Pentose phosphate pathway upregulation to increase production of non-native products of interest in transgenic microorganisms

A transgenic and microbial technology, applied in the direction of microorganisms, microorganisms, biochemical equipment and methods, etc., can solve problems that hinder the function, toxicity, and acceleration of the PP pathway

Inactive Publication Date: 2013-07-03
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Based on this observation, Miclet et al. concluded that 6PGL activity accelerates the hydrolysis of the delta form, thus preventing its conversion to the gamma form, and that 6PGL prevents the accumulation of delta-6-P-G-L, a product likely via its interaction with endogenous cellular Reaction of nucleophiles produces toxicity and hampers PP pathway function

Method used

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  • Pentose phosphate pathway upregulation to increase production of non-native products of interest in transgenic microorganisms
  • Pentose phosphate pathway upregulation to increase production of non-native products of interest in transgenic microorganisms
  • Pentose phosphate pathway upregulation to increase production of non-native products of interest in transgenic microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0275] Overexpression of glucose-6-phosphate dehydrogenase in Yarrowia lipolytica strain Y2107U ("G6PDH")

[0276] This example describes the construction of plasmid pZWF-MOD1 ( Figure 2A ; SEQ ID NO:7) to enable overexpression of the Yarrowia gene encoding glucose-6-phosphate dehydrogenase ["G6PDH"] under the control of the strong native Yarrowia promoter.

[0277] The PUFA-producing Yarrowia lipolytica strain Y2107U was transformed with the overexpression plasmid, and the effect of overexpression on cell growth and lipid synthesis was determined and compared. Specifically, overexpression of G6PDH caused decreased cell growth.

[0278] Construction of plasmid pZWF-MOD1 containing Yarrowia G6PDH

[0279] The Yarrowia lipolytica G6PDH ORF contains an intron near the 5' end (nucleotides 85-524 of SEQ ID NO: 10). The nucleotide sequence of the cDNA encoding G6PDH is shown in SEQ ID NO:1.

[0280] Primers YZWF-F1 (SEQ ID NO: 8) and YZWF-R (SEQ ID NO: 9) were designed to...

Embodiment 2

[0299] Construction of plasmid pZKLY-PP2 for glucose-6-phosphate dehydrogenase ["G6PDH"] and glucose-6-phosphate Co-regulated overexpression of glycolactonase ["6PGL"]

[0300] This example describes the construction of plasmid pZKLY-PP2 ( Figure 3A ; SEQ ID NO: 15) Overexpression of Yarrowia encoding glucose-6-phosphate dehydrogenase ["G6PDH"] and 6-phosphogluconolactonase ["6PGL"] in a co-regulated manner Gene. Specifically, the weak native Yarrowia promoter was chosen to drive expression of G6PD, while the strong native Yarrowia promoter was operably linked to 6PGL. This process was designed to ensure rapid conversion of 6-phosphogluconolactone to 6-phosphogluconate and thereby avoid accumulation of toxic levels of 6-phosphogluconolactone.

[0301] Construction of plasmid pZKLY-PP2 to overexpress G6PDH and 6PGL

[0302] Construction of plasmid pZKLY-PP2 first required separate amplification of the Yarrowia 6PGL and G6PDH genes and ligation of each corresponding ge...

Embodiment 3

[0310] Glucose-6-phosphate dehydrogenase ["G6PDH"] and 6- Co-regulated overexpression of phosphogluconolactonase ["6PGL"] increases accumulated total lipid

[0311] This example describes the transformation of PUFA producing Yarrowia lipolytica strain Y4305U with plasmid pZKLY-PP2 and the effect of co-regulated overexpression of G6PDH and 6PGL on cell growth and lipid synthesis. Specifically, co-regulated overexpression of G6PDH and 6PGL resulted in an increase in the total amount of lipids expressed as a percentage of DCW and the amount of PUFAs expressed as a percentage of TFA in transformed cells.

[0312] Y. lipolytica strain Y4305U (General Methods) was transformed with the 8.5 kB AscI / SphI fragment of pZKLY-PP2 (SEQ ID NO: 15; Example 2) (following General Methods). Transformants were selected on SD medium plates lacking uracil. The three pZKLY-PP2 transformants were referred to as strains PP12, PP13 and PP14.

[0313] For lipid analysis, pZKLY-PP2 transformants an...

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Abstract

Coordinately regulated over-expression of the genes encoding glucose 6-phosphate dehydrogenase ["G6PDH"] and 6-phospho-gluconolactonase ["6PGL"] in transgenic strains of the oleaginous yeast, Yarrowia lipolytica, comprising a functional polyunsaturated fatty acid ["PUFA"] biosynthetic pathway, resulted in increased production of PUFAs and increased total lipid content in the Yarrowia cells. This is achieved by increased cellular availability of the reduced form of nicotinamide adenine dinucleotide phosphate ["NADPH"], an important reducing equivalent for reductive biosynthetic reactions, within the transgenic microorganism.

Description

[0001] This application claims priority to US Provisional Patent Application 61 / 319473, filed March 31, 2010, which is hereby incorporated by reference in its entirety. field of invention [0002] The invention belongs to the field of biotechnology. More specifically, the present invention relates to co-regulation based pentose phosphate pathway genes (e.g., glucose-6-phosphate dehydrogenase ["G6PD"] and 6-phosphogluconolactonase ["6PGL"]) A method for manipulating the cellular availability of the reduced form of nicotinamide adenine dinucleotide phosphate ["NADPH"] in transgenic microorganisms. Background of the invention [0003] Cofactor vs. NADPH / NADP + Is required by all living organisms primarily because it serves as a donor and / or acceptor of reducing equivalents in multiple oxidation-reduction reactions during anabolism. For example, NADPH is essential for the production of amino acids, vitamins, aromatic compounds, polyols, polyamines, hydroxyesters, isoprenoids, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P1/00C12N1/00C12N1/13C12N1/19C12N1/15
CPCC12N9/0006C12N9/18C12P7/6427C12P7/6472C12P7/66C12P23/00C12P33/00C12N15/52C12P7/6431
Inventor S-P.洪Z.薛Q.Q.朱
Owner EI DU PONT DE NEMOURS & CO
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