2011	WO
2011038364	Mar 2011	WO
2011063304	May 2011	WO
2011063363	May 2011	WO
2011094457	Aug 2011	WO
2012017083	Feb 2012	WO
2012019175	Feb 2012	WO
2012050931	Apr 2012	WO
2012054400	Apr 2012	WO
2012129450	Sep 2012	WO
2012135760	Oct 2012	WO
2012177726	Dec 2012	WO
2013003608	Jan 2013	WO
2013039563	Mar 2013	WO
2013126855	Aug 2013	WO
2013152051	Oct 2013	WO
2013152052	Oct 2013	WO
2013192450	Dec 2013	WO
2013192451	Dec 2013	WO
2013192453	Dec 2013	WO
2014026162	Feb 2014	WO
2014042693	Mar 2014	WO
2014145096	Sep 2014	WO
2014145297	Sep 2014	WO
2014145332	Sep 2014	WO
2014145334	Sep 2014	WO
2014145343	Sep 2014	WO
2014145344	Sep 2014	WO
2014146026	Sep 2014	WO
2014146047	Sep 2014	WO
2014198831	Dec 2014	WO
2015010103	Jan 2015	WO
2015042626	Apr 2015	WO

Non-Patent Literature Citations (299)

Entry
Brosius, Jurgen , et al., “Spacing of the -10 and -35 Regions in the tac Promoter. Effect on its in vivo activity”, J Biol Chem. 260(6), Mar. 25, 1985, 3539-41.
Broun, Pierre , et al., “Catalytic Plasticity of Fatty Acid Modification Enzymes Underlying Diversity of Plant Lipids”, Science. 282(5392), Nov. 13, 1998, 1315-7.
Brown . et al., “Synthesis of labeled acrylamide and N-methylolacrylamide (NMA): 15N-acrylamide, 13C-NMA, 15N-NMA, and 13C, 15N-NMA”, Journal of labelled compounds & radiopharmaceuticals. 48(14):1031-1039., Nov. 14, 2005.
Brutlag, Douglas L., et al., “Improved sensitivity of biological sequence database searches”, Comput Appl Biosel. 6(3), Mar. 25, 1990, 237-45.
Bunch , et al., “The IdhA gene encoding the fermentative dehydrogenase of Escherichia coli”, Microbiology, Jan. 1997;143 ( Pt 1) 187-95.
Canada , et al., “Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation”, J Bacteriol. Jan. 2002;184(2):344-9.
Chang , et al., “Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival”, J Bacteriol. Nov. 1999;181(21): 6656-63.
Chang , et al., “Probable polyketide synthase/thioesterase. NCBI Direct Submission, Accession No. GI50082961”, Jun. 14, 2004.
Chao , et al., “Selective production of L-aspartic acid and L-phenylalanine by coupling reaction of aspartase and aminotransferase in Escherichia coli”, Enzyme Microb Technol, 27(1-2), Jul. 1, 2000, 19-25.
Cheng , et al., “Mammalian wax biosynthesis: I. Identification of two fatty acyl-Coenzyme A reductases with different substrate specificities and tissue distributions”, Journal of Biological Chemistry, Sep. 3, 2004, vol. 279, No. 36, pp. 37789-37797.
Chica, Roberto A., et al., “Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design”, Curr Opin Biotechnol. 16(4), Aug. 2005, 378-84.
Cho , et al., “Simultaneous synthesis of enantiomerically pure (S)-amino acids and (R)¬amines using coupled transaminase reactions”, Biotechnol Bioeng. Mar. 30, 2003;81(7):783-9.
Choi-Rhee, Eunjoo , et al., “The Biotin Caroxylase—Biotin Carboxyl Carrier Protein Complex of Escherichia coli Acetyl-CoA Carboxylase”, J Biol Chem. 278(33), Aug. 15, 2003, 30806-12.
Chotani , et al., “The commercial production of chemicals using pathway engineering”, Biochim Biophys Acta. Dec. 29, 2000;1543(2):434-455.
Cleusix , et al., “Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria”, BMC Microbiology, 7: 101, Nov. 12, 2007, 9 Pages.
Coleman, Rosalind A., et al., “Enzymes of triacylglycerol synthesis and their regulation”, Prog Lipid Res. 43(2), Mar. 2004, 134-76.
Cowan, Peter J., et al., “Characterization of the Major Promoter for the Plasmid-Encoded Sucrose Genes scrY, scrA, and scrB”, J Bacteriol. 173(23), Dec. 1991, 7464-70.
Crameri, et al., “DNA shuffling of a family of genes from diverse species accelerates directed evolution”, Nature. Jan. 15, 1998:391(6664) 288-91.
Cronan , et al., “Genetric and biochemical analyses of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium.”, J Bacteriol. Mar. 1982;149(3):916-22.
Cronan, J.E , “Beta-Alanine Synthesis in Escherichia coli”, J Bacteriol. Mar. 1980;141(3):1291-7.
Cronk , et al., “Cloning, crystallization and preliminary characterization of a beta-carbonic anhydrase from Escherichia coli”, Acta Crystallogr D Biol Crystallogr. Sep. 2000;56(Pt 9):1176-9.
Daley, Daniel O., et al., “Global Topology Analysis of the Escherichia coli Inner Membrane Proteome”, Science. 308(5726), May 27, 2005, 1321-3.
Daniel, Jaiyanth , et al., “Induction of a Novel Class of Diacylglycerol Acyltransferases and Triacylglycerol Accumulation in Mycobacterium tuberculosis as It Goes into a Dormancy-Like State in Culture”, J Bacteriol. 186(15), Aug. 2004, 5017-30.
Daruwala , et al., “Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli”, J. Bacteriol. 179(10), May 1997, 3133-8.
Datsenko, Kirill A., et al., “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products”, Proc Natl Acad Sci USA. 97(12), Jun. 6, 2000, 6640-5.
Datta, Simanti , et al., “A set of recombineering plasmids for gram-negative bacteria”, Gene, 379, Sep. 1, 2006, 109-15.
Davis, Mark S., et al., “Overproduction of Acetyl-CoA Carboxylase Activity Increases the Rate of Fatty Acid Biosynthesis in Escherichia coli”, The Journal of Biological Chemistry (2000), vol. 275, pp. 28593-28598, 2000, 28593-28598.
De Boer, Herman A., et al., “The tac promoter: A functional hybrid derived from the trp and lac promoters”, Proc Natl Acad Sci USA. 80(1), Jan. 1983, 21-5.
De Mendoza , et al., “Thermal regulation of membrane lipid fluidity in bacteria”, Trends Biochem. Sci. 1983; 8:49-52.
Dell'Aquila , et al., “Acid-base balance in peritoneal dialysis”, J Nephrol. Mar.-Apr. 2006;19 Suppl 9:S104-7.
Dellomonaco , et al., “Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals”, Nature Aug. 18, 2011, 476 (7360): 355-9.
Demmer, Ulrike , et al., “Structural Basis for a Bispecific NADP and CoA Binding Site in an Archaeal Malonyl-Coenzyme A Reductase”, J Biol Chem. 288(9), Mar. 1, 1990, 6363-70.
Den , et al., “Enzymatic Conversion of13-Hydroxypropionate to Malonic Semialdehyde*”, J Biol Chem Jul. 1959;234(7):1666-1671.
Denic , et al., “A Molecular Caliper Mechanism for Determining Very Long-Chain Fatty Acid Length”, vol. 130, Issue 4, Aug. 24, 2007, Aug. 24, 2007, 663-377.
Deshpande, Mukund V., “Ethanol Production from Cellulose by Coupled Saccharification/Fermenation using Saccharomyces cerevisiae and Cellulase Complez from Sclerotium rolfsii UV-8 Mutant”, Appl Biochem Biotechnol. 36(3), 1992, 227-34.
Devos, Damien , et al., “Practical Limits of Function Prediction”, Proteins. 41(1), Oct. 1, 2000, 98-107.
Dewick, P. , “Chapter 4, The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids”, Medicinal Natural Products: A Biosynthetic Approach. Second Edition (2002): 121-166.
Diaz , et al., “Characterization of the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid in Escherichia coli K-12”, J Bacteriol. Jun. 1998;180(11):2915-23.
Dittrich, Franziska , et al., “Fatty acid elongation in yeast. Biochemical characteristics of the enzyme system and isolation of elongation-defective mutants”, Eur J Biochem. 252(3), Mar. 15, 1998, 477-85.
Dohr, Olaf , et al., “Engineering of a functional human NADH-dependent cytochrome P450 system”, Proc Natl Acad Sci USA. 98(1), Jan. 2, 2001, 81-6.
Doroshenko, vera G., et al., “Pho regulon promoter-mediated transcription of the key pathway gene aroGFbr improves the performance of an L-phenylalanine-producing Escherichia coli strain”, Applied Microbiology and Biotechnology 86, 2010, 1287-1295.
Drake , et al., “Structure of the EntB Multidomain Nonribosomal Peptide Synthetase and Functional Analysis of Its Interaction with the EntE Adenylation Domain”, Chem Biol. Apr. 2006;13(4):409-19.
Duncan , et al., “Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product”, Appl Environ Microbiol. Oct. 2004;70(10):5810-7.
Duncan , et al., “The overexpression and complete amino acid sequence of Escherichia coli 3-dehydroquinase”, Biochem J. Sep. 1, 1986:238(2):475-83.
Elvin, Christopher M., et al., “Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli”, Gene. 87(1), Mar. 1, 1990, 123-6.
Eppink, Michel H. M., et al., “Switch of Coenzyme Specificity of p-Hydroxybenzoate Hydroxylase”, J Mol Biol. 292(1), Sep. 10, 1999, 87-96.
Epstein , et al., “Oil: A Life Cycle Analysis of its Health and Environmental Impacts”, The Center for Health and the Global Environment, Harvard Medical School. Mar. 2002. www.med.harvard.edu/chge/oil.html.
Erb , et al., “Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase”, Proc Natl Acad Sci U S A. Jun. 2, 2009: 106(22): 8871-8876. Published online May 20, 2009. doi: 10.1073/pnas.0903939106. 8871-8876.
Farmer , et al., “Improving lycopene production in Escherichia coli by metabolic control”, Nat Biotechnol. May 2000;18(5):533-7.
Felce, Jeremy , et al., “Carbonic Anhydrases Fused to Anion Transporters of SulP Family Evidence for Novel Type of Bicarbonate Transporter”, J Mol Microbiol Biotechnol. 8(3), 2004, 169-76.
“Agriculture Project Fact Sheet”, U.S. Department of Energy, Office of Industrial Technologies. Jul. 2001.
“Energetics Incorporated. 2003. Industrial Bioproducts: Today and Tomorrow. U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Office of the Biomass Program, Washington, D.C.”
“GenBank Accession No. AAC74497.1: Apr. 24, 2017. 2 pgs.”
“GenBank Accession No. NP 415816.1; available 1997”.
“GenBank Accession No. NP 415933.1; available 1997”.
“GenBank Accession No. NP 418045.4; available 1997”.
“GenBank Accession No. NP X81461”, AF473544, Sep. 7, 1994.
“GenBank Accession No. AAS20429.1”, Jan. 19, 2004.
“NCBI Reference Sequence: NP_414657.1”, Jan. 16, 1997.
“NCBI Reference Sequence: NP_415792.1”, Jan. 16, 1997.
“NCBI Reference Sequence: NP_416366.1”, Jan. 16, 1997.
“NCBI Reference Sequence: NP_418812.1”, Jan. 16, 1997.
“NCBI Reference Sequence: WP_011957906.1”, Jun. 6, 2007.
“NCBI Reference Sequence: WP_012121415.1”, Sep. 4, 2007.
“NCBI Reference Sequence: WP_012616528.1”, Dec. 29, 2008.
“NCBI Reference Sequence: YP_001636209.1”, Dec. 21, 2007.
“NCBI Reference Sequence: ZP_01039179.1”, Jan. 16, 2006.
“NCBI Reference Sequence: ZP_01626393.1”, Dec. 15, 2006.
“NCBI Reference Sequence: ZP_04957196.1”, Sep. 15, 2008.
“NCBI Reference Sequence: ZP_05125944.1”, Sep. 15, 2008.
“Nexant, Inc. Chemsystems Perp Program, Acrylic Acid. 08/09-3”, Jul. 2010.
Abdel-Hamid, Ahmed M., et al., “Coordinate Expression of the Acetyl Coenzyme A Carboxylase Genes, accB and accC, Is Necessary for Normal Regulation of Biotin Synthesis in Escherichia coli”, J Bacteriol. 189(2), Jan. 2007, 369-76.
Alber , et al., “Malonyl-coenzyme A reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archael metallosphaera and sulfolobus spp.”, J Bacteriol. Dec. 2006:188(24):8551-9.
Alberts, Bruce , et al., “Molecular Biology of the Cell”, 3rd Ed. Garland Publishing. New York, 1994, 42-45 66-74.
Anagnostopoulos, C. , et al., “Requirements for Transformation in Bacillus Subtilis”, J Bacteriol. 81(5), May 1961, 741-6.
Anton , et al., “Sequencing and overexpression of the Escherichia coli Aroe Gene Encoding Shikimate Dehydrogenase”, Biochem J. Jan. 15, 1988;249(2):319-26.
Armstrong, S. M., et al., “Abiotic conversion of dihydrophloroglucinol to resorcinol”, Canadian Journal of Microbiology. 39(9), 1993, 899-902.
Arthur , et al., “Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors”, Antimicrob Agents Chemother. 38(9), Sep. 1994, 1899-1903.
Asano , et al., “A new enzymatic method of acrylamide production”, Agricultural and Biological Chemistry. 46(5), 1982, 1183-1189.
Baek, Jong Hwan, et al., “Novel gene members in Pho regulon of Escherichia coli”, FEMS Microbiol Lett. 264(1), Nov. 2006, 104-9.
Bailey. James E., et al., “Biochemical Engineering Fundamentals”, 2nd Ed, McGraw Hill, New York, entire book for purposes indicated and Chapter 9, 1986, 533-657.
Bailey , et al., “Inverse metabolic engineering: A strategy for directed genetic engineering of useful phenotypes”, BBiotechnol Bioeng. 79(5), Sep. 5, 2002, 568-79.
Bailey , et al., “Toward a science of metabolic engineering”, Science;252(5013):, Jun. 21, 1991, 1668-75.
Barbin , et al., “induction of specific base-pair substitutions in E. coli trpA mutants by chloroethylene oxide, a carcinogenic vinyl chloride metabolite”, Mutat Res. Nov.-Dec. 1985;152(2-3):147-56.
Bastian , et al., “Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-l-ol production at theoretical yield in Escherichia coli”, Metab Eng. May 2011;13(3):345-52.
Beguin , et al., “The biological degradation of cellulose”, FEMS Microbiol Rev. Jan. 1994;13(1):25-58.
Beisson, Frederic , et al., “Arabidopsis Genes Involved in Acyl Lipid Metabolism. A 2003 Census of the Candidates, a Study of the Distribution of Expressed Sequence Tags in Organs, and a Web-Based Database”, Plant Physiol, 132(2), Jun. 2003, 681-97.
Bellion, Edward , et al., “Methylamine Utilization in Yeast and Bacteria: Studies Using in vivo NMR”, Micro b Growth C1 Compd. (Int. Symp.) 7th Editors: Murrell, J. Collin: Kelly, Don P. Publisher: Intercept. Andover, UK, 1993, 415-32.
Ben-Aroya, Shay , et al., “Toward a Comprehensive Temperature-Sensitive Mutant Repository of the Essential Genes of Saccharomyces cerevisiae”, Molecular Cell. 30, 2008. 248-258.
Bergler , et al., “Sequences of the envM gene and of two mutated alleles in Escherichia coli”, J Gen Microbiol. Oct. 1992;138(10):2093-100.
Bergler , et al., “The enoyl-[acyl-carrier-protein] reductase (FabI) of Escherichia coli, which catalyzes a key regulatory step in fatty acid biosynthesis, accepts NADH and NADPH as cofactors and is inhibited by palmitoyl-CoA”, Eur J Biochem. 242(3), Dec. 15, 1996, 689-94.
Bloch , et al., “Control mechanisms in the synthesis of saturated fatty acids”, Annu Rev Biochem. 46. 1977, 263-98.
Bonner , et al., “A core catalytic domain of the TyrA protein family: arogenate dehydrogenase from Synechocystis”, Biochem J. 382(Pt 1), Aug. 15, 2004, 279-91.
Bonner, William M., et al., “Purification and Properties of Fatty Acyl Thioesterase I from Escherichia coli”, J Biol Chem. 247(10), Mar. 25, 1972, 3123-33.
Borgaro, Janine G., et al., “Substrate Recognition by B-Ketoacyl-ACP Synthases”, Biochemistry. 50(49), Dec. 13, 2011, 10678-86.
Bowie, James U., et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions”, Science 247(4948), Mar. 16, 1990, 1306-10.
Branden, Carl . et al., “Introduction to Protein Stucture”, Garland Publishing Inc., New York, 1991, 247.
Bressler , et al., “Studies on the mechanism of fatty acid synthesis. XI. The product of the reaction and the role of sulfhydryl groups in the synthesis of fatty acids”, J. Biol Chem. vol. 237, May 1962, 1441-1448.
Brock, Thomas D., “Biotechnology: A Textbook of Industrial Microbiology”, Second Edition Sinauer Associates, Inc. Sunderland, Mass., 1989.
Brock , et al., “Naturally occurring adenines withing mRNA coding sequences affect ribosome binding and expression in Escherichia coli”, J Bacteriol. Jan. 2007;189(2):501-10, Epub Nov. 3, 2006.
“Extended European Search Report”, Patent Application 15182914.0, dated Mar. 7, 2016.
“Partial European Search Report”, Patent Application No. 15182914.0, dated Jan. 8, 2016.
Helge, Jans , et al., “Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels”, Biotechnology for Biofuels, vol. 7, No. 1, XP-021173667, Jan. 9, 2014.
Knothe, Gerhard , et al., “Biodiesel and renewable diesel: A comparison”, Progress in Energy and Combustion Science. vol. 36, No. 3 XP026919218, Jun. 1, 2010, 364-373.
Fernando , et al., “Biorefineries: current status, challenges and future direction”, Energ. Fuel. May 2006; 20:1727-1737.
Figge , “Methionine biosynthesis is Escherichia coli and Corynebacterium glutamicum”, Microbiol Monogro 2007; 5:163-193.
Fleming , et al., “Extracellular enzyme synthesis in a sporulation-deficient strain of Bacillus licheniformis”, Appl Environ Microbiol, Nov. 1995, 61 (11):3775-3780.
Fodor , et al., “Light-Directed, Spatially Addressable Parallel Chemical Synthesis”, Science. Feb. 15, 1991:251(4995):767-73.
Fowler, Zachary L , et al., “Increased Malonyl Coenzyme A Biosynthesis by Tuning the Escherichia coli Metabolic Network and Its Application to Flavanone Production”, Appl Environ Microbiol. 75(18), Sep. 2009, 5831-9.
Fujimoto , et al., “pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis”, doi: 10.1128/AEM.67.3.1262-1267.2001 Appl. Environ. Microbiol. Mar. 2001 vol. 67 No. 3 1262-1267.
Funa , et al., “A novel quinone-forming monooxygenase family involved in modification of aromatic polyketides”, J Biol Chem. Apr. 15, 2005;280(15):14514-23. Epub Feb. 8, 2005.
Gietz, R. Daniel , et al., “Transformation of Yeast by Lithium Acetate/Single-Stranded Carrier DNA/Polyethylene Glycol Method”, Methods Enzymol. 350, 2002, 87-96.
Giladi , et al., “FolM, a new chromosomally encoded dihydrofolate reductase in Escherichia coli.”, J Bacteriol. 185 (23), Dec. 2003, 7015-8.
Gilbert, Walter , et al., “Useful Proteins from Recombinant Bacteria”, Sci Am. 242(4), Apr. 1980, 74-94.
Gill , et al., “Genome-wide screening for trait conferring genes using DNA microarrays”, Proc Natl Acad Sci U S A. May 14, 2002;99(10):7033-8. Epub May 7, 2002.
Ginkel , et al., “Identification and cloning of the Mycobacterium avium folA gene, required for dihydrofolate reductase activity”, FEMS Microbiology Letters. vol. 156, Issue 1, Nov. 1, 1997, 69-78.
Gokarn , et al., “Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase”, Appl Environ Microbiol. May 2000;66(5):1844-50.
Goodwin , et al., “Purification and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver. Identity to malonate-semialdehyde dehydrogenase”, J Biol Chem. Sep. 5, 1989;264(25):14965-71.
Gray , et al., “Monofunctional chorismate mutase from Bacillus subtilis: purification of the protein, molecular cloning of the gene, and overexpression of the gene product in Escherichia coli”, Biochemistry. Jan. 16, 1990;29(2):376-83.
Gronenborn, Bruno , “Overproduction of Phage Lambda Repressor under Control of the Iac Promotor of Escherichia coli”, Mol Gen Genet. 148(3), Nov. 17, 1976, 243-50.
Gu , et al., “Polyketide Decarboxylative Chain termination Preceded by O-Sulfonation in Curacin A Biosynthesis”, J Am Chem Soc. Nov. 11, 2009: 131(44): 16033-16035. doi: 10.1021/ja9071578.
Gulmezian , et al., “Genetic Evidence for an Interaction of the UbiG O-Methyltransferase with UbiX in Escherichia coli Coenzyme Q Biosynthesis”, J Bacteriol. Sep. 2006;186(17):6435-9.
Guzman, L. M., et al., “Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter”, J Bacteriol. 177(14), Jul. 1995, 4121-30.
Haldimann, Andreas , et al., “Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of the Escherichia coli Phosphate Regulon”, J Bacteriol. 180(5), Mar. 1998, 1277-86.
Hall, Neil , et al., “Structure-function analysis of NADPH: nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo”, Microbiology. 146 (Pt.6), Jun. 2000, 1399-406.
Hatzimanikatis , et al., “Exploring the diversity of complex metabolic networks”, Bioinformatics. Apr. 15, 2005;21(8):1603-9. Epub Dec. 21, 2004.
He , et al., “A T42M Substitution in Bacterial 5-Enolpyruvylshikimate-3-phosphate Synthase (EPSPS) Generates Enzymes with Increased Resistance to Glyphosate”, Biosci Biotechnol Biochem. vol. 67, 2003—Issue 6. 1405-1409.
Heath, Richard J., et al., “Enoyl-Acyl Carrier Protein Reductase (fabl) Plays a Determinant Role in Completing Cycles of Fatty Acid Elongation in Escherichia coli”, J Biol Chem. 270(44), Nov. 3, 1995, 26538-42.
Henry , et al., “Discovery of novel routes for the biosynthesis of industrial chemicals: 3¬Hydroxypropanoate. Slides”, AICHE Annual Meeting. Nov. 8, 2007. Salt Lake City, UT.
Herter , “Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle”, J Bacteriol Jul. 2001;183(14):4305-4316.
Hondorp , et al., “Oxidation of cysteine 645 of cobalamin-independent methionine synthase causes a methionine limitation in Escherichia coli”, J Bacteriol. May 2009;191(10):3407-10. Epub Mar. 13, 2009.
Hugler , et al., “Malonyl-Coenzyme A Reductase from Chloroflexus aurantiacus, a Key Enzyme of the 3-Hydropropionate Cycle for Autotrophic CO2 Fixation”, J Bacteriol May 2002;184(9):2404-2410.
Ikuo Miyahisa , et al., “Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster”, Applied Microbiology and Biotechnology, Springer, Berlin, DE, (Sep. 1, 2005), vol. 68, No. 4. doi:10.1007/S00253-005-1916-3, ISSN 1432-0614, pp. 498-504, XP019331939.
Ivanova , et al., “Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis”, Nature. May 1, 2003;423(6935):87-91.
James, Ethan S., et al., “Expression of two Escherichia coli acetyl-CoA carboxylase subunits is autoregulated”, J Biol Chem. 279(4). Jan. 23, 2004, 2520-7.
Jan Podkowinski , et al., “Opinions Acetyl-coenzyme A carboxylase—an attractive enzyme for biotechnology”, Biotechnologia, PL. (Jan. 1, 2011), vol. 4, doi:10.5114/bta.2011.46549, ISSN 0860-7796, pp. 321-335, XP055303418.
Jenkins , et al., “Genetic and molecular characterization of the genes involved in short-chain fatty acid degradation in Escherichia coli: the ato system”, J Bacteriol. Jan. 1987; 169(1): 42-52.
Jiang , et al., “Biosynthetic pathways for 3-hydroxypropionic acid production”, Appl Microbiol Biotechnol. Apr. 2009;82(6):995-1003.
Jing , et al., “Phylogenetic and experimental characterization of an acyl-ACP thiosterase family reveals significant diversity in enzymatic and specificity and activity”, BMC Biochemistry201112:44. https://doi.org/10.1186/1471-2091-12-44.
Joike , et al., “Amino acid substitutions affecting catalytic activity and subunit interactions of aminodeoxychorismate synthase in E. coli”, Abstracts of the General Meeting of the American Society for Microbiology. 2002; 102:275-276, and 102nd General Meeting of the American Society for Microbiology; Salt Lake, UT, USA; May 19-23, 2002.
Juliano Alves , et al., “Cloning, expression and enzymatic activity ofandacetyl-coenzyme A carboxylases”, Analytical Biochemistry, Academic Press Inc. New York, vol. 417, No. 1, doi:10.1016/J.AB.2011.05.041, ISSN 0003-2697, (May 25, 2011), pp. 103-111, XP028245778.
Jung , et al., “Jung et al., Wax-deficient antherl is involved in cuticle and wax production in rice anther walls and is required for pollen development”, and is required for pollen development, Plant Cell, Nov. 2006, vol. 18. No. 11, pp. 3015-3032.
Kapol , et al., “Purification and characterization of 2-oxoglutarate decarboxylase of Leuconostoc oenos”, Journal of General Microbiology 136 (1990), 1497-1499.
Kavatavic, Vesna . et al., “Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate-induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity”, Plant Physiol. 108(1), May 1995, 399-409.
Katsuyama, Yohei , et al., “Production of curcuminoids by Escherichia coli carrying an artificial biosynthesis pathway”, Microbiology. 154(Pt 9), Sep. 2008. 2620-8.
Kern , et al., “Engineering primary metabolic pathways of industrial micro-organism”, J Biotechnol. Mar. 30, 2007;129(1):6-29. Epub Dec. 2, 2006.
Kiatpapan, Pornpimon , et al., “Molecular Characterization of Lactobacillus plantarum Genes for B-Ketoacyl-Acyl Carrier Protein Synthase III (fabH) and Acetyl Coenzyme A Carboxylase (accBCDA). Which Are Essential for Fatty Acid Biosynthesis”, Appl Environ Microbiol. 67(1), Jan. 2001, 426-33.
Kim. Youngnyun , et al., “Construction of an Escherichia coli K-12 Mutant for Homoethanologenic Fermentation of Glucose or Xylose without Foreign Genes”, Appl Environ Microbiol 73(6), Mar. 2007, 1766-71.
Kim. Youngnyun , et al., “Dihydrolipoamide Dehydrogenase Mutation Alters the NADH Sensitivity of Pyruvate Dehydrogenase Complex of Escherichia coli K-12”, J Bacteriol. 190(11), Jun. 2008, 3851-8.
Kim , et al., “Effect of Overexpression of Actinobacillus succinogenes Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli”, Appl. Environ. Microbiol. vol. 70 No. 2, Feb. 2004, 1238-1241.
Kim. Joong Kyun, et al., “Extractive Recovery of Products from Fermenation Broths”, Biotechnol. Bioprocess Eng, 4, 1999, 1-11.
Kim, Kwang-Seo , et al., “The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems”, J Bacteriol. 192(16), Aug. 2010, 4089-102.
Kimchi-Sarfaty , et al., “A “Silent” Polymorphism in the MDR1 Gene Changes Substrate Specificity”, Science. Science 315(5811):, Jan. 26, 2007, 525-8.
Kinney, Anthony J., “Manipilating flux through plant metabolic pathways”, Curr Opin Plant Biol. 1(2), Apr. 1998, 173-8.
Machado , et al., “A selection platform for carbon chain elongation using the CoA-dependent pathway to produce linear higher alcohols”, Metabolic Eng. 2012, 14, 504-11.
Okamura , et al., “Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of thiolase superfamily involved in the mevalonate pathway”, Proc. Natl. Acad. Sci. USA, 2010, 107, 11265-70.
Oliveira , et al., “Cloning and Overexpression in Soluble Form of Functional Shikimate Kinase and 5-Enolpyruvylshikimate 3-Phosphate Synthase Enzymes from Mycobacterium tuberculosis”, Protein Expr Purif. 22(3)., Aug. 2001, 430-5.
Orjuela , et al., “Presentation: Recovery of succinic acid from fermentative broth through esterification with ethanol”, Department of Chemical Engineering and Materials Science. Michigan State University. East Lansing. Michigan 48824, Jun. 29, 2010.
O'Sullivan , et al., “High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening”, Gene. vol. 137, Issue 2, Dec. 31, 1993, pp. 227-231.
Ozcelik , et al., “Metabolic engineering of aromatic group amino acid pathway in Bacillus subtilis for L-phenylalanine production”, Chemical Engineering Science. 59(22-23):, 2004, 5019-5026.
Papanikolaou, Seraphim , et al., “Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single-stage continuous culture”, Bioresour Technol. 82(1), Mar. 2002, 43-9.
Parikh , et al., “Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli”, Protein Eng Des Sel. 19(3), Mar. 2006, 113-9.
Park , et al., “Production of alternatives to fuel oil from organic waste by the alkane-producting”, Vibrio furnissii MI, Journal of Applied Microbiology, 2005, vol. 98, No. 2, pp. 324-331.
Patnaik , et al., “Genome shuffling of Lactobacillus for improved acid tolerance”, Nat Biotechnol. 20(7), Jul. 2002, 707-12.
Pohl , et al., “A new perspective on thiamine catalysis”, Curr Opin Biotechnol. 15(4), Aug. 2004, 335-42.
Ponce , et al., “Ioning of the Two Pyruvate Kinase Isoenzyme StructuralGenes from Escherichia coli: the Relative Roles of These Enzymes in Pyruvate Biosynthesis.”, J Bacteriol. 177(19), Oct. 1995, 5719-22.
Popp, J. , “Sequence and overexpression of the menD gene from Escherichia coli”, J Bacteriol. 171(8), Aug. 1989, 4349-54.
Prather, Kristala L, et al., “De novo biosynthetic pathways: rational design of microbial chemical factories”, Curr Opin Biotechnol 19(5), Oct. 19, 2008, 468-74.
Price-Carter , et al., “Polyphosphate kinase protects Salmonella enterica from weak organic acid stress”, Journal of Bacteriology. 187, 2005, 3088-3099.
Ramalinga , et al., “A mild and efficient method for esterification and transesterification catalyzed by iodine”, Tetrahedron Letters. 43(5), 2002, 879-882.
Ramey , et al., “Poster—Translation of genomics data into useful metabolic engineering strategies: construction of a 3-hydroxypropionic acid tolerant E. coli”, 2010.
Ramilo , et al., “Overexpression, purification, and characterization of tyrosine-sensitive 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase from Escherichia coli”, Protein Expr Purif. 9(2), Mar. 1997, 253-61.
Rathnasingh , et al., “Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3-hydroxypropionic acid from glycerol”, Biotechnol Bioeng. 104(4). doi: 10.1002/bit.22429., Nov. 1, 2009, 729-39.
Rathnasingh, Chelladurai , et al., “Production of 3-hydroxypropionic acid via malonyl-COA pathway using recombinant Escherichia coli strains”, J Biotechnol. 157(4), Feb. 20, 2012, 633-40.
Ray , et al., “Mutational analysis of the catalytic and feedback sites of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli”. J Bacteriol. 170(12), Dec. 1988. 5500-6.
Renault , et al., “Plasmid vectors for Gram-positive bacteria switching from high to low copy number”, Gene. vol. 183. Issues 1-2, 1996, pp. 175-182.
Rodriguez , et al., “Structure-cytoprotective activity relationship of simple molecules containing an alpha.beta-unsaturated carbonyl system”, J Med Chem. 40(12), Jun. 6, 1997, 1827-34.
Roe , et al., “Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity”, Microbiology. 148(Pt 7), Jul. 2002, 2215-2222.
Sadowski. M. I., et al., “The sequence-structure relationship and protein function prediction”, Current Opinion in Structural Biology 19, 2009, 357-362.
Saerens. S. M. G , et al., “Parameters Affecting Ethyl Ester Production by Saccharomyces cerevisiae during Fermentation”, Appl Environ Microbiol 74(2), Jan. 2008, 454-61.
Saier , et al., “The catabolite repressor/activator (Cra) protein of enteric bacteria”, J Bacteriol. 178(12), Jun. 1996, 3411-7.
Salis, Howard M., et al., “Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression”, Nat Biotechnol 27(10), Oct. 2009. 946-50.
Sambrook and Russell , “Molecular Cloning: A Laboratory Manual”. Third Edition (vol. 1-3). Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y., 2001.
Sauna , et al., “Silent polymorphisms speak: how they affect phharmacogenomics and the treatment of cancer”, Cancer Research. 67(20), Oct. 15, 2007, 9609-12.
Schmid, Katherine M., et al., “Lipid Metabolism in Plants”, Biochemistry of Lipids, Lipoproteins and memebranes. Ch 4, 2002, 93-126.
Schmidt-Dannert , et al., “Molecular breeding of carotenoid biosynthetic pathways”, Nat Biotechnol. 18(7), Jul. 2000. 750-3.
Seffernick, Jennifer L., et al., “Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different”, J Bacteriol. 183(8), Apr. 2001, 2405-10.
Sen, S. , et al., “Developments in Directed Evolution for Improving Enzyme Functions”, Appl Biochem Biotechnol. 143(3), Dec. 2007, 212-23.
Service , “Sugary Recipe Boosts Grow-Your-Own Plastics”, Science. 312(5782), Jun. 30, 2006, 1861.
Shelden, Megan C., et al., “Membrane topolgy of the cyanobacterial bicarbonate transporter, BicA, a member of the SulP (SLC26A) family”, Molecular Membrane Biology vol. 27(1), 2010, 12-22.
Singh , et al., “Genes restoring redox balance in fermentation-deficient E. coli NZN111”, Metabolic Engineering. vol. 11, Issue 6, Nov. 2009, 347-354.
Singh, Raushan Kumar, et al., “Protein Engineering Approaches in the Post-Genomic Era”, Curr Protein Pept Sci. 18. 2017. 1-11.
Skerra, Arne , “Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli”, Gene. 151(1-2), Dec. 30, 1994, 131-5.
Smirnova, N. , et al., “Engineered Fatty Acid Biosynthesis in Streptomyces by Altered Catalytic Function of B-Ketoacyl-Acyl Carrier Protein Synthase III”, Journal of Bacteriology, vol. 183, No. 7., Apr. 2001, 2335-2342 & 2335. 2336.
Sousa, Silvino , et al., “The ARO4 gene of Candida albicans encodes a tyrosine-sensitive DAHP synthase: evolution, functional conservation and phenotype of Aro3p-, Aro4p-deficient mutants”, Microbiology 148(Pt5), 2002, 1291-1303.
Stephanie C. Weatherly . “Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain”, Biochemical Journal, GB, (May 15, 2004), vol. 380, No. 1, doi:10.1042/bj20031960, ISSN 0264-6021, pp. 105-110, XP055302533, May 15, 2004.
Stephanopoulos , et al., “Challenges in engineering microbes for biofuels production”, Science. 315(5813), Feb. 9, 2007, 801-4.
Stephanopoulos , et al., “Network Rigidity and Metabolic Engineering in Metabolite Overproduction”, Science, 252(5013), Jun. 21, 1991, 1675-81.
Stephens , et al., “Mitochondrial fatty acid synthesis in Trypanosoma brucei”, Journal of Biological Chemistry, vol. 282, No. 7, Feb. 16, 2007, 4427-36.
Stim , et al., “Nucleotide sequence of the adi gene, which encodes the biodegradative acid-induced arginine decarboxylase of Escherichia coli”, J Bacteriol. 175(5), Mar. 1993, 1221-34.
Stone, Scot J., et al., “Lipids and Lipoproteins: Lipopenia and Skin Barrier Abnormalities in DGAT2-deficient Mice”, J Biol Chem. 279(12), Mar. 19, 2004, 11767-76.
Straathoff , et al., “Feasibility of acrylic acid production by fermentation”, Appl Microbiol Biotechnol.67(6), Jun. 2005, 727-34.
Strauss , et al., “Enzymes of a novel autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. the 3-hydroxypropionate cycle”, Eur J Biochem. 215(3). Aug. 1, 1993, 633-43.
Stryer , “Biochemistry”, 4th Ed. Freeman and Co , New York., 1995, 463-650.
Studier, William F., et al., “Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-level Expression of Cloned Genes”, J Mol Biol. 189(1), May 5, 1900, 113-30.
Subrahmanyam, Satyanarayana , et al., “Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli”, J Bacteriol. 180(17), Sep. 1998, 4596-602.
Kisselev , “Polypeptide Release Factors in Prokaryotes and Eukaryotes: Same Function. Different Structure”, Structure. 10(1), Jan. 2002, 8-9.
Kizer, Lance , et al., “Application of Functional Genomics to Pathway Optimization for Increased Isoprenoid Production”, Appl Environ Microbiol. 74(10), May 2008, 3229-41.
Kleerebezem , et al., “Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp”. Appl Environ Microbiol. Nov. 1997:63(11):4581-4584.
Kleerebezem , et al., “The qmeA (ts) mutation of Escherichia coli is localized in the fabl gene, which encodes enoyl-ACP reductase.”. Res Microbiol. 147(8), Oct. 1996, 609-13.
Kozliak , et al., “Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase”, J Bacteriol. 176(18), Sep. 1994, 5711-7.
Kozliak , et al., “Role of bicarbonate/CO2 in the inhibition of Escherichia coli growth by cyanate”, J. Bacteriol. vol. 177 No. 11, Jun. 1995, 3213-3219.
Kroeger, Jasmin K., et al., “A spectrophotometric assay for measuring acetyl-coenzyme A carboxylase”, Anal Biochem. 411(1), Apr. 1, 2011, 100-5.
Kunin , et al., “A comparative analysis of the inventive step standard in the European and Japanese patent office from an US perspective”, IP Litigator, Jan./Feb. 2008, 15-23.
Kurcok , et al., “Reactions of13-lactones with potassium alkoxides and their complexes with 18-crown-6 in aprotic solvents”, Journal of Organic Chemistry. 58(16), 1993, 4219-4220.
Kwon , et al., “A physiology study of Escherichia coli overexpressing phosphoenolpyruvate carboxykinase”, Biosci. Biotechnol. Biochem., 72 (4), 2008, 1138-1141.
Kwon , et al., “Influence of gluconeogenic phosphoenolpyruvate carboxykinase (PCK) expression on succinic acid fermentation in Escherichia coli under high bicarbonate condition”, Journal of Microbiology and Biotechnology 16(9)., Sep. 2006 , 1448-1452.
Lambert , et al., “Cre-lox-based System for Multiple Gene Deletions and Selectable-Marker Removal in Lactobacillus plantarum”, AEM, vol. 73, No. 4, Jan. 1, 1900, 1126-1135.
Langlois , et al., “A new peparation of trifluoromethanesulfinate salts”, Journal of Fluorine Chemistry. 128(7), 2007, 851-856.
Lassner. Michael W., et al., “Lysophosphatidic Acid Acyltransferase from Meadowfoam Mediates Insertion of Erucic Acid at the sn-2 Position of Triacylglycerol in Transgenic Rapeseed Oil”, Plant Physiol. 109(4), Dec. 1995, 1389-94.
Lee. Soo Hee. et al., “Fatty Acid Synthesis by Elongases in Trypanosomes”, Cell. 126(4), Aug. 25, 2006, 691-9.
Leeper, Stephen A., “Membrane Separations in the Recovery of Biofuels and Biochemicals: An Update Review”, Separation and Purification Technology, Norman N. Li and Joseph M. Calo, Eds., Marcel Dekker, 1992, 99-194.
Lennen , et al., “A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes”, Biotechnol Bioeng. vol. 106, Issue 2, Jun. 1, 2010, 193-202.
Leonard, Effendi , et al., “Engineering Central Metabolic Pathways for High-Level Flavonoid Production in Escherichia coli”, Appl Environ Microbiol. 73(12), Jun. 2007, 3877-86.
Li, Wang . et al., “Characterization of two temperature-inducible promoters newly isolated from B. subtilis”, Biochem Biophys Res Commun. 358(4), Jul. 13, 2007, 1148-53.
Li, Jianguo , et al., “Chronic intermittent hypoxia upregulates genes of lipid biosynthesis in obese mice”, J Appl Physiol. 99(5), Nov. 2005, 1643-8.
Li , et al., “Effect of poxB gene knockout on metabolism in Escherichia coli based on growth characteristics and enzyme activites”, World Journal of Microbiology and Biotechnology. vol. 23, Issue 4, Apr. 2007, 573-580.
Liang , et al., “Fe2(SO4)3.4H20/concentrated H2SO4: an efficient catalyst for esterification”, Journal of Chemical Research, Synopses. 3. 2004, 226-227.
Lilly, Mariska , et al., “The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates”, Yeast. 23(9), Jul. 15, 2006, 641-59.
Lioa , et al., “Metabolic engineering for a malonyl-CoA-dependent pathway for fatty acid production in Escherichia coli (abstract)”. SIMB Annual Meeting & Exhibition. Aug. 12-16, 2012. Washington Hilton, Washington, DC. Available at http://sim.confex.com/sim/2012/webprogram/Paper23197.html, Aug. 2012.
Lipscomb , et al., “Poster—Understanding production of 3-Hydroxypropionic Acid (3¬HP) in a genomic context.”, OPX Biotechnologies, Metabolic Engineering, Sep. 17, 2008.
Lu, Xuefeng , et al., “Overproduction of free fatty acids in E. coli: Implications for biodiesel production”, Metab Eng. 10(6), Nov. 2008. 333-9.
Lutke-Eversloh , et al., “Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants”, Appl Environ Microbiol. vol. 71 No. 11, Nov. 2005, 7224-8.
Lynch , “Rapid optimization of microorganisms for the cost superior production of chemicals & fuels”, OPX Biotechnologies, Sep. 15, 2008.
Lynch, M. , et al., “SCALEs: multiscale analysis of library enrichment. Nat Methods”, Nat Methods. 4(1)., Jan. 2007. 87-93.
Magnuson, Kelly , et al., “Regulation of fatty acid biosynthesis in Escherichia coli”. Microbiological Reviews, vol. 57, No. 3, 1993, 522-542.
Mandaokar, Ajin , et al., “Transcriptional regulators of stamen development in Arabidopsis identified by transcriptional profiling”, Plant J. 46(6), Jun. 2006, 984-1008.
Martin , et al., “Engineering a mevalonate pathway in Escherichia coli for production of terpenoids”, Nat Biotechnol. 21(7)., Jul. 2003, 796-802.
Masayuki , et al., “Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli”, Applied Microbiology and Biotechnology, Jan. 2008, vol. 77, Issue 6, pp. 1305-1316.
McCabe, Warren L., et al., “Unit Operations of Chemical Engineering”, 5th Ed., W.L. McGraw Hill, New York. 1993.
Meades, Glen , et al., “A tale of two functions: enzymatic activity and translational repression by carboxyltransferase”, Nucleic Acids Res. 38(4). Mar. 2010, 1217-27.
Mehta , et al., “Aminotransferases: demonstration of homology and division into evolutionary subgroups”, Eur J Biochem. 214(2), Jun. 1, 1993, 549-61.
Meng, Xin . et al., “Increasing fatty acid production in E. coli by simulating the lipid accumulation of oleaginous microorganisms”, Journal of Industrial Microbiology and Biotechnology. 38(8). 2011. 919-925.
Meng , et al., “Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH.”, J. Bacteriol. vol. 174 No. 8, Apr. 1992, 2659-2669.
Milton , et al., “In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha-subunits”, Biol Chem. 261(35). Dec. 15, 1986, 16604-15.
Mohan Raj , et al., “Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant Escherichia coli”, Appl Microbiol Biotechnol. 84(4), Sep. 2009, 649-57.
Moreau , et al., “Diversion of the metabolic flux from pyruvate dehydrogenase to pyruvate oxidase decreases oxidative stress during glucose metabolism in nongrowing Escherichia coli cells incubated under aerobic, phosphate starvation conditions”, J Bacteriol. 186(21), Nov. 2004, 7364-8.
Muday , et al., “The tyrosine repressor negatively regulates aroH expression in Escherichia coli”, 173(12), Jun. 1991, 3930-2.
Nackley , et al., “Human Catechol-O-Methyltransferase Haplotypes Modulate Protein Expression by Altering mRNA Scondary Structure”, Science. 314(5807)., Dec. 22, 2006, 1930-3.
Nelson, David L., et al., “Principles of Biochemistry 3rd Ed.”, Worth Publishers New York, 2000, 527-658.
Nichols , “Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase”, J Bacteriol. 174(16), Aug. 1992, 5309-16.
Nicholson, Donald , “Lipid Metabolism Graphic Design”, 2002, 1 page.
Nugent , Patricia , “Development of Improved Chemicals and Plastics from Oilseeds. Final technical report”, The Dow Chemical Company. DE-FC36-01ID14213. Jul. 31, 2006.
Ohmiya , et al., “Structure of Cellulases and Their Applications”, Biotechnol. Genet. Eng. Rev., vol. 14, 1997, 365-414.
Ohnishi . et al., “A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant”, Appl Microbiol Biotechnol. 58(2), Feb. 2002, 217-23.
Okamura, Eiji , et al., “Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway”, Proc. Natl. Acad. Sci. USA. 107., 2010, 11265-70.
Suh, Mi Chung, et al., “Cuticular Lipid Composition, Surface Structure, and Gene Expression in Arabidopsis Stem Epidermis”, Plant Physiol. 139(4), Dec. 13, 2005, 1649-65.
Sulter, G. J., et al., “Proliferation and metabolic significance of peroxisomes in Candida boldinii during growth on D-alanine or oleic acid as the sole carbon source”, Arch Microbiol. 153(5), 1990, 485-9.
Sun , et al., “ZrOC12 x 8H20: an efficient, cheap and reusable catalyst for the esterification of acrylic acid and other carboxylic acids with equimolar amounts of alcohol”. Molecules. 11(4):, Apr. 10, 2006, 263-71.
Taghavi , et al., “Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments”. Appl Environ Microbiol. Oct. 1994; 60(10): 3585-3591.
Takamizawa , et al., “Beta-Hydroxypropionic Acid Production by Byssochlamys sp. Grown on Acrylic Acid”, Appl Microbiol Biotechnol. 40. 1993. 196-200.
Takamura , et al., “Changes in the intracellular concentration of acetyl-CoA and malonyl-CoA in relation to the carbon and energy metabolism of Escherichia coli K12”, J Gen Microbiol. 134(8), Aug. 1988, 2249-53.
Tanimoto , et al., “Analysis of the Conjugal Transfer System of the Pheromone-Independent Highly Transferable Enterococcus Plasmid pMG1: Identification of a tra Gene (traA) Up-Regulated during Conjugation”, doi: 10.1128/JB.184.20.5800-5804.2002 J. Bacteriol. Oct. 2002 vol. 184 No. 20 5800-5804.
Tian , et al., “Mycobacterium tuberculosis appears to lack an alpha-ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes”, Mol Microbiol. 57(3), Aug. 2005, 859-68.
Tian , et al., “Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: Identification of alpha-ketoglutarate decarboxylase”. Proc Natl Acad Sci U S A. 102(30). Jul. 26, 2005, 10670-5.
Tomar, A. , “Master Thesis Prodution of Pyruvate by Escherichia coli Using Metabolic Engineering”, The University of Georgia, May 2002, 1-171.
Tunnicliff , et al., “The inhibition by substrate analogues of gamma-aminobutyrate aminotransferase from mitochondria of different subcellular fractions of rat brain”, Can J Biochem. 55(4), Apr. 1977, 479-84.
Turlin , et al., “3-phenylpropionate catabolism and the Escherichia coli oxidative stress response”, Res Microbiol. 156(3), Apr. 2005, 312-21.
Valentin H E , et al., “Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose”, Journal of Biotechnology, Elsevier Science Publishers, Amsterdam, NL. Elsevier Science Publishers, Amsterdam, NL, vol. 58, No. 1, doi:10.1016/S0168-1656(97)00127-2, ISSN 0168-1656, XP004126101, Oct. 2, 1997, 33-38.
Van Kranenburg , et al., “Functional Analysis of Three Plasmids from Lactobacillus plantarum”, doi: 10.1128/AEM.71.3.1223-1230.2005 Appl. Environ. Microbiol. Mar. 2005 vol. 71 No. 3 1223-1230.
Vedantam , et al., “Characterization of mutations contributing to sulfathiazole resistance in Escherichia coli”, Antimicrob Agents Chemother. 42(1), Jan. 1998, 88-93.
Vilcheze , et al., “Inactivation of the inhA-Encoded Fatty Acid Synthase II (FASII) Enoyl-Acyl Carrier Protein Reductase Induces Accumulation of the FASI End Products and Cell Lysis of Mycobacterium smegmatis”, doi: 10.1128/JB.182.14.4059-4067.2000 J. Bacteriol vol. 182 No. 14, Jul. 2000, 4059-4067.
Wankat, Phillip C., “Separation Process Engineering, Equilibrium Staged Separations”, P.C. Wankat, Prentice Hall, Englewood Cliffs. NJ. USA., 1988.
Warnecke . et al., “A genomics approach to improve the analysis and design of strain selections”, Metab Eng. 10(3-4), May-Jul. 2008, 154-65.
Warnecke . et al., “Engineering of Organix Acid Tolerance Genes in E. coli for Biorefinery Applications”, 2006 AlChE Annual meeting in San Francisco, California, Nov. 12-17, 2006, https://aiche.confex.comlaiche/2006/techprogram/P67122.HTM
Warnecke . et al., “Identification of a 21 amino acid peptide conferring 3-hydroxypropionic acid stress-tolerance to Escherichia coli”, Biotechnol Bioeng.109(5). doi: 10.1002/bit.24398., May 2012, 1347-52.
Warnecke . et al., “Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications.”, Microbial Cell Factories. 4(25). 2005. 1-8.
Warnecke , et al., “Rapid dissection of a complex phenotype through genomic-scale mapping of fitness altering genes”. Metab Eng. 12(3), May 2010. 241-50.
Wasewar , et al., “Fermenation of Glucose to Lactic Acid Coupled with Reactive Extraction: A Review.”, Ind. Eng. Chem. Res. 43, 2004, 5969-5982.
Waterson , et al., “Enoyl coenzyme A hydralase (crotonase). Catalytic properties of crotonase and its possible regulatory role in fatty acid oxidation”, J Biol Chem. 247(16). Aug. 25, 1972. 5258-65.
Weilbacher , et al., “A novel sRNA component of the carbon storage regulatory system of Escherichia coli.”, Moleculary Microbiology, vol. 48, No. 3, [online] [Retrieved on Jul. 11, 2011]. [Retrieved from the internet: http://www.blackwell-synergy.com/links/doi/10.1046/1.1365-2958.2003.03459.x/full/], May 2003, 657-670.
Welch , et al., “Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli.”, Proc Natl Acad Sci U S A. 99(26), Dec. 24, 2002, 17020-4.
Werpy . et al., “Top Value Added Chemicals From Biomass, Volume 1—Results of Screening for Potential candidates From Sugars and Synthesis Gas”, Pacific Northwest National Laboratory. U.S. Department of Energy, Aug. 2004.
Whisstock , et al., “Prediction of protein function from protein sequence and structure”, Q Rev Biophys. 36(3). Aug. 2003, 307-40.
White , et al., “The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa”, Biochem J. 251(2), Apr. 15, 1988, 313-22.
Winkler, Christoph K., et al., “Asymmetric bioreduction of activated alkenes to industrially relevant optically active compounds”. J Biotechnol. 162(4), Dec. 31, 2012, 381-9.
Wishart , et al., “A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase”, J Biol Chem. 270(45), Nov. 10, 1995, 26782-5.
Witkowski , et al., “Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.”, Biochemistry. 38(36), Sep. 7, 1999, 11643-50.
Wyckoff , et al., “Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector”, Appl Environ Microbiol. Apr. 1996; 62(4): 1481-1486.
Xie, Dongming , et al., “Microbial Synthesis of Triaetic Acid Lactone”, Biotechnol Bioeng. 93(4), Mar. 5, 2006, 727-36.
Xu , et al., “English Translation: Principles and Experiments of Biotechnology”, China Minzu University Press. (English Translation), Jul. 2006, 229-231.
Xu, Xiaowei , “Fatty acid synthase inhibitors: research advances”, Journal of international pharmaceutical research. vol. 36 (2). (English abstract), 2009, 105-108, 120.
Yee , et al., “On the role of helix 0 of the tryptophan synthase alpha chain of Escherichia coli.”, J Biol Chem. 271(25), Jun. 21, 1996, 14754-63.
Yiming Ren , et al., “Molecular Iodine in Ionic Liquid: A Green Catalytic System for Esterification and Transesterification”, Synthetic Communications. 40(11), 2010, 1670-1676.
Yoshida , et al., “Identification of PhoB binding sites of the yibD and ytfK promoter regions in Escherichia coli.”. J Microbiol. 49(2), Apr. 2011, 285-289.
Zha, Wenjuan , et al., “Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering”, Metab Eng. 11(3), May 2009, 192-8.
Zhang , et al., “Inhibiting bacterial fatty acid synthesis”, J. Biol. Chem. 281(26), Jun. 30, 2006, 17541-17544.
Zhao . “Binding of two flaviolin substrate molecules, oxidative coupling, and crystal structure of Streptomyces coellcolor A3(2) cytochrome P450 158A2.”, J Biol Chem. 280(12), Mar. 25, 2005, 11599-607.
Zhou , et al,, “Interdomain communication between the thiolation and thioesterase domains of EntF explored by combinatoral mutagenesis and selection”, Chem Biol. 13(8), Aug. 2006, 869-79.

Related Publications (1)

	Number	Date	Country
	20180273990 A1	Sep 2018	US

Provisional Applications (1)

	Number	Date	Country
	62044621	Sep 2014	US

Continuations (1)

	Number	Date	Country
Parent	14843525	Sep 2015	US
Child	15918806		US

Production of fatty acids esters

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

CPC

Field of Search

US

International Classifications

Disclaimer

Abstract