Genetically modified microorganisms having improved tolerance towards L-serine
Inventors
Mundhada, Hemanshu • Nielsen, Alex Toftgaard
Assignees
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Publication Number
US-11407976-B2
Publication Date
2022-08-09
Expiration Date
Abstract
The present invention generally relates to the microbiological industry, and specifically to the production of L-serine or L-serine derivatives using genetically modified bacteria. The present invention provides genetically modified microorganisms, such as bacteria, wherein the expression of genes encoding for enzymes involved in the degradation of L-serine is attenuated, such as by inactivation, which makes them particularly suitable for the production of L-serine at higher yield. The present invention also provides means by which the microorganism, and more particularly a bacterium, can be made tolerant towards higher concentrations of serine. The present invention also provides methods for the production of L-serine or L-serine derivative using such genetically modified microorganisms.
Core Innovation
The document relates to genetically modified Escherichia coli bacterium for producing L-serine and L-serine derivatives with improved tolerance and improved yield. It describes an Escherichia coli bacterium expressing a polypeptide having at least 95% sequence identity to the amino-acid sequence set forth in SEQ ID NO:12, wherein position 143 D is replaced by another amino acid, including D to G.
The document further describes attenuating L-serine degradation by inactivating serine degradation-associated genes, including sdaA, sdaB, tdcG, and glyA, and describes nucleotide substitutions within the lrp gene that cause an amino acid substitution at position 143 in the encoded polypeptide. It also describes isolated nucleic acids and expression vectors comprising the ThrA mutations, and genome-based engineering intended to improve L-serine tolerance.
The document also describes introducing additional serine-tolerant traits, including mutations in ThrA at specified positions and overexpression of an exporter, ydeD. It reports improved growth and higher specific productivity or yield under conditions that include minimal medium with high L-serine concentrations, with engineered strains assembled with multiple mutations and pathway-related gene alterations.
Claims Coverage
Two independent claim themes are present across the items. The inventive coverage centers on an Escherichia coli bacterium expressing a polypeptide with at least 95% sequence identity to SEQ ID NO: 12 and a position-143 D replacement, with dependent embodiments defining the D-to-G substitution, nucleotide substitutions in lrp, inactivation of serine degradation-related genes, and cultivation-based production of L-serine or L-serine derivatives.
Escherichia coli expressing a polypeptide with defined sequence identity and position-143 replacement
An Escherichia coli bacterium expresses a polypeptide having at least 95% sequence identity to the amino-acid sequence set forth in SEQ ID NO:12, wherein position 143 D is replaced by another amino acid.
Position-143 D to G substitution
The bacterium expresses a polypeptide having at least 95% sequence identity to SEQ ID NO:12, wherein the amino acid at position 143 D is replaced by G.
Nucleotide substitutions in the lrp gene causing the position-143 amino-acid substitution
The bacterium has one or more nucleotide substitutions within the lrp gene that cause an amino acid substitution at position 143 in the encoded polypeptide.
Inactivation of serine degradation-associated genes
The bacterium has at least one, and in some embodiments at least two, genes selected from sdaA, sdaB, tdcG, and glyA inactivated.
Cultivation to produce L-serine or L-serine derivatives
A method to produce L-serine or an L-serine derivative by cultivating the bacterium in a culture medium.
The claim set centers on an engineered Escherichia coli expressing a SEQ ID NO:12-like polypeptide with a defined position-143 substitution, including the D-to-G embodiment and the lrp nucleotide changes that create the substitution, together with optional inactivation of sdaA, sdaB, tdcG, and glyA and cultivation for L-serine or L-serine derivative production.
Stated Advantages
Improved L-serine production or yield in tolerant engineered strains.
Improved growth in minimal medium with high L-serine concentrations.
Higher specific productivity or higher yield.
Documented Applications
Production of L-serine or L-serine derivatives by cultivating the engineered Escherichia coli bacterium in a culture medium.
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