Method for the production of L-serine using genetically engineered microorganisms deficient in serine degradation pathways
Inventors
Mundhada, Hemanshu • Nielsen, Alex Toftgaard
Assignees
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Publication Number
US-10513682-B2
Publication Date
2019-12-24
Expiration Date
Abstract
The present invention generally relates to the microbiological industry, and specifically to the production of L-serine 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 invention relates to an engineered bacterium belonging to the Enterobacteriaceae family that is modified to inactivate the genes sdaA, sdaB, tdcG and glyA, so that serine degradation is attenuated while production of L-serine and L-serine derivatives is enabled. It further addresses serine toxicity and serine inhibition by overexpression of the serine exporter ydeD and by thrA mutations at positions Y356, S357 and S359, including Y356C, S357R and S359R.
The disclosed engineered strains further include specified gene mutations such as rpoB P520 replacement (P520L), fumB T218→P, gshA A178→V, and lamB truncation/stop at Q112*, together with additional genome mutations and structural genetic alterations such as deletions and IS insertions. The partial content also discusses strategies to attenuate gene expression, including zwf/G6PDH attenuation, as part of improving L-serine yield together with the tolerance-conferring modifications.
The document provides cultivation-based production concepts in a culture medium and a broad strain-genus range within Enterobacteriaceae-family bacteria. It further references producing L-serine or an L-serine derivative by modifying genetic content to improve both tolerance and production, including by identifying causative mutations through reverse engineering.
Claims Coverage
The independent claim set centers on an Enterobacteriaceae-family bacterium with inactivated serine-related genes and a cultivation method for producing L-serine or an L-serine derivative. Four inventive features are identified in the independent claims, with additional dependent refinements directed to overexpression targets, defined mutation patterns, and growth/tolerance constraints in minimal culture medium.
Enterobacteriaceae bacterium with serine degradation gene inactivation
A bacterium belonging to the Enterobacteriaceae family, which has been modified to inactivate the genes sdaA, sdaB, tdcG and glyA.
Additional enzymatic overexpression set
A modified bacterium that is additionally engineered to overexpress 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, and phosphoserine aminotransferase.
Gene ydeD overexpression
A modified bacterium is further engineered to overexpress the gene ydeD.
Defined nucleotide substitution-driven amino-acid and stop-codon mutations tied to SEQ ID references
A bacterium is characterized as comprising one or more specified gene mutations, defined as nucleotide substitutions in particular genes that produce the listed amino acid substitutions, including specified stop codons, in the amino acid sequences of the indicated SEQ ID NOs.
Cultivation to produce L-serine or an L-serine derivative
A method cultivates a bacterium in a culture medium to produce L-serine or an L-serine derivative.
Minimal-medium growth with L-serine concentration and exponential-growth rate constraint
A bacterium that can grow in minimal culture medium containing L-serine at a concentration of at least about 6.25 g/L with an exponential-growth rate of at least 0.1 hr−1 during exponential growth.
The claims cover an Enterobacteriaceae-family bacterium with inactivation of sdaA, sdaB, tdcG and glyA, further refined by overexpression targets including 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, phosphoserine aminotransferase, and ydeD, as well as specified nucleotide substitutions producing amino-acid changes and stop codons tied to SEQ ID references. The claims also cover cultivation-based production of L-serine or an L-serine derivative and a minimal-medium growth constraint.
Stated Advantages
Provides L-serine tolerance.
Improves L-serine yield.
Enables growth in minimal culture medium containing L-serine at specified concentration with a specified exponential-growth rate.
Enables attenuation of serine degradation while producing L-serine and L-serine derivatives.
Improves serine tolerance by overexpressing the serine exporter ydeD.
Reduces serine inhibition through thrA mutations at Y356, S357, and S359.
Supports improved growth/titer concepts in cultivation to produce L-serine or an L-serine derivative.
Documented Applications
Cultivation of an Enterobacteriaceae-family bacterium in a culture medium to produce L-serine or an L-serine derivative.
Producing L-serine by cultivating a modified bacterium in a culture medium.
Producing an L-serine derivative by cultivating the modified bacterium in a culture medium.
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