Engineered microorganisms with g3p---> 3pg enzyme and/or fructose-1,6-bisphosphatase including those having synthetic or enhanced methylotrophy

A technology of methyl nutrition and microorganisms, applied in the fields of biochemical equipment and methods, biofuels, enzymes, etc., can solve the problems of unsuitability for genetic engineering, unfavorable industrial production, and few metabolic intermediates.

Pending Publication Date: 2021-07-23
GENOMATICA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Methylotrophic microorganisms are generally not conducive to industrial production, because many methylotrophic microorganisms have strict aerobic requirements, produce relatively few metabolic intermediates, and are therefore far from suitable for facile genetic engineering
Therefore, it is challenging to develop synthetic methylnutrients in industrially useful microorganisms

Method used

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  • Engineered microorganisms with g3p---> 3pg enzyme and/or fructose-1,6-bisphosphatase including those having synthetic or enhanced methylotrophy
  • Engineered microorganisms with g3p---> 3pg enzyme and/or fructose-1,6-bisphosphatase including those having synthetic or enhanced methylotrophy
  • Engineered microorganisms with g3p---> 3pg enzyme and/or fructose-1,6-bisphosphatase including those having synthetic or enhanced methylotrophy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0303] Example 1-GAPN and GAPA dynamics contrast

[0304] The catalytic constant of E. coli GAPA and the GAPN of Bacillus Methanol was measured and shown in Table 18.

[0305] Table 18.

[0306]

Embodiment 2

[0307] Example 2 - Due to GAPN expression to obtain intracellular metabolism

[0308] The intracellular metabolic production spectrum of Escherichia coli, of which GAPN enzyme (SEQ ID: 1 having 95% ID) and hex ketosyl-6-phosphatase (HPS) with SEQ ID: 1-phosphatase (HPS) (SEQ ID: 2) Combination, 6-phosphate-3-hexazone isomerase (PHI) (SEQ ID: 3) was compared to strains expressing Gapa and expressed the same HPS and PHI enzymes. The strain also lacks GAPA, expresses methanol dehydrogenase, and expresses FBA, GLPX, RPE, and TKTT from Bacillurbium. The expression of GAPN results in an increase in the metabolites of the related RUMP circulating metabolites compared to strains with GAPA expression without GAPN. This suggests that GAPN expression is the benefit of RUMP cycle activity and methyl nutrition. Table 19 shows the multiple variations of the key RUMP circulating metabolies needed to be related to methyl nutrition.

[0309] Table 19: The multiplication of RUMP circulating metabol...

Embodiment 3

[0311] Example 3 - Synthetic Methyl Nutrition in Culture

[0312] Escherichia coli strain (overexpression of GAPN (login number WP_003351798; with SEQ ID: 1 with 95% ID), Gapa deletion, other enzymes, methanol dehydrogenase, HPS (SEQ ID: 2), PHI (SEQ ID: 3 ), FBA, GLPX, TKT) and its parent strains expressing GAPA culture were cultured in a constant medium containing glucose and methanol. The constant bioreactor is initiated in a batch culture mode and operated under aerobic conditions, with glucose is carbon source, temperature control at 35 degrees Celsius, and the oxygen is controlled in 20% or more, and the gas flow is changed and stirred. The pH is constant at 6.95 by automatically adding base or acid when necessary. By a dilution rate of 0.1H-1, a medium containing methanol as a single carbon source is performed, and the fermenter is continuously operated, and the transition to methanol is performed. The methanol feed was started before glucose depletion, and the methanol gro...

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Abstract

Described herein are engineered cells including ones having synthetic methylotrophy which include an NADH-dependent enzyme capable of converting G3P to 3PG (e.g.,6. methanolicus gapN) and / or fructose-1, 6-bisphosphatase, along with hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, a phosphoketolase, or a combination thereof. Engineered cells of the disclosure beneficially maintain adequate pool sizes of phosphorylated C3 and / or C4 compounds, and / or provide increased levels of NADPH. As such, the modifications allow for the generation of C6 compounds from Cl (e.g. a methanol feedstod) and C5 compounds, the regeneration of C5 compounds from C6 compounds by carbon rearrangement, and an improved balance between regeneration of C5 compounds and lower glycolysis.

Description

[0001] priority claim [0002] This application claims Serial Number 62 / 690,209, filed on June 26, 2018, titled "Including those engineered microorganisms with synthetic or enhanced methylotrophy, having G3P → 3PG enzymes and / or fructose-1,6 -Engineered Microorganism for Bisphosphatase," the disclosure of which is incorporated herein by reference. At the same time, the entire contents of the 29.2 kilobyte ASCII text file titled "GNO0088WO_Sequence_Listing.txt" created on June 25, 2019 are incorporated herein by reference. Technical field [0003] The present invention relates to engineered microorganisms with synthetic or enhanced methylotrophy and engineered microorganisms utilizing NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and / or fructose-1,6-bisphosphatase. Background technique [0004] Methylotrophy involves the ability of microorganisms to utilize one-carbon (C1) compounds such as methanol and methane as energy and carbon sources. Carbon feedstocks cont...

Claims

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Application Information

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IPC IPC(8): C12P5/00C12P5/02C12P7/04C12P7/16C12P7/18C12P7/24C12P7/40C12P7/42C12P7/46C12P7/64C12P13/00C12N9/02C12N9/16C12N9/88C12N9/90
CPCC12P5/007C12P5/026C12P7/04C12P7/16C12P7/18C12P7/24C12P7/40C12P7/46C12P7/42C12P7/6409C12P13/005C12P13/001C12N9/0008C12N9/88C12N9/90C12N9/16C12Y102/01012C12Y301/03011C12Y401/02009C12Y401/02043C12Y503/01027C12N15/52
Inventor 哈里什·纳加拉让杨泰勋艾利·阔达雅里
Owner GENOMATICA INC
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