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Method of producing succinic acid and other chemicals using sucrose-containing feedstock

一种有机化学品、蔗糖磷酸化酶的技术，应用在遗传修饰领域，能够解决没有提及生产非氨基酸、不是最佳、未提及生产非氨基酸的化学品等问题，达到增强蔗糖运输和代谢的能力的效果

Active Publication Date: 2013-10-09
PTT GLOBAL CHEMICAL PUBLIC COMPANY LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In US 6,960,455 there is no intentional mention of correcting any inherent defect in the csc operon of EC3132
Also, the patent does not mention chemicals that produce non-amino acids, such as succinic acid
[0016] US 7,179,623 discloses a strain constructed from a sucrose-differentiated strain having a sucrose PTS gene (scrKYABR) from E. The PTS-dependent system claimed in the disclosure is not optimal for the production of chemicals derived from phosphoenolpyruvate (PEP)

Method used

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  • Method of producing succinic acid and other chemicals using sucrose-containing feedstock
  • Method of producing succinic acid and other chemicals using sucrose-containing feedstock
  • Method of producing succinic acid and other chemicals using sucrose-containing feedstock

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Construction of SD14, a KJ122 derivative containing the cscBKA gene cluster from Escherichia coli W

[0096] A gene cluster encoding cscBKA for sucrose utilization uptake and utilization was amplified from genomic DNA of the W strain of Escherichia coli (ATCC 9637) using polymerase chain reaction. The PCR primers were designed so that the obtained PCR products contained only the cscB, cscK and cscA genes from the original csc operon in the W strain, but did not contain the functional cscR gene encoding the repressor protein. In addition to the sequence homologous to the csc operon, the primers used for PCR amplification included a 50 base (bp) sequence at the 5' end homologous to the site used for integration into the chromosome of E. coli KJ122 . The PCR primer sequences used in this example are listed in Table 1. The target site for integration was 291 bp upstream of the rrnC gene. This site does not encode any known genes, thus minimizing the possibility of disrup...

Embodiment 2

[0101] Growth and sucrose utilization analysis of the recombinant strain SD14 in a 7-liter fermenter

[0102] The SD14 strain was grown in basal medium supplemented with 10% sucrose. Ammonium hydroxide and ammonium bicarbonate (7N NH 4 OH and 3M NH 4 HCO 3 ) to neutralize the succinic acid produced in the fermentor at 39°C. The initial volume of 3 liters contained potassium dihydrogen phosphate (18 mM), magnesium sulfate (2 mM), betaine (1.33 mM), trace elements (Jantama et al., 2008a,b), antifoam 204 (8 ppm) and batch of 98g / l sucrose. The pH was initially adjusted to pH 7.0 and then maintained at pH 6.5 by addition of the above ammonium hydroxide / ammonium bicarbonate solution. Air was delivered at 5ml / min. A 150 ml inoculum was grown aerobically and contained basal medium with 2% sucrose supplemented with 0.1 mM calcium chloride.

[0103] For comparison, KJ122 was fermented in the same medium with the same neutralizing solution, except that the carbon source was gluco...

Embodiment 3

[0106] Cloning and expression of sucrose phosphorylase in SD14

[0107] The sucrose phosphorylase gene from Leuconostoc enteritidis strain DSM 20193 (Goedl et al., 2007) was cloned by PCR amplification using primers BY107 and BY108 (see Table 1) and genomic DNA as template. The obtained 1594 bp PCR product was purified on a Quiagen QIAquick PCR purification column, cut with Xbal and BamHI restriction enzymes (New England Biolabs), and purified by agarose gel electrophoresis. The obtained fragment was ligated into the Xbal to BamHI backbone of pOM324 (US Patent Application 2009 / 0311756) to generate plasmid pRY801, which places the sucrose phosphorylase gene under the control of a strong constitutive promoter. The promoter-sucrose phosphorylase gene-terminator cassette was excised from pRY801 by cutting with XhoI and BamHI, and the sticky ends of the resulting cassette were filled in with the Quick Blunting Kit (New England Biolabs), and the blunt-ended fragments were ligated int...

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Abstract

This invention relates to the production of chemicals by fermentation with a microorganism in which the fermentation medium contains the sugar sucrose. As a specific example, succinic acid is produced from a sucrose-containing renewable feedstock through fermentation using a biocatalyst. Examples of such a biocatalyst include microorganisms that have been enhanced in their ability to utilize sucrose 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 other than sucrose. The genetic manipulations of the present invention involve the introduction of exogenous genes involved in the transport and metabolism of sucrose into the parental strains. The genes involved in the transport and metabolism of sucrose 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 sucrose transport and metabolism by strains already known to have some ability for sucrose utilization in biological fermentation.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 61 / 459,446, filed December 13, 2010. field of invention [0003] The present invention is in the field of the production of specialty and commodity organic chemicals using biocatalysts that have been modified to increase their ability to use feedstocks containing sucrose. More specifically, the present invention relates to genetic modifications necessary for sucrose transport and metabolism in the biological production of succinate and other chemicals. Background of the invention [0004] Currently, a large number of organic chemicals are derived from petrochemical feedstocks. There is growing interest in the production of many of these petrochemically derived organic compounds by biological fermentation methods using renewable feedstocks. The list of organic compounds that can be derived from renewable raw materials includes 1,4-diacids (succi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/70C12P7/46C12P7/02
CPCC12P7/46C12R1/01C12P7/18C12N9/16C12Y301/03024C12P7/42C12N9/1051C12N1/205C12R2001/01
Inventor S·多勒R·R·约卡姆T·赫尔曼X·于
Owner PTT GLOBAL CHEMICAL PUBLIC COMPANY LIMITED
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