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Metabolic evolution of escherchia coli strains that produce organic acids

A technology of Escherichia coli and Bacillus, applied in the field of metabolic evolution of Escherichia coli strains producing organic acids, can solve problems such as reducing growth rate

Active Publication Date: 2012-09-19
PTT GLOBAL CHEMICAL PUBLIC COMPANY LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Additionally, it has been reported that ablation of ptsG gene function can reduce the growth rate of microorganisms metabolically engineered to produce organic acids (Sanchez et al., 2005)

Method used

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  • Metabolic evolution of escherchia coli strains that produce organic acids
  • Metabolic evolution of escherchia coli strains that produce organic acids
  • Metabolic evolution of escherchia coli strains that produce organic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1C5

[0099] Embodiment 1C5 utilizes

[0100] E. coli strain KJ122 (E. coli C, ΔldhA, ΔadhE, ΔackA, ΔfocA-pflB, ΔmgsA, ΔpoxB, ΔtdcDE, ΔcitF, ΔaspC, ΔsfcA) was able to grow aerobically on glucose, xylose and arabinose. The purpose of the present invention is to aerobically cultivate Escherichia coli KJ122 strain in a medium containing hexoses and other pentoses and to select organisms that can simultaneously utilize two types of sugars.

[0101] Initial screens for C5 utilization were performed by aerobic incubation on NBS inorganic media plates supplemented with 2% xylose. Plates were incubated overnight at 37°C. Colonies appearing on xylose plates were streaked three times consecutively on fresh plates. At the end of the third transfer on solid NBS inorganic medium with 2% xylose, cells from the plate were scraped off and inoculated directly into fermentation flasks containing 3 , 1 mM betaine and 8% xylose in AM1 inorganic medium. Fermentation medium with 1.2N KOH and 2.4M K ...

Embodiment 2

[0104] Metabolic evolution of embodiment 2 KJ122

[0105] In another embodiment of the invention, the KJ122 strain has undergone metabolic evolution. Micro-aerobically grown KJ122 cultures in liquid AM1 medium supplemented with xylose were transferred every 24 hours to fresh liquid AM1 medium containing 8% xylose for a period of 2 weeks. At the end of these multiple transfers, the KJ122 strain was transferred to a new fermenter with AM1 medium supplemented with 8% xylose. The anaerobic growth rate and kinetics of succinate production and xylose utilization of KJ122 in the fermentor were monitored. The succinic acid production in the fermentor started immediately without any lag period and also produced a higher final titer, and this strain was termed the "metabolically evolved strain". In our strain collection, this metabolically evolved strain has been named TG400.

[0106] To determine whether the KJ122 "adapted strain" and the "metabolically evolved" TG400 strain have an...

Embodiment 3C5

[0109] Example 3C5+C6 co-fermentation

[0110] In KJ122 under anaerobic growth conditions, C5 sugars and C6 sugars were not metabolized simultaneously. C6 sugars are usually metabolized first and show a lag period before C5 metabolism. Therefore, it was important to determine the fermentation characteristics of TG400 in the presence of equal amounts of C6 and C5 sugars. like Figure 4 As shown in , TG400 was able to utilize glucose and xylose at the same rate and produce succinate without any lag period. KJ122 is also able to utilize xylose and glucose. In the KJ122 strain, however, xylose utilization started only after a large drop in glucose concentration. As further shown in Table 3, when compared to KJ122's utilization of xylose and glucose, TG400 utilized more xylose than glucose on a molar basis.

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Abstract

This invention relates to the metabolic evolution of a microbial organism previously optimized for producing an organic acid in commercially significant quantities under fermentative conditions using a hexose sugar as sole source of carbon in a minimal mineral medium. As a result of this metabolic evolution, the microbial organism acquires the ability to use pentose sugars derived from cellulosic materials for its growth while retaining the original growth kinetics, the rate of organic acid production and the ability to use hexose sugars as a source of carbon. This invention also discloses the genetic change in the microorganism that confers the ability to use both the hexose and pentose sugars simultaneously in the production of commercially significant quantities of organic acids.

Description

[0001] Cross-references to related applications [0002] This application claims priority to US Provisional Application Serial No. 61 / 281,483, filed November 18, 2009. [0003] Government funding [0004] This invention was made with US Government support under contract awarded by the US Department of Energy under Award No. DE-EE0002878 / 001. The US Government has certain rights in this invention. Background technique [0005] A 2004 US Department of Energy report titled "Advanced Value-Adding Chemicals from Biomass" has identified 12 building block chemicals that can be produced from renewable feedstocks. The 12 building block chemicals are 1,4-diacids (succinic acid, fumaric acid, and maleic acid), 2,5-furandicarboxylic acid, 3-hydroxypropionic acid, aspartic acid, glucaric acid, Glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerin, sorbitol and xylitol / arabitol. [0006] Building block chemicals are molecules with multiple functional groups that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12P7/00C12R1/01C12R1/19
CPCC12P7/54C12P7/46C07K14/245C12P7/40
Inventor T·格拉巴龚巍R·R·约卡姆
Owner PTT GLOBAL CHEMICAL PUBLIC COMPANY LIMITED
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