Method of producing succinic acid and other chemiclas using facilitated diffusion for sugar import

a technology of facilitated diffusion and succinic acid, which is applied in the direction of bacteria peptides, biofuels, peptide sources, etc., can solve the problems of poor efficiency of atp production from sugars such as glucose, severe limitations of 030 to use glf, and inability to show that the facilitated diffusion system could support anaerobic growth, etc., to achieve the effect of improving fermentation performance parameters, facilitating diffusion, and improving the balance of facilitated diffusion

Inactive Publication Date: 2016-06-09
PTT GLOBAL CHEMICAL PUBLIC COMPANY LIMITED
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  • Description
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Benefits of technology

[0021]In another embodiment, the present invention provides for methods for improving fermentation performance parameters (titer, yield, specific productivity, minimizing byproduct formation) of strains engineered to use facilitated diffusion.
[0022]In another embodiment, the present invention provides for methods for achieving an improved balance of facilitated diffusion and sugar kinase activity leading to improved growth and fermentation parameters in genetically engineered microorganism that used facilitated diffusion to import a sugar.
[0023]According to the present invention, one approach is to genetically transfer a facilitated diffusion system for importing a sugar from a seco

Problems solved by technology

However, under anaerobic conditions, production of ATP from sugars such as glucose is much less efficient than under aerobic conditions, and the so-called “substrate level” phosphorylation, for example, by pyruvate kinase, becomes a larger portion of the ATP production budget than under aerobic conditions where oxidative phosphorylation provides the majority of the ATP budget.
However, wild type E. coli using the native PTS for glucose import has an aerobic specific growth rate of 1.0 to 1.2 hr-1), so the strains engineered in U.S. Pat. No. 5,602,030 to use glf appear to be severely limited by glucose uptake.
Moreover, the disclosures did not show that the facilitated diffusion system could support anaerobic growth.
Finally, U.S. Pat. No. 5,602,030 did not demonstrate that a glf-based system could support high titer production of a commodity chemical such as ethanol or succinate in E. coli or any other organism that does not natively use facilitated diffusion.
However, the best succinate production in this system was modest, only 220 mM (26 g/l) in 96 hours.
To further complicate the potential replacement of

Method used

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  • Method of producing succinic acid and other chemiclas using facilitated diffusion for sugar import
  • Method of producing succinic acid and other chemiclas using facilitated diffusion for sugar import
  • Method of producing succinic acid and other chemiclas using facilitated diffusion for sugar import

Examples

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example 1

Construction of AC15, a Derivative of KJ122 that Contains the Glf and Glk Genes from Gene Cluster from Zymomonas mobilis CP4

[0074]All manipulations of DNA and plasmids, polymerase chain reaction (PCR), transformation, and chromosomal integration were accomplished by standard methods that are well known in the art, It is well known that DNA sequences can be cloned and joined together to form new combinations that cannot be easily found in nature. In addition to the more traditional methods involving restriction enzymes and DNA ligase, newer methods using recombineering in yeast, the so-called “Gibson Method” of in vitro splicing of DNA, or any other appropriate method can be used to construct such novel DNA sequences. The DNA fragments needed can be obtained from libraries of clones or by PCR from appropriate template DNA. It is also understood that many desired DNA sequences can be designed and synthesized from chemical precursors. Such a service is supplied by a number of commercia...

example 2

Strain AC15 Produces Succinate as Well as Parent KJ122

[0079]Strains KJ122 and AC15 were grown under microaerobic condition in 7 liter fermentors (New Brunswick Scientific) at 39° C. using a minimal medium with glucose fed batch system. The starting volume of 3 liters contained potassium phosphate monobasic (18 mM), magnesium sulfate (2 mM), betaine (1.33 mM), trace elements, Antifoam 204 (8 ppm) and 25 g / l glucose. The pH was adjusted initially to pH 7.0 and thereafter was maintained at pH 6.5 as acid was produced by addition of the ammonium hydroxide / ammonium bicarbonate solution described below. The 150 ml inocula were grown aerobically and contained a minimal medium similar to the above described medium, except that glucose was at 20 g / l and calcium chloride was added to a final concentration of 0.1 mM. Agitation was set at 750 RPM (revolutions per minute). When glucose decreased to 5 g / l, a 650 g / l glucose feed was started and maintained at a rate aimed to keep the glucose conce...

example 3

Spontaneous “Red Mutants” Derived from AC15

[0080]KJ122 is able to ferment lactose, as evidenced by formation of red colonies on MacConkey lactose plates (Beckton-Dickinson, Franklin Lakes, N.J.). However, AC15 does not ferment lactose, as evidence by producing “white” (beige) colonies on MacConkey lactose plates. This white colony phenotype of AC15 results from binding and inhibition of lactose permease (LacY) by unphosphorylated EIIAGlc protein. This white colony phenotype is present in all strains deleted for ptsHI, since the enzymes required to phosphorylate EIIAGlc are absent, and as a result, all EIIAGlc present in the cells remains unphosphorylated. Thus, ironically, E. coli ptsHI mutants are phenotypically Lac−, even though lactose is not imported by the PTS system in E. coli.

[0081]The inventors noticed by chance that when MacConkey lactose plates were streaked with AC15 and allowed to incubate overnight at 37° C., and then for an extra day at room temperature (about 22° C.)...

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Abstract

This invention relates to the production of succinic acid and other chemicals derived from phosphoenolpyruvate (PEP) by fermentation with a microorganism in which the fermentation medium contains one or more sugars, and in which one or more of the sugars is imported into the cell by facilitated diffusion. As a specific example, succinic acid is produced from a glucose-containing renewable feedstock through fermentation using a biocatalyst. Examples of such a biocatalyst include microorganisms that have been enhanced in their ability to utilize glucose as a carbon and energy source. The biocatalysts of the present invention are derived from the genetic manipulation of parental strains that were originally constructed with the goal to produce one or more chemicals (for example succinic acid and/or a salt of succinic acid) at a commercial scale using feedstocks that include, for example, glucose, fructose, or sucrose. The genetic manipulations of the present invention involve the introduction of exogenous genes involved in the transport and metabolism of glucose or fructose into the parental strains. The genes involved in the transport and metabolism of glucose or fructose can also be introduced into a microorganism prior to developing the organism to produce a particular chemical. The genes involved in the transport and metabolism of sucrose can also be used to augment or improve the efficiency of sugar transport and metabolism by strains already known to have some ability for glucose utilization in biological fermentations.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims the priority of the U.S. Provisional Application Ser. No. 61 / 857,300, filed on Jul. 23, 2013.FIELD OF THE INVENTION[0002]The present invention is in the field of producing specialty and commodity organic chemicals using biocatalysts (bacteria and other microorganisms) that can be modified to increase their efficiency in using sugar-containing feedstocks. More specifically, the present invention is related to the genetic modifications of genes that encode functions involving transport and metabolism of sugars for the biological production of succinic acid and other chemicals.BACKGROUND OF THE INVENTION[0003]A large number of organic chemicals are currently derived from petrochemical feedstocks. There is a growing interest in producing many of these petrochemical-derived organic compounds through biological fermentation processes using renewable feedstocks. The list of organic compounds that can be derived from renewa...

Claims

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Application Information

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IPC IPC(8): C12P7/46C07K14/195
CPCC07K14/195C12P7/46C12N1/36C12N15/01C12P7/44Y02E50/10
Inventor YOCUM, R. ROGERSCOLLARD, ANDREW CHRISTOPHERHERMANN, THERONYU, XIAOHUIGONG, WEI
Owner PTT GLOBAL CHEMICAL PUBLIC COMPANY LIMITED
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