Archer Daniels Midland Company

P John Rayapati

Corey M Crafton

Beth Fatland-Bloom

Nyerhovwo John Tonukari

Gyan P Rai

Paul D Hanke

Holly J Walsh

Lhing-Yew Li-D'Elia

Nucleotide and protein sequences that encode enzymes that change carbon flux through metabolic pathways that lead to lactic acid or fumarate production in a host cell, such as acell, are provided. Methods of manipulating carbon flux in a cell also are provided.

Compositions and methods for manipulating carbon flux in cells

The present invention is directed towards the fermentative production of amino acids, providing microorganisms, methods and processes useful therefor. Microorganisms of the invention are capable of converting glucose to amino acids other than L-isoleucine, L-leucine and L-valine with greater efficiency than the parent strain. The efficiency of conversion may be quantified by the formula: [(g amino acid produced/g dextrose consumed)*100]=% Yield and expressed as yield from dextrose. The invention provides microorganisms that are made auxotrophic or bradytrophic for the synthesis of one or more branched chain amino acids by mutagenesis and selected for their ability to produce higher percent yields of the desired amino acid than the parental strain. Preferred microorganisms areorproducing L-lysine. Mutagenesis is performed by classical techniques or through rDNA methodology. Methods of the invention are designed to increase the production of an amino acid by mutagenizing a parental strain, selecting cells auxotrophic or bradytrophic for the synthesis of one or more branched chain amino acids and selecting branched chain amino acid auxotrophs or bradytrophs that produce a higher percent yield from dextrose of the desired amino acid than the parental strain. Processes of the invention are designed for the production an amino acid comprising culturing in a medium a microorganism obtained by mutagenizing a parent strain to be auxotrophic or bradytrophic for branched chain amino acid synthesis and selecting variants that are capable of converting glucose to amino acids other than L-isoleucine, L-leucine and L-valine with greater efficiency than the parent strain.

Metabolic engineering of amino acid production

Nucleotide sequences and genetic constructs that can be used to regulate genes encoding enzymes that change carbon flux through metabolic pathways that lead to lactic acid or fumarate production in a host cell, such as acell, are provided. Methods of manipulating carbon flux in a cell also are provided.

The present invention is directed towards the fermentative production of amino acids, providing microorganisms, methods and processes useful therefor. Microorganisms of the invention are capable of converting glucose to amino acids other than L-isoleucine, L-leucine and L-valine with greater efficiency than the parent strain. The efficiency of conversion may be quantified by the formula: [(g amino acid produced/g dextrose consumed)*100]=% Yield and expressed as yield from dextrose. The invention provides microorganisms that are made bradytrophic for the synthesis of valine by mutagenesis.

The invention relates to isolated polynucleotides fromwhich are useful in the regulation of gene expression. In particular, the invention relates to isolated polynucleotides comprisingpromoters which may be used to regulate, i.e., either increase or decrease, gene expression. In certain embodiments, isolated promoter sequences of the present invention regulate gene expression through the use of exogenous or endogenous induction. The invention further provides recombinant vectors and recombinant cells comprising isolated polynucleotides of the present invention, preferably in operable association with heterologous genes. Also provided are methods of regulating bacterial gene expression comprising growth of a recombinant cell of the present invention. In particular, the present invention provides methods to regulate genes involved in amino acid production comprising growth of a recombinant cell of the present invention. In certain embodiments, the present invention provides methods of regulating gene expression in bacteria, particularlyspecies, especially of the genus, comprising fermentation growth of a recombinant cell of the present invention, where metabolite concentrations, temperature, or oxygen levels are manipulated to regulate gene expression.

Nucleotide sequences for transcriptional regulation in corynebacterium glutamicum

The invention provides methods to increase the production of an amino acid from Corynebacterium species by way of the amplification of amino acid biosynthetic pathway genes in a host cell chromosome. In a preferred embodiment, the invention provides methods to increase the production of L-lysine inby way of the amplification of L-lysine biosynthetic pathway genes in a host cell chromosome. The invention also provides novel processes for the production of an amino acid by way of the amplification of amino acid biosynthetic pathway genes in a host cell chromosome and/or by increasing promoter strength. In a preferred embodiment, the invention provides processes to increase the production of L-lysine inby way of the amplification of L-lysine biosynthetic pathway genes in a host cell chromosome. The invention also provides novel isolated nucleic acid molecules for L-lysine biosynthetic pathway genes ofsuch as a naturally occurring, feedback-sensitive form of aspartokinase (ask) resulting from a threonine to isoleucine mutation at amino acid residuein the ask gene of ATCC 21529, aspartate-semialdehyde dehydrogenase (asd), dihydrodipicolinate synthase (dapA), dihydrodipicolinate reductase (dapB), diaminopimelate dehydrogenase (ddh), and diaminopimelate decarboxylase (lysA).

Increased lysine production by gene amplification using coryneform bacteria

The present invention provides a method of increasing the productivity of a microorganism by improving the assimilation of carbon dioxide. Specifically, the invention provides a polypeptide having phosphoenolpyruvate carboxylase activity which does not require acetyl coenzyme A for activation and is desensitized to feedback inhibition by aspartic acid, and to genes coding for this polypeptide. A gene encoding a PEP carboxylase that is not regulated by acetyl-CoA or aspartic acid can improve carbon flow from the three carbon intermediate PEP to the four carbon intermediate OAA, contribute to compounds derived from OAA, and increase amino acid biosynthesis. The invention further provides recombinant DNA molecules containing these genes, bacteria transformed with these genes, and a method of producing amino acids using the transformed bacteria.

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Monticello, IL, United States of America

P John Rayapati