Methods and compositions for preventing erroneous incorporation of n-leucine into proteins

Abstract

The present invention relates to methods and compositions for preventing erroneous incorporation of n-leucine into a protein during the production of a recombinant protein in a bacterium. The invention also provides microbial host cells and nucleic acid molecules for use in the methods and compositions provided herein.

Description

Methods and compositions for preventing the erroneous incorporation of oroleucine into proteins.

[0001] This application is a divisional application of application CN 202011466081.6, filed on September 19, 2013, entitled "Method and Composition for Preventing Erroneous Incorporation of Leucine into Proteins".

[0002] Related applications

[0003] This application claims the benefits of U.S. Provisional Application No. 61 / 777,700, filed March 12, 2013, and U.S. Provisional Application No. 61 / 703,142, filed September 19, 2012, both of which are incorporated herein by reference in their entirety.

[0004] sequence list

[0005] This application contains a sequence list submitted via EFS-Web in ASCII format, and is incorporated herein by reference in its entirety. The ASCII copy was created on September 17, 2013, named P4967R1_WO_SequenceList.txt, and is 45,847 bytes in size. Technical Field

[0006] This invention relates to methods and compositions for preventing the erroneous incorporation of ortholeucine into proteins during the production of recombinant proteins in bacteria. The invention also provides microbial host cells and nucleic acid molecules for use in the methods and compositions provided herein. Background Technology

[0007] Leucine, an analogue of the amino acid methionine, can be incorrectly incorporated into proteins in place of methionine residues. When expressed in *Escherichia coli*, many heterologous proteins have leucine incorrectly incorporated into the positions where methionine residues should be. The incorrect incorporation of leucine into proteins, particularly in heterologous proteins produced through recombinant methods, is generally considered undesirable, partly because it results in proteins with altered, undesirable properties.

[0008] The misincorporation of ortholeucine at the methionine position during the production of recombinant proteins in *E. coli* has been observed for over 50 years (see, for example, Munier and Cohen (1959) *Biochim Biophys Acta 31:378-391; Cohen and Munier (1956) *Biochim Biophys Acta 21:592-593; Cohen and Munier (1959) *Biochim Biophys Acta 31:347-356; and Cowie et al., (1959) *Biochim Biophys Acta 34:39-46). For instance, during the recombinant production of methionyl bovine growth hormone (MBS) protein in *E. coli*, approximately 14% of the methionine residues in this protein exhibited misincorporation of ortholeucine, compared to approximately 6% of the methionine residues in native *E. coli* proteins which are also replaced by ortholeucine. (See Bogosian et al., (1989) J Biol Chem 264:531-9). In another instance, the production of interleukin-2 in basal E. coli fermentation resulted in the substitution of approximately 19% of methionine residues with ortholeucine in the recombinant protein (see Tsai et al., (1988) Biochem Biophys Res Commun 156:733-739). Other studies have shown that the misincorporation of ortholeucine residues into proteins can occur at both internal methionine residues and N-terminal methionine residues (see Brown (1973) Biochim Biophys Acta 294:527-529; and Barker and Bruton (1979) J Mol Biol 133:217-231).

[0009] Due to the generalized nature of the enzyme methionyl-tRNA synthetase (MetG), ortholeucine competes with methionine for incorporation into proteins (see Trupin et al., (1966) Biochem Biophys Res Commun 24:50-55; and Fersht and Dingwall (1979) Biochemistry 18:1250-1256). Kinetic studies using E. coli MetG enzymes have shown that MetG acylation using methionine is approximately four times more efficient than using ortholeucine (see van Hest et al., (2000) Am Chem Soc 122:1282-1288). Due to the non-strict substrate specificity of MetG, ortholeucine can substitute for methionine in acylation reactions, leading to the erroneous incorporation of ortholeucine into proteins instead of methionine.

[0010] In recombinant protein production, the misincorporation of leucine residues replacing methionine residues is generally considered undesirable. Recombinant proteins or peptides containing misincorporated leucine residues can exhibit altered structural and functional characteristics, such as, for example, altered sensitivity to proteolysis, reduced biological activity, or increased immunogenicity.

[0011] Various strategies have been developed to reduce or prevent the erroneous incorporation of ortholeucine during recombinant protein production. For example, the probability of incorrect ortholeucine loading by methionine-tRNA has been reduced by ensuring that cells have an excess of methionine through continuous or bolus-feed / addition of methionine during fermentation (see, for example, U.S. Patent No. 5,599,690). While continuous or bolus-feed / addition of methionine reduces the degree of erroneous ortholeucine incorporation into recombinant proteins, it can increase the operational complexity and cost of the fermentation process. Additionally, continuous or bolus-feed / addition of methionine during fermentation can lead to undesirable dilution of the fermenter contents, resulting in lower cell density and lower product yield.

[0012] The erroneous incorporation of ortholeucine into proteins has been reduced by deleting genes involved in the ortholeucine biosynthesis pathway, such as deleting genes for leucine operons (leuA, leuB, leuC, and leuD) or deleting transaminase-encoding genes such as ilvE or tyrB (see Bogosian et al., (1989) J Biol Chem 264:531-539; Tsai et al., (1989) Biochem Biophys Res Commun 156:733-739; and Randhawa et al., (1994) Biochemistry 33:4352-4362). However, in order to prevent the deletion of genes involved in the biosynthetic pathway of ortholeucine erroneously incorporated, it may be necessary to add other amino acids (such as leucine or isoleucine) to the culture medium during fermentation, because many genes involved in ortholeucine biosynthesis are also involved in the biosynthesis of branched-chain amino acids (see Bogosian et al., (1989) J Biol Chem 264:531-539; see Figure 8 in this specification).

[0013] Another strategy to prevent the mis-incorporation of ortholeucine involves the co-expression of enzymes that degrade ortholeucine, including, for example, amino acid dehydrogenases and amino acid oxidases. However, this approach requires overexpression of these enzymes, which can be undesirable in recombinant protein production and may result in lower recombinant protein yields (see, for example, U.S. Patent Application Publication No. US2007 / 0009995). Furthermore, overexpression of these enzymes may lead to the degradation of other similar amino acids during fermentation. The mis-incorporation of ortholeucine has also been prevented by altering the primary amino acid sequence of the polypeptide to be expressed by substituting other codons for the methionine codon (see, for example, U.S. Patent No. 5,698,418). However, such substitutions can lead to reduced activity or structural changes in the resulting protein, which is a highly undesirable outcome for recombinant protein production in the biotechnology industry.

[0014] As noted above, current methods for preventing the erroneous incorporation of ortholeucine during the production of recombinant proteins in microorganisms have various drawbacks; therefore, a new method is needed to prevent or reduce the erroneous incorporation of ortholeucine into proteins, particularly during the production of recombinant proteins in microorganisms such as Escherichia coli.

[0015] This invention addresses this need by providing engineered microbial host cells, such as bacteria, that effectively prevent the erroneous incorporation of ortholeucine during the production of recombinant proteins in microorganisms. Among other things, the invention provides *E. coli* host cells containing mutated metA and metK alleles (i.e., altered metA and metK nucleic acid sequences), resulting in methionine production by the microorganism to a degree or extent sufficient to reduce or prevent the erroneous incorporation of ortholeucine into proteins and peptides. Analysis of recombinant proteins produced using such host cells shows the elimination of erroneous incorporation of ortholeucine residues replacing methionine residues. The invention also demonstrates that the fermentation process performance using such *E. coli* host cells, including host cell growth and the titer of recombinant protein products using such *E. coli* host cells, is comparable to that observed in control host cells. Summary of the Invention

[0016] A portion of this invention provides methods and compositions for preventing or reducing the erroneous incorporation of oroleucine into proteins and peptides. The methods and compositions of this invention can be used to prevent or reduce the erroneous incorporation of oroleucine into heterologous (e.g., recombinant) proteins and peptides expressed by microorganisms such as bacteria (e.g., *Escherichia coli*).

[0017] In some embodiments, the present invention provides a method for preventing or reducing the erroneous incorporation of oroleucine into proteins or peptides expressed by microorganisms, wherein the microorganisms produce methionine to a degree or extent sufficient to prevent or reduce the erroneous incorporation of oroleucine into the protein or peptide. In some embodiments, the microorganisms are feedback-resistant or feedback-insensitive homoserine succinyltransferase microorganisms. In other embodiments, the microorganisms are microorganisms containing a mutated metA allele, a mutated metK allele, or both a mutated metA allele and a mutated metK allele.

[0018] In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into proteins or peptides, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism comprises a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence encoding an amino acid substitution in MetA, the MetA amino acid substitution being selected from an arginine-cysteine ​​substitution at amino acid position 27, a glutamine-glutamate substitution at amino acid position 64, a tyrosine-cysteine ​​substitution at amino acid position 294, an isoleucine-serine substitution at amino acid position 296, and a proline-leucine substitution at amino acid position 298. In some embodiments, the mutated metA allele comprises a nucleic acid sequence encoding an amino acid substitution in MetA, the amino acid substitution in MetA including an isoleucine-serine substitution at amino acid position 296 and a proline-leucine substitution at amino acid position 298. The MetA amino acid positions described herein are referenced to the wild-type MetA amino acid sequence shown in Figure 7A and SEQ ID NO: 29.

[0019] In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of oroleucine into a protein or polypeptide, the method comprising expressing the protein or polypeptide in a microorganism, wherein the microorganism comprises a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

[0020] As described above, the present invention provides a method for preventing or reducing the erroneous incorporation of oroleucine into proteins or peptides expressed by microorganisms, wherein the microorganisms produce methionine to a degree or range sufficient to prevent or reduce the erroneous incorporation of oroleucine into the protein or peptide. In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of oroleucine into proteins or peptides expressed by microorganisms, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism is a microorganism that relieves methionine production inhibition. In some embodiments, the microorganism relieves methionine production inhibition due to partial loss of S-adenosylmethionine synthase function. In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of oroleucine into proteins or peptides expressed by microorganisms, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism includes a mutated metK allele. In some embodiments, the mutated metK allele results in partial loss of MetK function.

[0021] In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or peptide, the method comprising expressing the protein or peptide in a microorganism comprising a mutated metK allele, wherein the mutated metK allele comprises a nucleic acid sequence encoding an amino acid substitution in MetK, the amino acid substitution in MetK comprising a substitution of valine to glutamic acid at amino acid position 185. In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or peptide, the method comprising expressing the protein or peptide in a microorganism comprising a mutated metK allele, wherein the mutated metK allele comprises a nucleic acid sequence containing a cytosine base deletion at nucleic acid residue position 1132 of the metK allele. The MetK amino acid position described herein is referenced to the wild-type MetK amino acid sequence shown in Figure 8A and SEQ ID NO: 30. The metK nucleic acid position described herein is referenced to the wild-type metK nucleic acid sequence shown in Figure 8B and SEQ ID NO: 32.

[0022] In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of oroleucine into a protein or polypeptide, the method comprising expressing the protein or polypeptide in a microorganism, wherein the microorganism comprises a mutated metK allele, wherein the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28.

[0023] In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into proteins or peptides, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism comprises a mutated metA allele and a metK allele. In some embodiments, the mutated metA allele comprises a nucleic acid sequence encoding a tyrosine-cysteine ​​substitution at amino acid position 294 of MetA, and the mutated metK allele comprises a nucleic acid sequence encoding a valine-glutamic acid substitution at amino acid position 185 of MetK. In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into proteins or peptides, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism comprises a mutated metA allele and a metK allele, wherein the mutated metA allele comprises a nucleic acid sequence encoding a tyrosine-cysteine ​​substitution at amino acid position 294 of MetA, and wherein the mutated metK allele comprises a nucleic acid sequence containing a cytosine base deletion at nucleic acid residue position 1132 of the metK allele.

[0024] In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or peptide, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism comprises a mutated metA allele and a mutated metK allele, and wherein the mutated metA allele comprises the nucleic acid sequence of SEQ ID NO: 24, and the mutated metK allele comprises the nucleic acid sequence of SEQ ID NO: 27. In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or peptide, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism comprises a mutated metA allele and a mutated metK allele, wherein the mutated metA allele comprises the nucleic acid sequence of SEQ ID NO: 24, and the mutated metK allele comprises the nucleic acid sequence of SEQ ID NO: 28.

[0025] The present invention also provides microbial host cells that are useful for preventing or reducing the erroneous incorporation of ortholeucine into proteins and peptides expressed by microbial host cells. The present invention also provides microbial host cells for expressing proteins or peptides by microbial host cells, wherein the expressed protein or peptide does not contain the erroneous incorporation of ortholeucine. In some embodiments, the microbial host cell is a bacterium. In other embodiments, the microbial host cell is *Escherichia coli*.

[0026] This invention provides microorganisms (e.g., microbial host cells) that produce methionine to a degree or range sufficient to prevent or reduce the erroneous incorporation of ortholeucine into proteins or peptides expressed by the microorganism. In some embodiments, this invention provides microorganisms that are feedback-insensitive homoserine succinyltransferase microorganisms. In other embodiments, this invention provides microorganisms comprising a mutated metA allele. In some embodiments, this invention provides microorganisms comprising a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence encoding an amino acid substitution in MetA, said amino acid substitution being selected from a substitution of arginine to cysteine ​​at amino acid position 27, a substitution of glutamine to glutamate at amino acid position 64, a substitution of tyrosine to cysteine ​​at amino acid position 294, a substitution of isoleucine to serine at amino acid position 296, and a substitution of proline to leucine at amino acid position 298. In some embodiments, the microbial host cell is a bacterium. In other embodiments, the microbial host cell is *Escherichia coli*.

[0027] In some embodiments, the present invention provides a microorganism comprising a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence encoding a substitution of one or more amino acids in MetA. In some embodiments, the mutated metA allele comprises a nucleic acid sequence encoding a substitution of isoleucine to serine at amino acid position 296 of MetA and a substitution of proline to leucine at amino acid position 298 of MetA. In some embodiments, the microbial host cell is bacteria. In other embodiments, the microbial host cell is *Escherichia coli*.

[0028] In some embodiments, the present invention provides a microorganism comprising a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In some embodiments, the microbial host cell is a bacterium. In other embodiments, the microbial host cell is *Escherichia coli*.

[0029] This invention provides microorganisms (e.g., microbial host cells) that produce methionine to a degree or extent sufficient to prevent or reduce the erroneous incorporation of ortholeucine into proteins or peptides expressed by the microorganism. In some embodiments, the invention provides microorganisms that are methionine production inhibitory microorganisms. In some aspects, the methionine production inhibitory microorganisms are generated from partial functional loss of S-adenosylmethionine synthase. In other embodiments, the invention provides microorganisms comprising a mutated metK allele. In some embodiments, the invention provides microorganisms comprising a mutated metK allele, wherein the mutated metK allele comprises a nucleic acid sequence encoding an amino acid substitution in MetK, the amino acid substitution in MetK comprising a substitution of valine to glutamic acid at amino acid position 185. In other embodiments, the invention provides microorganisms comprising a mutated metK allele, wherein the mutated metK allele comprises a nucleic acid sequence containing a cytosine base deletion at nucleic acid residue position 1132 of the metK allele. In some embodiments, the microbial host cell is a bacterium. In other embodiments, the microbial host cell is *Escherichia coli*.

[0030] In some embodiments, the present invention provides a microorganism comprising a mutated metK allele, wherein the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28. In some embodiments, the microbial host cell is a bacterium. In other embodiments, the microbial host cell is *Escherichia coli*.

[0031] The present invention also provides microbial host cells comprising different combinations of mutated metA alleles and mutated metK alleles. In some embodiments, the present invention provides microorganisms comprising mutated metA alleles and mutated metK alleles, wherein the mutated metA allele comprises a nucleic acid sequence encoding a MetA amino acid substitution, the MetA amino acid substitution comprising a tyrosine to cysteine ​​substitution at amino acid position 294, and wherein the mutated metK allele comprises a nucleic acid sequence encoding a MetK amino acid substitution, the MetK amino acid substitution comprising a valine to glutamic acid substitution at amino acid position 185. In other embodiments, the present invention provides microorganisms comprising mutated metA alleles and mutated metK alleles, wherein the mutated metA allele comprises a nucleic acid sequence encoding an amino acid substitution in MetA, the MetA amino acid substitution comprising a tyrosine to cysteine ​​substitution at amino acid position 294, and wherein the mutated metK allele comprises a deletion of cytosine at nucleic acid residue 1132 of the metK allele. In some embodiments, the present invention provides a microorganism comprising a mutated metA allele and a mutated metK allele, wherein the mutated metA allele comprises the nucleic acid sequence of SEQ ID NO: 24, and wherein the mutated metK allele comprises the nucleic acid sequence of SEQ ID NO: 27. In other embodiments, the present invention provides a microorganism comprising a mutated metA allele and a mutated metK allele, wherein the mutated metA allele comprises the nucleic acid sequence of SEQ ID NO: 24, and wherein the mutated metK allele comprises the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the microbial host cell is bacteria. In other embodiments, the microbial host cell is *Escherichia coli*.

[0032] The present invention also provides isolated nucleic acid molecules for use in the methods of the present invention. In some aspects, the present invention provides isolated metA nucleic acid molecules (i.e., isolated nucleic acid molecules encoding MetA). In some embodiments, the present invention provides isolated metA nucleic acid molecules comprising nucleic acid sequences encoding amino acid substitutions in MetA, wherein the amino acid substitutions in MetA are selected from the substitution of arginine to cysteine ​​at amino acid position 27, the substitution of glutamine to glutamate at amino acid position 64, the substitution of tyrosine to cysteine ​​at amino acid position 294, the substitution of isoleucine to serine at amino acid position 296, and the substitution of proline to leucine at amino acid position 298. In other embodiments, the isolated metA nucleic acid molecules provided by the present invention comprise nucleic acid sequences selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In particular, the present invention provides the use of these isolated metA nucleic acid molecules and their sequences for the production of microorganisms, wherein the microorganisms are used to prevent or reduce the erroneous incorporation of ortholeucine into proteins or peptides.

[0033] This invention also provides isolated metK nucleic acid molecules (i.e., isolated nucleic acid molecules encoding MetK). In some embodiments, this invention provides metK nucleic acid molecules comprising a nucleic acid sequence encoding an amino acid substitution in MetK, wherein the amino acid substitution in MetK includes a substitution of valine to glutamic acid at amino acid position 185. In other embodiments, this invention provides metK nucleic acid molecules comprising a deletion of cytosine at nucleic acid residue 1132 of the metK allele. In other embodiments, the metK nucleic acid molecules provided by this invention comprise nucleic acid sequences selected from SEQ ID NO: 27 and SEQ ID NO: 28. In particular, this invention provides the use of these isolated metK nucleic acid molecules and their sequences for the production of microorganisms for preventing or reducing the erroneous incorporation of ortholeucine into proteins or peptides.

[0034] This invention provides microorganisms comprising nucleic acids, said nucleic acids comprising a mutated metA allele, wherein the microorganism further comprises nucleic acids encoding an anti-VEGF antibody or an anti-VEGF antibody fragment. In some embodiments, this invention provides microorganisms comprising nucleic acids comprising a mutated metA allele, wherein the microorganism further comprises nucleic acids encoding amino acid sequence SEQ ID NO: 46 and nucleic acids encoding amino acid sequence SEQ ID NO: 47. In some embodiments, the nucleic acid encoding amino acid sequence SEQ ID NO: 46 is the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid encoding amino acid sequence SEQ ID NO: 47 is the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, this invention provides microorganisms comprising nucleic acids comprising a mutated metA allele, wherein the microorganism further comprises nucleic acids having a nucleic acid sequence corresponding to SEQ ID NO: 33 and nucleic acids having a nucleic acid sequence corresponding to SEQ ID NO: 34. In some embodiments, the mutated metA allele comprises nucleic acid sequences selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In some respects, the microorganisms are bacteria, such as Escherichia coli.

[0035] This invention provides microorganisms comprising nucleic acids containing a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding an anti-VEGF antibody or an anti-VEGF antibody fragment. In some embodiments, the invention provides microorganisms comprising nucleic acids containing a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding amino acid sequence SEQ ID NO: 46 and nucleic acids encoding amino acid sequence SEQ ID NO: 47. In some embodiments, the nucleic acid encoding amino acid sequence SEQ ID NO: 46 is the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid sequence encoding amino acid sequence SEQ ID NO: 47 is the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the invention provides microorganisms comprising nucleic acids containing a mutated metK allele, wherein the microorganism further comprises nucleic acids having a nucleic acid sequence corresponding to SEQ ID NO: 33 and nucleic acids having a nucleic acid sequence corresponding to SEQ ID NO: 34. In some embodiments, the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28. In some aspects, the microorganism is a bacterium, for example, *Escherichia coli*.

[0036] This invention provides microorganisms comprising nucleic acids, said nucleic acids comprising a mutated metA allele and a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding an anti-VEGF antibody or an anti-VEGF antibody fragment. In some embodiments, this invention provides microorganisms comprising nucleic acids comprising a mutated metA allele and a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding amino acid sequence SEQ ID NO: 46 and nucleic acids encoding amino acid sequence SEQ ID NO: 47. In some embodiments, the nucleic acid encoding amino acid sequence SEQ ID NO: 46 is the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid sequence encoding amino acid sequence SEQ ID NO: 47 is the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, this invention provides microorganisms comprising nucleic acids comprising a mutated metA allele and a mutated metK allele, wherein the microorganism further comprises nucleic acids having a nucleic acid sequence corresponding to SEQ ID NO: 33 and nucleic acids having a nucleic acid sequence corresponding to SEQ ID NO: 34. In some embodiments, the mutated metA allele comprises nucleic acids selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and the mutated metK allele comprises nucleic acid sequences selected from SEQ ID NO: 27 and SEQ ID NO: 28. In some aspects, the microorganism is a bacterium, such as Escherichia coli.

[0037] This invention provides a microorganism comprising nucleic acid, said nucleic acid comprising a mutated metA allele, wherein the microorganism further comprises nucleic acid encoding an anti-factor D antibody or an anti-factor D antibody fragment. In some embodiments, the invention provides a microorganism comprising nucleic acid including a mutated metA allele, wherein the microorganism further comprises nucleic acid encoding amino acid sequence SEQ ID NO: 48 and nucleic acid encoding amino acid sequence SEQ ID NO: 49. In some embodiments, the mutated metA allele comprises nucleic acid selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26. In some aspects, said microorganism is a bacterium, for example, *Escherichia coli*.

[0038] This invention provides microorganisms comprising nucleic acids, said nucleic acids comprising a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding anti-factor D antibodies or fragments of anti-factor D antibodies. In some embodiments, the invention provides microorganisms comprising nucleic acids comprising a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding amino acid sequence SEQ ID NO: 48 and nucleic acids encoding amino acid sequence SEQ ID NO: 49. In some embodiments, the mutated metK allele comprises nucleic acid sequences selected from SEQ ID NO: 27 and SEQ ID NO: 28. In some aspects, said microorganisms are bacteria, for example, *Escherichia coli*.

[0039] This invention provides a microorganism comprising nucleic acids, said nucleic acids comprising a mutated metA allele and a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding an anti-factor D antibody or an anti-factor D antibody fragment. In some embodiments, this invention provides a microorganism comprising nucleic acids comprising a mutated metA allele and a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding the amino acid sequence SEQ ID NO: 48 and nucleic acids encoding the amino acid sequence SEQ ID NO: 49. In some embodiments, the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28. In some aspects, said microorganism is a bacterium, for example, *Escherichia coli*.

[0040] This invention provides microorganisms comprising nucleic acids, said nucleic acids comprising a mutated metA allele, wherein the microorganism further comprises nucleic acids encoding anti-MET antibodies or anti-MET antibody fragments. In some embodiments, the invention provides microorganisms comprising nucleic acids comprising a mutated metA allele, wherein the microorganism further comprises nucleic acids encoding amino acid sequence SEQ ID NO: 50, amino acid sequence SEQ ID NO: 51, and amino acid sequence SEQ ID NO: 52. In some embodiments, the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In some aspects, said microorganism is a bacterium, for example, *Escherichia coli*.

[0041] This invention provides microorganisms comprising nucleic acids, said nucleic acids comprising a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding an anti-MET antibody or an anti-MET antibody fragment. In some embodiments, the invention provides microorganisms comprising nucleic acids comprising a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding amino acid sequence SEQ ID NO: 50, amino acid sequence SEQ ID NO: 51, and amino acid sequence SEQ ID NO: 52. In some embodiments, the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28. In some aspects, said microorganism is a bacterium, for example, *Escherichia coli*.

[0042] This invention provides a microorganism comprising nucleic acids, said nucleic acids comprising a mutated metA allele and a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding an anti-MET antibody or an anti-MET antibody fragment. In some embodiments, the invention provides a microorganism comprising nucleic acids comprising a mutated metA allele and a mutated metK allele, wherein the microorganism further comprises nucleic acids encoding amino acid sequence SEQ ID NO: 50, amino acid sequence SEQ ID NO: 51, and amino acid sequence SEQ ID NO: 52. In some embodiments, the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28. In some aspects, said microorganism is a bacterium, for example, *Escherichia coli*.

[0043] The present invention further provides a method for producing a protein or polypeptide free of leucine misincorporation in a bacterial host cell, the method comprising expressing a nucleic acid encoding the protein or polypeptide in a bacterial host cell, wherein the bacterial host cell contains a mutated metA allele, a mutated metK allele, or a mutated metA allele and a mutated metK allele, thereby producing a protein or polypeptide free of leucine misincorporation. In some embodiments, the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28.

[0044] The present invention also provides a method for producing antibodies or antibody fragments in bacterial host cells, wherein the antibodies or antibody fragments are free from the misincorporation of leucine. The method comprises expressing a nucleic acid encoding the antibody or antibody fragment in a bacterial host cell, wherein the bacterial host cell contains a mutated metA allele, a mutated metK allele, or a mutated metA allele and a mutated metK allele, thereby producing an antibody or antibody fragment free from the misincorporation of leucine. In some aspects, the method for producing the antibody or antibody fragment free from the misincorporation of leucine according to the present invention in bacterial host cells includes expressing a nucleic acid encoding an antibody heavy chain polypeptide and a nucleic acid encoding an antibody light chain polypeptide in the bacterial host cell. In some aspects, the antibody heavy chain polypeptide is an antibody Fab fragment heavy chain polypeptide, and the antibody light chain polypeptide is an antibody Fab fragment light chain polypeptide. In some embodiments, the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28.

[0045] In some embodiments, the present invention provides a method for generating anti-VEGF antibodies or anti-VEGF antibody fragments in bacterial host cells, wherein the anti-VEGF antibodies or anti-VEGF antibody fragments are free from the misincorporation of leucine. The method comprises expressing nucleic acids encoding anti-VEGF antibodies or anti-VEGF antibody fragments in bacterial host cells containing mutated metA alleles, mutated metK alleles, or mutated metA alleles and mutated metK alleles, thereby generating anti-VEGF antibodies or anti-VEGF antibody fragments free from the misincorporation of leucine. In some embodiments, the method comprises expressing nucleic acids encoding anti-VEGF antibody heavy chain polypeptides or anti-VEGF antibody fragment heavy chain polypeptides or fragments thereof in bacterial host cells, and nucleic acids encoding anti-VEGF antibody light chain polypeptides or anti-VEGF antibody fragment light chain polypeptides or fragments thereof. In some aspects, the anti-VEGF antibody heavy chain and anti-VEGF antibody light chain are full-length heavy chain and light chain anti-VEGF antibody polypeptides. In other aspects, the anti-VEGF antibody heavy chain is a heavy chain polypeptide of the antibody Fab fragment, and the anti-VEGF antibody light chain is a light chain polypeptide of the antibody Fab fragment. In some embodiments, the anti-VEGF antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 47, and the anti-VEGF antibody light chain comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the nucleic acid encoding the amino acid sequence SEQ ID NO: 47 is the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the nucleic acid sequence encoding the amino acid sequence SEQ ID NO: 46 is the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In some embodiments, the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28.

[0046] The present invention also provides anti-VEGF antibodies or anti-VEGF antibody fragments produced by any of the methods described herein, wherein the anti-VEGF antibodies or anti-VEGF antibody fragments are free from the mis-incorporation of ortholeucine.

[0047] In some embodiments, the present invention provides a method for producing anti-factor D antibodies or anti-factor D antibody fragments in bacterial host cells, wherein the anti-factor D antibodies or anti-factor D antibody fragments are free from leucine misincorporation. The method comprises expressing nucleic acids encoding the anti-factor D antibody or anti-factor D antibody fragment in bacterial host cells containing a mutated metA allele, a mutated metK allele, or both a mutated metA allele and a mutated metK allele, thereby producing anti-factor D antibodies or anti-factor D antibody fragments free from leucine misincorporation. In some embodiments, the method comprises expressing nucleic acids encoding an anti-factor D antibody heavy chain polypeptide or an anti-factor D antibody fragment heavy chain polypeptide or a fragment thereof, and nucleic acids encoding an anti-factor D antibody light chain polypeptide or an anti-factor D antibody fragment light chain polypeptide or a fragment thereof, in bacterial host cells. In some aspects, the anti-factor D antibody heavy chain and the anti-factor D antibody light chain are full-length heavy chain and light chain anti-factor D antibody polypeptides. In other aspects, the anti-factor D antibody heavy chain is an antibody Fab fragment heavy chain polypeptide, and the anti-factor D antibody light chain is an antibody Fab fragment light chain polypeptide. In some embodiments, the heavy chain of the anti-factor D antibody comprises the amino acid sequence of SEQ ID NO: 49, and the light chain of the anti-factor D antibody comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In some embodiments, the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28.

[0048] The present invention further provides an anti-factor D antibody or anti-factor D antibody fragment produced by any of the methods described herein, wherein the anti-factor D antibody or anti-factor D antibody fragment is free from the mis-incorporation of ortholeucine.

[0049] In some embodiments, the present invention provides a method for generating anti-MET antibodies or anti-MET antibody fragments in bacterial host cells, wherein the anti-MET antibody or anti-MET antibody fragment is free from the misincorporation of leucine. The method comprises expressing nucleic acids encoding the anti-MET antibody or anti-MET antibody fragment in bacterial host cells containing a mutated metA allele, a mutated metK allele, or both a mutated metA allele and a mutated metK allele, thereby generating anti-MET antibodies or anti-MET antibody fragments free from the misincorporation of leucine. In some embodiments, the method comprises expressing nucleic acids encoding an anti-MET antibody heavy chain polypeptide or an anti-MET antibody fragment heavy chain polypeptide or a fragment thereof, and nucleic acids encoding an anti-MET antibody light chain polypeptide or an anti-MET antibody fragment light chain polypeptide or a fragment thereof, in bacterial host cells. In some aspects, the anti-MET antibody heavy chain and anti-MET antibody light chain are full-length heavy chain and light chain anti-MET antibody polypeptides. In other aspects, the anti-MET antibody heavy chain is a heavy chain polypeptide of an antibody Fab fragment, and the anti-MET antibody light chain is a light chain polypeptide of an antibody Fab fragment. In some embodiments, the anti-MET antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 51, the anti-MET antibody heavy chain fragment comprises the amino acid sequence of SEQ ID NO: 52, and the anti-MET antibody light chain comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In some embodiments, the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28.

[0050] The present invention further provides anti-MET antibodies or anti-MET antibody fragments produced by any of the methods described herein, wherein the anti-MET antibodies or anti-MET antibody fragments are free from the mis-incorporation of ortholeucine.

[0051] In various aspects, the mutant microorganism comprising any one or more nucleic acid sequences provided by this invention is a bacterium; in other aspects, the microorganism is *Escherichia coli*. This invention particularly provides the use of the mutant microorganisms described herein for the production of heterologous (e.g., recombinant) polypeptides and heterologous (e.g., recombinant) proteins, wherein the erroneous incorporation of ortholeucine into the heterologous polypeptides and heterologous proteins is reduced, substantially reduced, substantially eliminated, or prevented. Attached Figure Description

[0052] Figure 1 shows the nucleic acid sequence metA (R27C) corresponding to SEQ ID NO: 23.

[0053] Figure 2 shows the nucleic acid sequence metA (Y294C) corresponding to SEQ ID NO: 24.

[0054] Figure 3 shows the nucleic acid sequence metA (I296S / P298L) corresponding to SEQ ID NO: 25.

[0055] Figure 4 shows the nucleic acid sequence metA (Q64E) corresponding to SEQ ID NO: 26.

[0056] Figure 5 shows the nucleic acid sequence metK (V185E) corresponding to SEQ ID NO: 27.

[0057] Figure 6 shows the nucleic acid sequence metK (c1132del) corresponding to SEQ ID NO: 28.

[0058] Figures 7A and 7B show the amino acid and nucleic acid sequences of wild-type MetA corresponding to SEQ ID NO: 29 and SEQ ID NO: 31, respectively.

[0059] Figures 8A and 8B show the amino acid and nucleic acid sequences of wild-type MetK corresponding to SEQ ID NO: 30 and SEQ ID NO: 32, respectively.

[0060] Figure 9 shows the structures of ortholeucine and its analogues. Ortholeucine is a structural analogue of methionine in which the sulfur (S) atom is replaced by a methylene group (i.e., -CH2).

[0061] Figure 10 shows a schematic diagram of the biosynthetic pathway of ortholeucine in *E. coli*. Dashed arrows indicate multiple steps involved. Pyruvate is converted to α-ketocaproate via a three-step ketoacid chain elongation process catalyzed by enzymes encoded by the leuABCD operon. The intermediate α-ketocaproate is then transaminated to ortholeucine via transaminases IlvE or TyrB.

[0062] Figure 11 illustrates the biosynthesis and regulation of methionine in *E. coli*. Dashed arrows indicate feedback inhibition, and open arrows indicate repression. Methionine and S-adenosylmethionine (SAM) are feedback inhibitors of the enzyme MetA. The repressor MetJ and its co-repressor SAM inhibit the transcription of the enzyme in the methionine regulator.

[0063] Figures 12A, 12B, and 12C illustrate the growth trend, such as the OD measured by fermentation with 10L of E. coli. 550 (Figure 12A) and iOD 550(Figure 12B). Control host (60E4) fermentation was carried out with continuous methionine (■) or continuous water feed (□) (Figure 12A). Continuous water feed (△) or no feed (…) Fermentation was carried out using the 60E4metA (Y294C) host (Figure 12A). Figure 12C shows the growth trends of the control host 60E4 without feed (squares), the control host 60E4 with methionine feed (circles), and the host 60E4metA (Y294C) without feed (triangles). Fermentation was carried out using all other mutants with continuous water feed.

[0064] Figures 13A and 13B show the extracellular (Figure 13A) and intracellular (Figure 13B) methionine levels in the fermentation medium of control host cells (□) and 60E4metA (Y294C) (△) fermented with continuous water feed. The phosphate levels in the extracellular medium of control host cells (□) and 60E4metA host cells (Y294C) (△) fermented with continuous water feed are also shown as dashed curves (Figure 13A) and (Figure 13B).

[0065] Figures 14A and 14B show the extracellular (Figure 14A) and intracellular (Figure 14B) methionine levels in the mutant host cell line of the present invention. The extracellular and intracellular methionine levels in two control host cell fermentations with either continuous methionine (■) or continuous water feed (□) are also shown.

[0066] Figure 15 shows the level of extracellular phosphate during fermentation.

[0067] Figures 16A and 16B show the run-end titer (Figure 16A) and time-process titer (Figure 16B) of *E. coli* host cell fermentation. Using continuous methionine ( Fermentation of control host cells (60E4) was performed using either continuous water feed (□) or no feed (△). Fermentation was performed using 60E4metA host cells (Y294C). Fermentation using all other mutant host cells was carried out with a continuous water feed.

[0068] Figures 17A and 17B illustrate the results of Western blotting of whole-cell culture samples obtained during fermentation of 60E4 host cells (control host cells) and 60E4metA host cells (Y294C).

[0069] Figures 18A and 18B show the nucleic acid sequences of the light and heavy chains of the Fab fragments of the anti-vascular endothelial growth factor (anti-VEGF) antibodies corresponding to SEQ ID NO: 33 and SEQ ID NO: 34, respectively.

[0070] Figures 19A and 19B illustrate how OD can be used.550 The growth trend of 10 liters of E. coli fermentation was measured. Control hosts (66F8 or 64B4) were fermented in steps AF2 or AF3 with no feed (square) or continuous methionine feed (circle). Host fermentation of 66F8metA (Y294C) and 64B4metA (Y294C) was carried out with no feed (triangle).

[0071] Figures 20A, 20B, and 20C illustrate the yields of recombinant protein products using host strains 60E4 (control host) and 60E4metA (Y294C), 66F8 (control host) and 66F8metA (Y294C), and 64B4 (control host) and 64B4metA (Y294C), respectively.

[0072] Figures 21A and 21B show the amino acid sequences of the light and heavy chains of the Fab fragments of the anti-vascular endothelial growth factor (anti-VEGF) antibodies corresponding to SEQ ID NO: 46 and SEQ ID NO: 47, respectively.

[0073] Figures 22A and 22B show the amino acid sequences of the light and heavy chains of the anti-factor D antibody Fab fragments corresponding to SEQ ID NO: 48 and SEQ ID NO: 49, respectively.

[0074] Figures 23A, 23B, and 23C show the amino acid sequences of the light chain (SEQ ID NO: 50), heavy chain (SEQ ID NO: 51), and heavy chain fragment (SEQ ID NO: 52) of the anti-MET antibody. Detailed Implementation

[0075] Detailed description of the invention

[0076] Among other things, the present invention provides methods and compositions for preventing the erroneous incorporation of ortholeucine into proteins and peptides, particularly during the production of recombinant proteins in microorganisms. The present invention also provides microbial host cells and nucleic acid molecules for use in the methods of the present invention.

[0077] General methods

[0078] Unless otherwise stated, the practice of this invention will employ conventional techniques of cell biology, cell culture, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are known and available to those skilled in the art. These techniques are described in references such as, *Molecular Cloning: A laboratory Manual, third edition* (Sambrook et al., 2001), Cold Spring Harbor Press; *Oligonucleotide Synthesis* (edited by P. Herdewijn, 2004); *Animal Cell Culture* (edited by R.Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., 1987); *PCR: The Polymerase Chain Reaction* (edited by Mullis et al., 1994); *Current Protocols in Immunology* (edited by J.E. Coligan et al., 1991); and *Short Protocols in Molecular Biology* (Wiley and Sons, 1999). The expression of antibody fragments and peptides in bacteria is described, for example, in U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli.)

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0080] definition

[0081] The terms "heterologous protein" or "heterologous polypeptide" refer to proteins or polypeptides that are not naturally synthesized or produced by the cells or organisms of interest (such as microorganisms). For example, *E. coli* cells can produce human proteins or human polypeptides, and the human proteins or human polypeptides produced therefrom are heterologous proteins or heterologous polypeptides. In the context of this invention, those heterologous proteins or heterologous polypeptides containing methionine are of particular interest. As used herein, heterologous protein or heterologous polypeptide also refers to recombinant proteins or recombinant polypeptides.

[0082] The term "incorrect incorporation of ortholeucine" refers to the incorporation of ortholeucine residues into a protein or polypeptide, while the corresponding nucleic acid encoding the protein or polypeptide encodes a methionine residue.

[0083] The term “mutated allele” or “mutated allele” refers to an allele that has a different nucleic acid sequence than the wild-type allele, or an allele that has a nucleic acid sequence that is altered from the nucleic acid sequence of the wild-type allele (i.e., naturally found in the cells or microorganisms of interest).

[0084] The term "mutated microbe" or "mutant microbe" refers to a microbe that contains one or more mutated alleles or mutant alleles.

[0085] As used herein, the phrases “substantially reduced” or “substantially different” refer to a difference between two values ​​(typically one related to a molecule and the other to a reference / comparison molecule) that is high enough that a person skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of a biological characteristic measured by the values ​​(e.g., the content of ortholeucine in a protein or polypeptide).

[0086] "Isolated" nucleic acids refer to nucleic acid molecules that have been isolated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules normally found in cells, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0087] "Isolated metA nucleic acid molecule" or "isolated metK nucleic acid molecule" refers to one or more nucleic acid molecules that encode MetA or MetK, respectively, contained in a single vector or different vectors, or present at one or more locations in a host cell. "Isolated metA nucleic acid molecule" or "isolated metK nucleic acid molecule" also refers to a mutated metA allele or a mutated metK allele.

[0088] The phrase "protein or polypeptide that does not contain erroneously incorporated oroleucine" refers to a protein or polypeptide that does not contain detectable levels of oroleucine residues.

[0089] As used herein, the singular forms of “a,” “one,” and “the” include references to the plural unless otherwise stated.

[0090] As used herein, “about” a value or “about” a parameter refers to a typical range of error for the corresponding value known to those skilled in the art. “About” a value or “about” a parameter as used herein includes (and describes) the value or parameter itself. For example, a description referring to “about X” includes a description of “X”.

[0091] Methods to prevent or reduce the erroneous incorporation of oroleucine

[0092] This invention relates in part to methods and compositions for preventing or reducing the erroneous incorporation of oroleucine into proteins and peptides, particularly during the production of recombinant proteins in microorganisms.

[0093] The misincorporation of ortholeucine residues in place of methionine residues during the production of recombinant proteins in *E. coli* has been previously described. One current approach to prevent or reduce ortholeucine misincorporation is to continuously or in concentrated feed of methionine into the culture medium during fermentation. While this strategy is effective in reducing ortholeucine misincorporation, several operational drawbacks associated with continuous or concentrated feeding or the addition of methionine during fermentation exist. For example, continuous or concentrated feeding of the culture medium increases operational complexity and the overall cost of the fermentation process. Furthermore, methionine feeding leads to undesirable dilution of the fermentation medium, resulting in lower cell densities and potentially reduced product yields.

[0094] To overcome these drawbacks, the inventors of this invention provide alternative methods for continuous or concentrated methionine feeding to prevent or reduce the erroneous incorporation of ortholeucine in the production of heterologous proteins or peptides. In particular, this invention provides engineered microbial (e.g., *E. coli*) host cells to produce methionine to a degree or extent sufficient to prevent or reduce the erroneous incorporation of ortholeucine during recombinant protein production, including recombinant protein production at high host cell densities.

[0095] Mutants of *E. coli* host cells used for large-scale methionine production have been previously reported (see, for example, Chattopadhyay et al., (1991) J Gen Microbiol 137:685-691; Nakamori et al., (1999) Appl Microbiol Biotechnol 52:179-185; Usuda and Kurahashi (2005) Appl EnvironMicrobiol 71:3228-3234; International Patent Application Publication No. WO2005 / 111202 2005; and U.S. Patent Application Publication No. US2009 / 0298135). Many of these mutant *E. coli* strains contain mutations in the following three genes related to the regulation of methionine biosynthesis: metJ, metA, and metK.

[0096] Transcriptional regulation of methionine biosynthesis in *E. coli* involves the enzyme MetJ (a product of the metJ gene). MetJ is a transcriptional repressor that, when bound to its co-repressor S-adenosylmethionine (SAM), inhibits transcription of genes in the methionine regulator, thereby regulating methionine levels in the cell (see, for example, Marinacs et al. (2006), Biochem J396:227-234). As previously reported, chemical mutagenesis of *E. coli*, through selection for growth on ethionine (a toxic methionine analog), resulted in the isolation of a serine-to-asparagine mutation (S54N) at amino acid position 54 of MetJ. This mutation led to the release of inhibition of the methionine biosynthetic enzyme and increased methionine production (see Nakamori et al., (1999) Appl Microbiol Biotechnol 52:179-185). Complete disruption of the metJ gene also leads to the release of inhibition of enzymes involved in the methionine biosynthesis pathway and the overproduction of methionine (see Usuda and Kurahashi (2005) Appl Environ Microbiol 71:3228-3234).

[0097] Methionine biosynthesis in *E. coli* is also regulated by feedback inhibition (via methionine and SAM) of homoserine succinyltransferase (metA gene product), an enzyme involved in the first step of methionine biosynthesis (see, for example, Born and Blanchard (1999) Biochemistry 38:14416-14423). Previously, feedback-resistant MetA (metA gene product) mutants leading to deregulated methionine synthesis were isolated in *E. coli* by selecting for the growth of the toxic methionine analog α-methylmethionine (see Usuda and Kurahashi (2005) Appl Environ Microbiol 71:3228-3234; and International Patent Application Publication No. WO2005 / 111202).

[0098] The metK gene encodes the enzyme S-adenosylmethionine synthase, which converts methionine to S-adenosylmethionine. (See Markham et al., (1980) J Biol Chem 255:9082-9092). Previously, partially nonfunctional MetK mutants were isolated by selecting growth on toxic methionine analogs, oroleucine, and ethionine, resulting in low levels of SAM, thus relieving the inhibition of methionine biosynthetic enzyme (SAM is a co-repressor of MetJ). (See Chattopadhyay et al., (1991) Gen Microbiol 137:685-691; Usuda and Kurahashi (2005) Appl Environ Microbiol 71:3228-3234; and International Patent Application Publication No. WO2005 / 111202).

[0099] In this invention, specific nucleic acid residues in the wild-type metA gene are mutated, resulting in the following amino acid substitutions in MetA (see Figure 7A and the wild-type MetA amino acid sequence of SEQ ID NO: 29): substitution of arginine to cysteine ​​at amino acid position 27 (R27C); substitution of glutamine to glutamate at amino acid position 64 (Q64E); substitution of tyrosine to cysteine ​​at amino acid position 294 (Y294C); substitution of isoleucine to serine at amino acid position 296 (I296S); and substitution of proline to leucine at amino acid position 298 (P298L). E. coli host cells comprising one or more of these MetA amino acid substitutions produce methionine to a degree or range sufficient to prevent the erroneous incorporation of ortholeucine into the expressed heterologous protein.

[0100] In some embodiments, the present invention provides various mutant metA alleles encoding amino acid substitutions of R27C, Q64E, Y294C, I296S, and P298L in MetA (compared to the wild-type MetA amino acid sequence; Figure 7 and SEQ ID NO: 29). Such mutant metA alleles result in feedback resistance to the MetA enzyme. Mutant metA alleles were introduced into *E. coli* host cells (60E4) using an allele exchange method (see Materials and Methods below) to obtain mutant *E. coli* host cell strains 66H6 (60E4metA(R27C)), 66H8 (60E4metA(Y294C)), 67B8 (60E4metA(Q64E)), and 67B9 (60E4metA(I296S P298L)). The study evaluated the incorrect incorporation of ortholeucine into the resulting mutant E. coli host cells during recombinant protein production without a continuous methionine feed (see Example 4 below).

[0101] The positions of all mentioned amino acids in MetA were constructed based on homoserine succinyltransferase encoded by the *E. coli* metA gene (shown in Figures 7A and 7B, corresponding to SEQ ID NO: 29 and SEQ ID NO: 31). The mentioned amino acid positions were constructed with the first amino acid, methionine, counted as amino acid position 1. The corresponding positions in homoserine succinyltransferases from other organisms can be identified by those skilled in the art through, for example, simple sequence alignment.

[0102] In this invention, the nucleic acid in the wild-type metK gene is mutated, resulting in an amino acid substitution from valine to glutamate at amino acid position 185 in MetK (V185E) (see Figure 8A and SEQ ID NO: 30 for the amino acid sequence of wild-type MetK). Additionally, a specific nucleic acid at cytosine base position 1132 in the metK gene is deleted (c1132del). E. coli host cells containing one or more of these mutated metK alleles produce methionine to a degree or extent sufficient to prevent the erroneous incorporation of ortholeucine into the expressed heterologous protein.

[0103] In some embodiments, the present invention also provides various mutated metK alleles encoding amino acids that substitute for the cytosine base deletion at position 1132 (c1132del) in the V185E or metK alleles. Such mutated metK alleles result in partial loss of function of the MetK enzyme. The mutated metK alleles were introduced into various *E. coli* host cells (66H8; 60E4metA (Y294C), see above) using an allele exchange method (see Materials and Methods below) to obtain *E. coli* host cell strains 67C2 (66H8metK (V185E)) and 67C3 (66H8metK (c1132del)), respectively. The resulting mutated *E. coli* host cells were evaluated for leucine misincorporation during recombinant protein production without a continuous methionine feed (see Example 4 below).

[0104] The positions of all mentioned amino acids in MetK were constructed based on the S-adenosylmethionine synthase encoded by the *E. coli* metK gene, as shown in Figures 8A and 8B, corresponding to SEQ ID NO: 30 and SEQ ID NO: 32. The mentioned amino acid positions were constructed with the first amino acid, methionine, counted as amino acid position 1. The corresponding positions in the corresponding regions of S-adenosylmethionine synthases from other organisms can be identified by those skilled in the art through, for example, simple sequence alignment.

[0105] nucleic acid molecules metA and metKde

[0106] By way of example, the present invention uses isolated nucleic acid molecules containing metA and metK nucleic acid sequences that are different from wild-type metA and metK nucleic acid sequences. The metA and metK nucleic acid sequences provided by this invention encode various substitutions for the amino acids encoded by wild-type metA (replacing arginine with cysteine ​​at amino acid position 27 (R27C); replacing glutamine with glutamic acid at amino acid position 64 (Q64E); replacing tyrosine with cysteine ​​at amino acid position 294 (Y294C); replacing isoleucine with serine at amino acid position 296 (I296S); replacing proline with leucine at amino acid position 298 (P298L); and replacing isoleucine with serine at amino acid position 296 (I296S) and replacing proline with leucine at amino acid position 298 (P298L)); and substitutions for the amino acids encoded by wild-type metK (replacing valine with glutamic acid at amino acid position 185 (V185E) and a nucleic acid sequence containing a cytosine base deletion at position 1132 (cdel1132del)). The use of any nucleic acid sequence encoding the metA or metK allele that results in these amino acid substitutions is specifically considered for use in the methods of the present invention.

[0107] The present invention also provides isolated metA nucleic acid molecules that encode a variety of modified MetA enzymes (i.e., enzymes encoding a variety of mutated homoserine succinyltransferases). In some embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence selected from SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

[0108] The present invention also provides isolated metK nucleic acid molecules that encode various modified MetK enzymes (i.e., encoding various mutant S-adenosylmethionine enzymes). In some embodiments, the present invention provides isolated nucleic acid molecules comprising nucleic acid sequences selected from SEQ ID NO: 27 and SEQ ID NO: 28.

[0109] By way of example, the present invention also provides various combinations of mutated metA alleles and corresponding isolated nucleic acid molecules comprising nucleic acid sequences encoding amino acid substitutions in the following MetA: arginine replaced by cysteine ​​at amino acid position 27 (R27C); glutamine replaced by glutamic acid at amino acid position 64 (Q64E); tyrosine replaced by cysteine ​​at amino acid position 294 (Y294C); isoleucine replaced by serine at amino acid position 296 (I296S); proline replaced by leucine at amino acid position 298 (P298L); and isoleucine replaced by serine at amino acid position 296 (I296S) and proline replaced by leucine at amino acid position 298 (P298L). In some aspects, the mutated metA alleles provided by the present invention result in feedback resistance (i.e., feedback insensitivity) to the MetA enzyme. The amino acid positions are referenced to the wild-type MetA amino acid sequence shown in Figure 7A and SEQ ID NO: 29.

[0110] By way of example, the present invention also provides various combinations of mutated metK alleles and corresponding isolated nucleic acid molecules comprising nucleic acid sequences encoding amino acid substitutions in MetK, where valine is replaced by glutamic acid at amino acid position 185 (V185E). The present invention also provides nucleic acid sequences containing a cytosine base deletion (c1132del) at metK allele position 1132. In some aspects, the mutated metK alleles provided by the present invention result in partial loss of function of the MetK enzyme. The amino acid positions are referenced to the wild-type MetK amino acid sequence, as shown in Figure 8A and SEQ ID NO: 30.

[0111] Microorganisms used in the method of the present invention

[0112] As described herein and by way of example, *E. coli* host cells are engineered to produce methionine sufficient to prevent or reduce the degree or extent of erroneous incorporation of ortholeucine during recombinant protein production, including recombinant protein production at high host cell densities. Therefore, in some embodiments provided herein, the present invention provides mutant microbial strains (i.e., mutant microbial host cells) that produce methionine to a degree or extent sufficient to reduce or prevent the erroneous incorporation of ortholeucine into proteins or peptides (e.g., to a degree or extent sufficient to reduce or prevent the erroneous incorporation of ortholeucine into recombinant proteins or recombinant peptides, or to a degree or extent sufficient to reduce or prevent the erroneous incorporation of ortholeucine into heterologous proteins or heterologous peptides).

[0113] Suitable starting E. coli host cells for use in this method include, for example, (but not limited to) E. coli W3110, E. coli 294, E. coli X1776, etc. These examples of E. coli host cells are illustrative and not limiting. E. coli strain W3110 is a common host strain used for fermentation of recombinant DNA products. Mutated E. coli host cells of any of the above-mentioned E. coli host cell lines can also be used as starting host cells, which are then further modified to contain the mutated metA and / or metK alleles described herein.

[0114] This invention demonstrates that using E. coli host cells containing multiple metA and metK mutant alleles and combinations of metA and metK mutant alleles during recombinant protein production effectively prevents the erroneous incorporation of ortholeucine into the expressed recombinant protein (see Example 4 below).

[0115] This invention provides microorganisms that produce methionine to a degree or extent sufficient to prevent or reduce the erroneous incorporation of ortholeucine into proteins or peptides. In some aspects, this invention provides microorganisms that are feedback-insensitive homoserine succinyltransferases. In some embodiments, this invention provides microorganisms comprising a mutated metA allele. In other embodiments, this invention provides microorganisms comprising a mutated metA allele, wherein the mutated metA allele encodes an R27C amino acid substitution, a Q64E amino acid substitution, a Y294C amino acid substitution, an I296S amino acid substitution, or a P298L amino acid substitution in MetA. In some embodiments, this invention provides microorganisms comprising a mutated metA allele that encodes one or more of the above-mentioned amino acid substitutions, including, for example, mutated metA alleles encoding an I296S amino acid substitution and a P298L amino acid substitution in MetA. In many respects, the microorganisms comprising any one or more nucleic acid sequences provided by the present invention are bacteria; in other respects, the microorganisms are *Escherichia coli*. The present invention particularly provides the use of the microorganisms described herein for the production of heterologous (e.g., recombinant) peptides and heterologous (e.g., recombinant) proteins, wherein the erroneous incorporation of ortholeucine into the heterologous peptides and heterologous proteins is reduced or prevented.

[0116] As described above, the present invention provides microorganisms comprising one or more mutated metA alleles. In some embodiments, mutated metA alleles encoding R27C amino acid substitution, Q64E amino acid substitution, Y294C amino acid substitution, or I296S and P298L amino acid substitutions in MetA are encoded by nucleic acid sequences comprising SEQ ID NO: 23 (R27C), SEQ ID NO: 26 (Q64E), SEQ ID NO: 24 (Y294C), or SEQ ID NO: 25 (I296S and P298L), respectively. In other embodiments, the microorganisms provided by the present invention comprise mutated metA alleles encoded by nucleic acid sequences comprising SEQ ID NO: 23 (R27C), SEQ ID NO: 26 (Q64E), SEQ ID NO: 24 (Y294C), or SEQ ID NO: 25 (I296S and P298L). In several respects, the microorganisms comprising any one or more nucleic acid sequences provided by the present invention are bacteria; in other respects, the microorganisms are *Escherichia coli*. The present invention particularly provides the use of the microorganisms described herein for the production of heterologous (e.g., recombinant) peptides and heterologous (e.g., recombinant) proteins, wherein the erroneous incorporation of ortholeucine into the heterologous peptides and heterologous proteins is reduced or prevented.

[0117] As described above, the present invention provides a method for preventing or reducing the incorporation of ortholeucine into proteins and peptides expressed by microorganisms, wherein the microorganisms are microorganisms that produce methionine to a degree or extent sufficient to prevent or reduce the erroneous incorporation of ortholeucine into the protein or peptide. In some aspects, the present invention provides microorganisms wherein the microorganisms are microorganisms that relieve inhibition of methionine production. In some embodiments, the present invention provides microorganisms comprising a mutated metK allele. In other embodiments, the present invention provides microorganisms comprising a mutated metK allele, wherein the mutated metK allele encodes a V185E amino acid substitution in MetK. In some embodiments, the present invention provides microorganisms comprising a mutated metK allele, wherein the mutated metK allele comprises a deletion of cytosine at nucleic acid residue 1132 of the metK allele. In various aspects, microorganisms comprising any one or more nucleic acid sequences provided by the present invention are bacteria; in other aspects, the microorganisms are *Escherichia coli*. The present invention specifically provides the use of the microorganisms described herein for the production of heterologous (e.g., recombinant) peptides and heterologous (e.g., recombinant) proteins, wherein the erroneous incorporation of ortholeucine into the heterologous peptides and heterologous proteins is reduced or prevented.

[0118] As described above, the present invention provides microorganisms comprising one or more mutated metK alleles. In some embodiments, the mutated metK allele encoding the V185E amino acid substitution in MetK or containing a cytosine deletion at nucleic acid residue 1132 of the metK allele is encoded by a nucleic acid sequence comprising SEQ ID NO: 27 (V185E) or a nucleic acid sequence comprising SEQ ID NO: 28 (c1132del), respectively. In other embodiments, the microorganisms provided by the present invention comprise mutated metK alleles encoded by nucleic acid sequences comprising SEQ ID NO: 27 (V185E) or SEQ ID NO: 28 (c1132del). In all aspects, the microorganisms comprising any one or more nucleic acid sequences provided by the present invention are bacteria; in other aspects, said microorganisms are *Escherichia coli*. The present invention particularly provides the use of the microorganisms described herein for the production of heterologous (e.g., recombinant) peptides and heterologous (e.g., recombinant) proteins, wherein the erroneous incorporation of ortholeucine into heterologous peptides and heterologous proteins is reduced or prevented.

[0119] The use of any nucleic acid sequence encoding the metA or metK allele that leads to the amino acid substitutions described herein is particularly intended for use in the methods of the present invention.

[0120] In other embodiments, the present invention provides microorganisms comprising a mutated metA allele and a mutated metK allele. In some embodiments, the microorganism provided by the present invention is a microorganism comprising a mutated metA allele and a mutated metK allele, wherein the mutated metA allele encodes a Y294C amino acid substitution in MetA, and the mutated metK allele encodes a V185E amino acid substitution in MetK. In some embodiments provided by the present invention, the microorganism is a microorganism comprising a mutated metA allele and a mutated metK allele, wherein the mutated metA allele encodes a Y294C amino acid substitution in MetA, and the mutated metK allele comprises a deletion of cytosine at nucleic acid residue 1132 of the metK allele. In various aspects, microorganisms comprising any one or more nucleic acid sequences provided by the present invention are bacteria; in other aspects, said microorganism is *Escherichia coli*. The present invention specifically provides the use of any microorganism described herein for the production of heterologous (e.g., recombinant) peptides and heterologous (e.g., recombinant) proteins, wherein the erroneous incorporation of ortholeucine into heterologous peptides and heterologous proteins is reduced or prevented.

[0121] Production of microbial strains

[0122] This invention provides a method for producing host cells of microorganisms (e.g., *Escherichia coli*), wherein the microorganism produces methionine to a degree or extent sufficient to reduce or prevent the erroneous incorporation of ortholeucine into polypeptides and proteins. By way of example, *E. coli* host cells containing mutated metA alleles and / or mutated metK alleles are generated using allele exchange methods as known in the art and previously described (see Metcalf et al., (1994) Gene 138:1-7; and Bass et al., (1996) J Bacteriol 178: 1154-61; see the Materials and Methods section of this specification). This invention is not limited to methods for generating *E. coli* host cells containing mutated metA alleles and mutated metK alleles. Various methods for introducing mutated alleles or otherwise generating microbial strains (e.g., bacteria, *E. coli*) containing mutated alleles are well known to those skilled in the art.

[0123] Prevent or reduce the erroneous incorporation of oroleucine

[0124] The methods and compositions of the present invention can be used to produce heterologous or recombinant proteins or peptides, and can be used for both large-scale and small-scale protein or peptide production. The methods and compositions of the present invention are particularly useful for high-density microbial fermentation, for example, using *E. coli* host cells for the production of recombinant proteins and peptides. The methods and compositions provided by the present invention are useful for the recombinant production of proteins and peptides, particularly in the production of recombinant proteins and peptides where the erroneous incorporation of ortholeucine is undesirable, for example, in recombinant proteins and peptides for various research and therapeutic applications.

[0125] In some embodiments, the present invention provides a method for preventing or reducing the erroneous incorporation of ortholeucine into a protein or peptide, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism is a feedback-resistant or feedback-insensitive homoserine succinyl transferase. In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or peptide, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism is a methionine production inhibitor. In some embodiments, the feedback-resistant or feedback-insensitive homoserine succinyl transferase microorganism is a microorganism containing a mutated metA allele. In some embodiments, the methionine production inhibitor is a microorganism containing a mutated metK allele. In other embodiments, the microorganism for preventing or reducing the erroneous incorporation of ortholeucine contains both a mutated metA allele and a mutated metK allele.

[0126] In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or polypeptide, the method comprising expressing the protein or polypeptide in a microorganism, wherein the microorganism contains a mutated metA allele, wherein the mutated metA allele contains a nucleic acid sequence selected from: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In other embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or polypeptide, the method comprising expressing the protein or polypeptide in a microorganism, wherein the microorganism contains a mutated metA allele, wherein the mutated metA allele contains a nucleic acid sequence encoding MetA, wherein the nucleic acid sequence encodes an amino acid substitution selected from R27C, Q64E, Y294C, I296S, and P298L in MetA. In other embodiments, the nucleic acid sequence encodes an amino acid substitution in MetA composed of both I296S and P298L. The amino acid positions are referenced to the wild-type MetA amino acid sequence, as shown in Figure 7A and SEQ ID NO: 29.

[0127] In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or peptide, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism contains a mutated metK allele, wherein the mutated metK allele contains a nucleic acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 28. In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or peptide, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism contains a mutated metK allele, wherein the mutated metK allele contains a nucleic acid sequence encoding MetK, wherein the nucleic acid sequence encodes a V185E amino acid substitution in MetK. In other embodiments, the nucleic acid sequence contains a cytosine base deletion at position 1132 of the nucleic acid residue of the metK allele. The amino acid position refers to the wild-type MetK amino acid sequence, as shown in Figure 8A and SEQ ID NO: 30.

[0128] In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or polypeptide, the method comprising expressing the protein or polypeptide in a microorganism, wherein the microorganism comprises a mutated metA allele and a mutated metK allele, and further, wherein the mutated metA allele comprises the nucleic acid sequence of SEQ ID NO: 24, and the mutated metK allele comprises the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into a protein or polypeptide, the method comprising expressing the protein or polypeptide in a microorganism, wherein the microorganism comprises a mutated metA allele and a mutated metK allele, wherein the mutated metA allele comprises the nucleic acid sequence of SEQ ID NO: 24, and the mutated metK allele comprises the nucleic acid sequence of SEQ ID NO: 28.

[0129] In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into proteins or peptides, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism comprises a mutated metA allele and a metK allele, wherein the mutated metA allele comprises a nucleic acid sequence encoding an amino acid substitution Y294C in MetA, and the mutated metK allele comprises a nucleic acid sequence encoding an amino acid substitution V185E in MetK. In some embodiments, the present invention provides a method for reducing or preventing the erroneous incorporation of ortholeucine into proteins or peptides, the method comprising expressing the protein or peptide in a microorganism, wherein the microorganism comprises a mutated metA allele and a metK allele, wherein the mutated metA allele comprises a nucleic acid sequence encoding an amino acid substitution Y294C in MetA, and the mutated metK allele comprises a nucleic acid sequence containing a cytosine base deletion at position 1132 of the nucleic acid residue in the metK allele. The amino acid positions are shown in Figure 7A and SEQ ID NO: 29 for wild-type MetA amino acid sequence and in Figure 8A and SEQ ID NO: 30 for wild-type MetK amino acid sequence.

[0130] In some aspects of the method for reducing or preventing the erroneous incorporation of ortholeucine into proteins or peptides using microorganisms provided by the present invention, the microorganisms are bacteria, particularly *Escherichia coli*. In other aspects, the protein or peptide is a heterologous protein or peptide, or a recombinant protein or peptide. For example, the microorganism may include a nucleic acid encoding a protein or peptide that is heterologous to the microorganism; for example, the microorganism may be transformed with a nucleic acid encoding a protein or peptide that is heterologous to the microorganism by using a recombinant expression vector, such nucleic acid being, for example, DNA (e.g., cDNA or genomic DNA). In other aspects, the method further includes culturing the microorganism under conditions suitable for expressing the protein or peptide. In some embodiments, the microorganism is cultured in a culture medium containing a low concentration of methionine. The protein or peptide can then be recovered, purified, etc.; recovery may be from, for example, the periplasm of the microorganism or the culture medium. In some aspects, the culture occurs in a fermenter, for example, under high cell density fermentation conditions.

[0131] Heterogeneous nucleic acids encoding heterologous proteins or polypeptides are appropriately inserted into reproducible vectors under the control of suitable promoters for expression in microorganisms. Many vectors are available for this purpose, and the choice of a suitable vector will depend, for example, on the size of the nucleic acid to be inserted into the vector or the specific microbial host cell to be transformed with that vector. Suitable vectors are well known to those skilled in the art.

[0132] The methods and compositions provided by this invention are particularly useful for the production of recombinant proteins and peptides in which the mis-incorporation of ortholeucine is undesirable, for example, in the production of recombinant proteins and peptides for various therapeutic, medical, research, and diagnostic applications. For example, the methods and compositions of this invention can be applied to the recombinant production of therapeutic antibodies, such as polyclonal and monoclonal antibodies for medical and pharmaceutical applications. Examples of polyclonal and monoclonal antibodies for medical and pharmaceutical uses include, but are not limited to, anti-VEGF antibodies, anti-factor D antibodies, anti-hepatocyte growth factor receptor antibodies (e.g., anti-Met antibodies), etc.

[0133] The methods and compositions of the present invention are also useful for the production of antibody fragments. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. The production of recombinant antibodies provided by the methods, compositions, and microorganisms of the present invention can be carried out by expressing nucleic acids encoding antibody heavy chain polypeptides and expressing nucleic acids encoding antibody light chain polypeptides in the aforementioned microorganisms (e.g., bacterial host cells, *Escherichia coli*). In some aspects, the antibody heavy chain and antibody light chain are full-length heavy chain and light chain antibody polypeptides. In other aspects, the antibody heavy chain is the heavy chain of an antibody Fab fragment, and the antibody light chain is the light chain of an antibody Fab fragment.

[0134] The methods and compositions of the present invention are also useful for the production of multispecific antibodies, for example, for the production of bispecific antibodies. A multispecific antibody is an antibody that has binding specificity to at least two different epitopes. Exemplary multispecific antibodies may bind to two different epitopes of a protein, or may bind to two different epitopes of two different proteins. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody). Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983); International Application Publication No. WO 93 / 08829; Traunecker et al., EMBO J. 10: 3655 (1991); and “knob-in-hole” engineering (see, for example, U.S. Patent No. 5,731,168).

[0135] In addition, the methods and compositions of the present invention are useful for the production of other biomolecules for therapeutic and research applications, such as human growth hormone (GPH), insulin, etc.

[0136] Example

[0137] The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be implemented in conjunction with the general description provided above.

[0138] Materials and methods

[0139] Bacterial strains, plasmids, and growth conditions

[0140] The bacterial strains used in the examples described herein are derivatives of Escherichia coli strain W3110 (see Bachmann (1972) Bacteriol Rev 36:525-557). Antibiotic selection was maintained for all markers at the following concentrations: carbenicillin (plasmid or chromosomal), 50 μg / ml; kanamycin (chromosomal), 30 μg / ml; tetracycline (plasmid or chromosomal), 10 μg / ml.

[0141] Construction of strains and plasmids

[0142] The oligonucleotides used in plasmid and bacterial strain (i.e., *E. coli*) construction are listed in Table 1 below. Cloning, DNA analysis, PCR amplification, transformation, electroporation, and P1 transduction were performed using standard techniques. Chromosomal alleles were transduced by P1. metJ::Kan R Alleles were derived from bacterial strain JW3909-1, obtained from the Coli Genetic Stock Center (CGSC, Yale University). All allele substitutions were confirmed by PCR analysis.

[0143] Table 1

[0144] a Underlined nucleic acid residues introduce amino acid mutations. Lowercase nucleic acid residues indicate residues that differ from the wild-type nucleic acid sequence.

[0145] The metA gene was amplified by PCR from bacterial strain W3110 (Bachmann (1972) Bacteriol Rev 36:525-557) using primers SacI-metAflank-F and SalI-metAflank-R. The sample was digested with SacI and SalI and ligated into the SacI and SalI-digested plasmid pS1080 to obtain plasmid pS1080-metAflank. Plasmids pS1080-metAflank (R27C), pS1080-metAflank (Q64E), pS1080-metAflank (Y294C), and pS1080-metAflank (I296SP298L) were constructed by mutagenizing plasmid pS1080-metAflank using the QuikChange kit (Stratagene) and the following primer sets (QC-metAR27C-F; QC-metAR27C-R), (QC-metAQ64E-F; QC-metAQ64E-R), (QC-metAY294C-F; QC-metAY294C-R), and (QC-metAI296SP298L-F; QC-metAI296SP298L-R), respectively.

[0146] The metK gene was amplified by PCR from bacterial strain W3110 using primers SacI-metKflank-F and SalI-metKflank-R, digested with SacI and SalI, and ligated into the SacI and SalI-digested plasmid pS1080 to obtain plasmid pS1080-metKflank. Plasmids pS1080-metKflank (V185E) and pS1080-metKflank (c1132del) were constructed by mutagenizing plasmid pS1080-metKflank using the QuikChange kit (Stratagene) and the following primer sets, respectively: (QC-metKV185E-F; QC-metKV185E-R) and (QC-metKc1132del-F; QC-metKc1132del-R).

[0147] Allelic exchange was performed using the methods described previously (see Metcalf et al., (1994) Gene 138:1-7; and Bass et al., (1996) J Bacteriol 178: 1154-61).

[0148] As described above, allele exchange was performed using protocols such as those described by Metcalf et al. (ibid.) and modified by Bass et al. (ibid.). Cointegrate was transferred to either a 60E4 or 66H8 host cell background. Following sucrose anti-selection, sucrose-resistant colonies were screened for carbenicillin sensitivity by streaking onto LB agar plates containing carbenicillin. Carbenicillin-sensitive colonies were then isolated, and allele exchange was confirmed by PCR amplification of the complete metA or metK reading frames and subsequent DNA sequencing. The suicide plasmid vector pS1080 contained a conditional R6Kγ initiation and carbenicillin resistance selection marker, as well as a reselectible sacB gene that conferred sucrose sensitivity.

[0149] The bacterial strains and plasmids used in the experiments described in this article are listed in Table 2 below.

[0150] Table 2

[0151] Fermentation

[0152] Escherichia coli host strain 60E4 was transformed with a pBR322-based expression plasmid containing a light chain and a heavy chain polynucleotide encoding an antigen-binding (Fab) fragment of an anti-VEGF antibody (SEQ ID NO: 33 and SEQ ID NO: 34, respectively). (See anti-VEGF antibody Y0317 in International Application Publication No. WO1998 / 45331; International Application Publication No. WO2002 / 40697 (Example 2, describing fermentation of anti-VEGF antibody Y0317); and Chen et al., (1999) J Mol Biol 293:865-881, anti-VEGF antibody Y0317, each incorporated herein by reference in its entirety).

[0153] The expression plasmid was transformed into Escherichia coli host strain 66F8, the expression plasmid containing light and heavy chains of polynucleotides encoding antigen-binding (Fab) fragments of anti-factor D antibodies corresponding to amino acid sequences SEQ ID NO: 48 and SEQ ID NO: 49, respectively (see anti-factor D antibody number 238-1 in International Application Publication No. WO2009 / 134711 and anti-factor D antibody number 111 in International Application Publication No. WO2008 / 055206, each of which is incorporated herein by reference in its entirety).

[0154] The expression plasmid was transformed into Escherichia coli host strain 64B4. The expression plasmid contains polynucleotides encoding light chain, heavy chain, and heavy chain fragments of anti-Met antibody corresponding to amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively.

[0155] Expression of the recombinant heavy and light chain Fab fragment peptides was controlled by the phoA promoter and induced when inorganic phosphate in the culture medium was depleted (see Laird et al., (2005) Protein Expr Purif 39:237-246). The heavy and light chain Fab fragment peptides were directed to the periplasm of *E. coli* by the STII signal sequence, where the products were assembled. High-density fermentation at a working volume (WV) of 10 liters was performed as previously described (see Simmons et al., (2002) J Immunol Methods 263:133-147). At a cell density of approximately 200 OD... 550 At this time, start a continuous 3% methionine or water feed, and the feed continues throughout the rest of the fermentation process.

[0156] Three different fermentation processes were examined using host cell 60E4 (fermentation process AF1), host cell 66F8 (fermentation process AF2), and host cell 64B4 (fermentation process AF3) (see Example 5 and Table 4 below).

[0157] purification

[0158] After fermentation, the whole-cell culture medium was cooled to <15°C in the fermenter and used for protein purification. One volume of the cooled culture medium was mixed with 0.06 volumes of MgSO4 (final concentration 60 mM) and titrated to pH 3.8 with citric acid (1 M). The cells were then disrupted using a microfluidizer at approximately 12,000 psi (Microfluidics, Redwood Shores, CA), and the disrupted cells were incubated at 35°C for 3 hours with continuous shaking. The homogenate was diluted 3-fold with cold pure water and centrifuged at 6000 × g for 20 minutes at 4°C using a fixed-angle rotor. The supernatant was filtered through a 0.22 μm filter and titrated to pH 7.5 with 1.5 M Tris base.

[0159] The recombinant Fab protein was purified using protein G affinity chromatography as described below. A poly-prep column (Bio Rad) was packed with Protein G Sepharose 4 Fast Flow resin (GE Healthcare) and equilibrated with at least 5 column volumes of PBS (pH 7.2). The filtered supernatant was loaded onto the protein G-packed column, washed twice with PBS, and eluted with 50 mM citric acid. The final Fab protein aggregate was titrated to pH 7 with 1.5 M Tris base, and the ortholeucine content was analyzed as described below. This corresponds to the purification of AF1 from the fermentation process.

[0160] Three different recombinant protein product purification processes were used, each specific to fermentation process AF1 (for host cell 60E4), AF2 (for host cell 66F8), or AF3 (for host cell 64B4) (see Example 7 and Table 6 below).

[0161] Amino acid analysis

[0162] To determine the intracellular methionine level, a sample containing 87.6 × 10⁻⁶ methionine was used. 9 Whole-cell culture samples were precipitated at 17000×g for 5 min at 4 °C, washed once in PBS, and then resuspended in extraction buffer (10 mM Tris, 5 mM EDTA, 5 mM iodoacetamide (IAM), 0.2 mg / ml lysozyme, pH 6.8). Cells were then lysed by two cycles of sonication and centrifuged at 13500 rpm for 20 min to remove cell debris. The supernatant was transferred to a 0.2 μm microcentrifuge tube filter (Bio Rad) and centrifuged at 17000×g for 5 min at 4 °C. The filtrate was diluted and amino acids were analyzed as previously described (Feeney et al., (2013) Biotechnology and Bioengineering, 110:1087-1097). To determine extracellular methionine levels, supernatant samples prepared from whole-cell culture collected during fermentation were diluted after centrifugation at 14,000 rpm for 3 minutes and amino acids were analyzed as described below (see Feeney et al., (2013) Biotechnology and Bioengineering, 110:1087-1097).

[0163] Amino acid concentrations were analyzed using reversed-phase HPLC. Samples containing amino acids were treated with 6-aminoquinolinyl-N-hydroxysuccinimide carbamate to produce highly fluorescent derivatives (see Cohen and Michaud (1993) AnalBiochem 211:279-287). The following amino acids were detected by HPLC analysis with a detection limit of 0.01 mM: histidine, asparagine, serine, glutamine, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, ornithine, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine, and tryptophan.

[0164] Phosphate levels

[0165] Phosphate levels were measured using the COBAS Integra 400 (Roche Diagnostics) according to previously published methods (see Taussky and Shorr (1953) J Biol Chem 202:675-685).

[0166] titer determination

[0167] Whole-cell culture samples were diluted 6-fold with extraction buffer (10 mM Tris, 5 mM EDTA, 5 mM IAM, 0.2 mg / ml lysozyme, pH 6.8) and incubated on ice for 10 minutes. After two rounds of sonication, the samples were centrifuged at 17000 × g for 20 minutes at 4 °C. The product titer from the supernatant was determined by HPLC.

[0168] Overall OD550 measurement

[0169] The global OD is determined using the trapezoidal integral using the following formula. 550 :

[0170] Where j = the index of the first measurement taken at or after 24 hours of incubation; k = the OD value performed. 550 The total number of measurements; t i =Incubation time elapsed at measurement i, in hours; OD 550，i =OD when measuring i 550 .

[0171] Ortholeucine analysis

[0172] To analyze ortholeucine content, purified recombinant protein samples were digested with trypsin according to previously described methods (see Yu et al., (2009) Anal Chem 81:9282-9290). Peptide profiling was performed using reversed-phase HPLC and online liquid chromatography-tandem mass spectrometry (LC / MS) as previously described (see Yu et al., (2009) Anal Chem 81:9282-9290; and Yu et al., (2011) Anal Chem 83:5912-5919). High-resolution mass determination was performed using an LTQ-Orbitrap XL instrument (Thermo Scientific, San Jose, US), with full MS measurements at a resolution set at 400 m / z 60,000, followed by scanning of the ions of interest via an ion trap MS2. To determine the relative levels of ortholeucine in the peptides, extractive ion chromatography was performed using the most abundant charge state with an extraction window of m / z ± 10 ppm for a single isotope, generating peptides containing methionine and ortholeucine. The relative amount of the product containing oroleucine relative to the product containing methionine was calculated using their respective overall peak areas.

[0173] Protein blot

[0174] The whole-cell culture samples obtained during fermentation were diluted 6-fold with extraction buffer (10 mM Tris, 5 mM EDTA, 5 mM IAM, 0.2 mg / ml lysozyme, pH 6.8) and incubated on ice for 10 min. After two rounds of sonication, the samples were centrifuged at 17000 × g for 25 min at 4 °C. The samples were loaded onto 4–12% Tris-glycine gels under non-reducing conditions. Proteins were transferred to nitrocellulose membranes using the iBlot system (Invitrogen). The membranes were blocked for 30 min with NET buffer (150 mM sodium chloride, 5 mM EDTA, 50 mM Tris, 0.05% Triton X-100) containing 0.5% gelatin, followed by incubation in blocking buffer containing peroxidase-conjugated goat IgG fractionated anti-human IgG Fab (MP Biomedical) diluted 1:300,000. After washing three times with NET buffer, the imprint became visible after 5 seconds of exposure on X-ray film using Western Lightning ECL substrate (PerkinElmer).

[0175] Example 1: Incorrect incorporation of ortholeucine during E. coli fermentation

[0176] As mentioned above, erroneous incorporation of ortholeucine often occurs during the production of recombinant proteins in E. coli. The extent of erroneous incorporation of ortholeucine during recombinant protein production depends on a variety of factors, such as, for example, the nature of the recombinant protein, the fermentation method used, and the contents of the fermentation medium (see, for example, Bogosian et al., (1989) Biol Chem 264:531-539).

[0177] To examine the erroneous incorporation of ortholeucine during the fermentation process for recombinant protein expression, the following study was conducted. *E. coli* host strain 60E4 was transformed with a plasmid containing nucleic acid sequences encoding the light and heavy chains of the Fab antibody fragment (SEQ ID NO: 31 and SEQ ID NO: 32, respectively), and used in the fermentation studies described below, which employed either a water-feed or methionine-feed method according to the above procedures. The ortholeucine content of the expressed recombinant protein was analyzed using the methods described above.

[0178] As shown in Table 3 below, approximately 5-10% erroneous incorporation of ortholeucine was observed in the recombinant peptides expressed in *E. coli* host cells 60E4 without a continuous methionine feed (i.e., water feed). As expected, no ortholeucine (ND) was detected in the expressed recombinant peptides with a continuous methionine feed.

[0179] Table 3

[0180] These results confirm that ortholeucine is mistakenly incorporated into the production of recombinant proteins in bacteria fed with methionine-free feed.

[0181] Example 2: Construction of Escherichia coli host cells with mutations in the methionine biosynthesis pathway

[0182] As mentioned above, continuous methionine feeding during recombinant protein fermentation is often used to prevent the erroneous incorporation of ortholeucine. As shown in Example 1 above, continuous methionine feeding ensures sufficient methionine is available to the host cells, thereby reducing or preventing erroneous incorporation of ortholeucine during recombinant protein production. To examine the effect of using *E. coli* host cells containing mutated metA and / or metK alleles on erroneous incorporation of ortholeucine, instead of continuous methionine feeding, the following study was conducted.

[0183] In this study, using the allele exchange method (see Materials and Methods above), metA alleles containing mutations of R27C, Q64E, Y294C, I296S, and P298L (these metA alleles produce feedback resistance to MetA) were introduced into 60E4 host cells, thereby obtaining bacterial host cell lines 66H6 (60E4metA(R27C)), 66H8 (60E4metA(Y294C)), 67B8 (60E4metA(Q64E)), and 67B9 (60E4metA(I296S P298L)) (see Tables 2 and 3 above).

[0184] Using the allele exchange method (see Materials and Methods above), the metK allele containing the V185E and c1132del (a deletion of a cytosine base at position 1132 of the metK gene) mutation (which leads to partial loss of MetK enzyme function) was introduced into the host cell of 66H8 (60E4metA(Y294C)) to obtain bacterial host cell strains 67C2 (66H8metK(V185E)) and 67C3 (66H8metK(c1132del)) (see Tables 2 and 3 above).

[0185] The study assessed the erroneous incorporation of ortholeucine during recombinant protein production in these E. coli host cells during fermentation without a continuous methionine feed (see Example 3 below).

[0186] Example 3, Fermentation Results

[0187] Small-scale fermentations (10L) without continuous methionine feed were conducted using bacterial strains with mutated methionine biosynthesis pathways constructed in this study (see Table 1). Water feed or no feed was used instead of methionine feed during these fermentations. Three 10L fermentations were performed using the control host cell line 60E4 as follows: 1) continuous methionine feed, 2) continuous water feed, and 3) no feed.

[0188] Through OD 550The monitored cell growth fermentation trends are shown in Figure 12A. Regardless of feed properties (methionine, water, or no feed), the growth of bacterial host cells 60E4metA(R27C), 60E4metA(Y294C), 60E4metA(Y294C)metK(V185E), and 60E4metA(Y294C)metK(c1132del) with methionine biosynthesis pathway mutations was comparable to that observed in control host cells during the growth phase of fermentation (5–28 hours). However, the double mutant host cells 60E4metA(Y294C)metK(V185E) and 60E4metA(Y294C)metK(c1132del) had lower iOD compared to that observed in control host cell fermentation. 550 (Area under the growth curve from 24 hours of fermentation to the end) (see Figure 12B). Fermentation using host cells 60E4 and 60E4metA (Y294C) with water as feed showed slightly higher iOD compared to control host cell fermentation with methionine feed and 60E4metA (Y294C) host cell fermentation without feed. 550 Mutant cell 60E4ΔmetJ:: kan R 60E4metA (I296SP298L) has a longer adaptation phase, and therefore exhibits a lower iOD compared to that observed in control host cell fermentation. 550 (See Figures 12A and 12B). The mutant host cell 60E4metA (Q64E) grew poorly in the fermenter, reaching its maximum OD. 550 The value was 150, which is higher than the maximum OD observed in fermentation using other mutant host cells. 550 The growth rate was approximately 30-40% lower (see Figure 12A). After 20 hours, the mutant host cell 60E4metA (Q64E) reached saturation; therefore, fermentation using this mutant host cell exhibited the lowest iOD. 550 (See Figures 12A and 12B).

[0189] The presence or absence of methionine feed during fermentation does not affect the growth of 60E4 host cells (see Figure 12C).

[0190] Fermentation using the methionine biosynthesis pathway mutant resulted in higher levels of methionine accumulation in both in vivo (i.e., intracellular) and extracellular media compared to fermentation in control host cells (see Figures 13A, 13B, 14A, and 14B). At the start of fermentation, an excess of methionine (>3 mM) was present in the fermentation medium. As cells began to grow, they took up methionine for protein synthesis, methyl donor function, and other functions. As a result, the extracellular methionine concentration gradually decreased as cells continued to grow, reaching below-detectable levels (<10 μM) at approximately 16 hours (see Figures 13A and 14A).

[0191] At 16 hours, intracellular methionine concentrations varied between different hosts, ranging from 0.5–2.5 mM (concentration based on cell volume) (see Figures 13B and 14B). At such high intracellular methionine concentrations, wild-type MetA was strongly inhibited; however, feedback-resistant MetA mutants may have been only weakly inhibited, allowing mutant host cells to produce methionine via biosynthetic pathways (see Usuda and Kurahashi (2005) Appl Environ Microbiol 71:3228–3234). However, intracellular methionine levels continued to decline until approximately 28 hours, at which point cell growth slowed significantly. It is plausible that during the bacterial growth phase of fermentation (5–28 hours), the rate of methionine utilization for protein synthesis and other cellular functions may exceed the rate of methionine synthesis in vivo. This could explain the gradual decrease in intracellular methionine levels until the end of the growth phase.

[0192] During the fermentation of recombinant protein (28 hours until the end of fermentation), the host cells that overproduce methionine continue to synthesize methionine in vivo, and intracellular methionine levels continue to increase during this phase of fermentation (see Figures 13B and 14B). These results indicate that during the fermentation of recombinant protein, the rate of methionine biosynthesis exceeds the rate of methionine utilization for various intracellular functions.

[0193] During fermentation of control host cells with a continuous water feed, both extracellular and intracellular methionine levels continued to decrease, reaching below the detectable limit (10 μM) at approximately 16 hours for extracellular methionine and below the detectable limit (10 μM) at approximately 24 hours for intracellular methionine. For control host fermentation with a continuous methionine feed, the feed ensured an excess of methionine intracellularly at approximately 26 hours (the time point at which the feed began). During the production phase of fermentation, the double-mutant host cells 60E4metA (Y294C)metK (V185E) accumulated more intracellular methionine compared to that observed in control host cell fermentation with a continuous methionine feed.

[0194] Compared to control host cells, host cells 60E4metA (I296S P298L) and 60E4ΔmetJ:: kan R The longer adaptation phase and poor growth of host cells 60E4metA (Q64E) are likely due to the accumulation of high levels of homocysteine, a toxic intermediate in the methionine biosynthesis pathway (see Roe et al., (2002) Microbiology 148:2215-2222; see Figure 12A). Homocysteine ​​has previously been shown to inhibit threonine deaminase, an enzyme involved in the first step of the isoleucine biosynthesis pathway, leading to growth inhibition (see Tuite et al., (2005) J Bacteriol 187:4362-4371). This theory was validated by measuring intracellular isoleucine levels in mutant host cells. Analysis showed that intracellular isoleucine levels were comparable to those observed in control host cells during fermentation (data not shown). The possibility of other toxic effects of homocysteine ​​on cell growth cannot be completely ruled out. However, these growth differences between mutants are not fully understood at present.

[0195] The time progression of protein product titers and immunoblotting data are shown in Figures 16A, 16B, and 17. Fermentation from host cells 60E4metA (Q64E) produced less product than observed in other host cells. Except for a brief period between 45 and 50 h, phosphate levels were never depleted during fermentation in the 60E4metA (Q64E) host cells (Figure 15); therefore, recombinant protein synthesis was low. Mutant host cells 60E4metA (I296S P298L) and 60E4ΔmetJ::kan RThe extended adaptation phase resulted in phosphate depletion after 40 hours, approximately 12 hours later than typically observed; consequently, fermentation using these host cells yielded lower protein product titers compared to other mutant host cells that depleted phosphate earlier (see Figures 12A, 15, and 16A). Fermentation using host cells 60E4metA (R27C) and 60E4metA (Y294C) produced the highest protein product titers among all mutant host cells examined.

[0196] Fermentation using host cells with the metA-metK double mutants, 60E4metA(Y294C)metK(V185E) and 60E4metA(Y294C)metK(c1132del), produced somewhat low protein product titers, despite comparable growth to control host cells. These double-mutant host cells have a mutation in the metK gene, resulting in partial loss of MetK function. The product of MetK is S-adenosylmethionine (SAM), a methyl donor for many reactions in bacterial cells. However, it is not yet known why reduced SAM levels affect protein product titers. Continuous feeding during fermentation may lead to dilution of the culture medium, which could result in lower cell density and lower product titers. The growth and titers of fermentation using the host cell 60E4metA(Y294C) without any feed were comparable to those observed in fermentation of the same host cells using a continuous water feed.

[0197] Example 4: Incorrect Incorporation of Leucine

[0198] As described above, the misincorporation of ortholeucine into proteins occurs because methionine levels in cells are sufficiently low, allowing ortholeucine to compete with methionine residues for loading methionyl-tRNA during protein synthesis. As described in Example 1 above, fermentation of control host cells without a methionine feed resulted in high levels of ortholeucine misincorporation in the recombinant protein (Table 3). The low intracellular methionine levels in the production phase of the control host cell fermentation without a methionine feed indicate that ortholeucine residues can compete with methionine residues in the recombinant protein. However, high levels of extracellular and intracellular methionine were observed in the production phase of the mutant host cell fermentation (see Figures 13B and 14B). Due to the elevated intracellular methionine levels, it can be expected that the misincorporation of ortholeucine can be minimized or eliminated when using such host cell fermentations.

[0199] Trypsin digestion of the recombinant protein yielded two methionine-containing peptides: Peptide 1: And peptide 2: Peptide profiling analysis revealed that the recombinant protein assemblies purified from mutant host cell fermentation contained less than detectable levels of leucine misincorporation, while control host cell fermentation without methionine feed accumulated high levels of leucine in both methionine-containing peptides (see Table 3). These results indicate that using the *E. coli* host cell strain of the present invention leads to a reduction or prevention of leucine incorporation into heterologous (e.g., recombinant) peptides.

[0200] Example 5: Other bacterial host cells

[0201] In addition to using host cell 60E4 or host cells derived from 60E4 for experiments, two other bacterial host cells were developed and their growth, leucine misincorporation, and recombinant protein production were examined, as follows.

[0202] Bacterial host cells 66F8 and 64B4 (and bacterial host cell 60E4) are described in Table 2 above. As shown in Table 2, host cell 60E4 has some different genotypes compared to host cells 66F8 and 64B4 (which share similar genotypes).

[0203] Three different fermentation processes were examined using host cells 60E4 (fermentation process AF1), 66F8 (fermentation process AF2), and 64B4 (fermentation process AF3). Table 4 below shows the differences in various fermentation parameters (pH, agitation, culture duration, and feed start time) in each of the examined fermentation processes (AF1, AF2, and AF3).

[0204] Table 4

[0205] a Cells reach OD 550 After setting the temperature to 200, reduce the stirring speed to 800, and then gradually reduce it to 500 rpm every 2 hours by 100 rpm.

[0206] Using the method described in Example 1 above for host cell 60E4, the metA (Y294C) allele was introduced into host cells 66F8 and 64B4. Fermentation was performed using 66F8metA (Y294C) and 64B4metA (Y294C) via fermentation processes AF2 and AF3, respectively, demonstrating that the growth of the host cells was comparable to that observed in their parental host cells (see Figures 19A and 19B; and Table 5 below).

[0207] Example 6: Comparison of the growth rate of E. coli host cells and the yield of recombinant protein products.

[0208] The growth rate and yield of recombinant protein products of various Escherichia coli host strains 60E4metA (Y294C), 66F8metA (Y294C), and 64B4metA (Y294C) were tested using fermentation processes AF1, AF2, and AF3, respectively. As described in Example 3 above, 10L of fermentation was performed on host cells of strain 60E4 using the fermentation process improvements listed in Table 4 above.

[0209] The growth rate and yield of recombinant protein products observed in various host cells of strain 60E4 are discussed in detail in Example 3 above.

[0210] As shown in Figures 20A, 20B, and 20C, the yields of recombinant protein products obtained using host strains 60E4metA (Y294C), 66F8metA (Y294C), and 64B4metA (Y294C) are comparable to those observed using host strains 60E4, 66F8, and 64B4 (see also Table 5 below). The presence or absence of methionine feed does not affect the yield of recombinant proteins obtained from fermentation of 60E4 host cells.

[0211] Table 5

[0212] a For the 60E4metJ and 60E4 metAmetA (I296S P298L) hosts, μ was calculated using time intervals of 6–14 hours and 14–22 hours, respectively. For all other hosts, μ was calculated using intervals of 2–10 hours. The μ values ​​shown are averages over n = 2 runs.

[0213] b The values ​​shown are the average values ​​for n = 2 rounds.

[0214] c During fermentation, when the cells reach OD... 550 Add 0.15mM of ortholeucine concentrate at a final concentration of 200.

[0215] Example 7. Comparison of incorrect incorporation of oroleucine

[0216] Three different purification processes were used for the recombinant protein products, each specific to the fermentation processes AF1 (for host cells 60E4), AF2 (for host cells 66F8), and AF3 (for host cells 64B4). Table 6 below shows the differences detected in the purification methods used for each fermentation process (i.e., AF1, AF2, and AF3).

[0217] Table 6

[0218] a The final concentration is expressed as coagulant.

[0219] The recombinant protein products described above were quantified using LC-MS analysis on trypsin peptides.

[0220] Trypsin digestion of recombinant proteins produced by the 60E4 host yielded two methionine-containing peptides (Table 7). Trypsin digestion of recombinant proteins produced by the 66F8 host yielded three methionine-containing peptides (Table 8). Trypsin digestion of recombinant proteins produced by the 64B4 host yielded six methionine-containing peptides (Table 9).

[0221]

[0222] Recombinant proteins purified from AF1 fermentation using the 60E4 host under methionine-free feed showed accumulations of 5.1% and 10% ortholeucine at two methionine residues in the protein (Table 7). Ortholeucine was not detected in recombinant proteins purified from AF1 fermentation using the hosts 60E4metA(Y294C) (Table 7), 60E4metA(R27C), 60E4metA(Y294C)metK(V185E), and 60E4metA(Y294C)metK(c1132del) under methionine-free feed (data not shown).

[0223] When the 60E4 metA (Y294C) host was fermented, leucine (0.15 mM final concentration) was added to the fermentation medium. No leucine was observed in the recombinant protein, indicating that the bacterial host cell of the present invention produces sufficient methionine in the cell to prevent the erroneous incorporation of leucine during the synthesis of the recombinant protein.

[0224] The AF2 fermentation process using the 66F8 host and a methionine-free feed produced recombinant proteins, and approximately 2.7%, 0.7%, and 1% erroneous leucine incorporation was observed in the three methionine-containing trypsin peptides obtained from these recombinant proteins (see Table 8). Similarly, the AF3 process using the 64B4 host and a methionine-free feed produced recombinant proteins, and approximately 1.3%, 1.3%, 2.4%, 2.2%, 1.5%, and 1.3% erroneous leucine incorporation was observed in the six methionine-containing trypsin peptides obtained from these recombinant proteins (see Table 9). However, no leucine was detected in the recombinant proteins purified from the AF2 and AF3 fermentation processes using the 66F8metA (Y294C) and 64B4metA (Y294C) hosts, respectively (see Tables 8 and 9 above).

[0225] Trypsin peptide profiling analysis revealed that the recombinant protein assemblies purified from the fermentation of mutated host cells contained erroneous incorporation of ortholeucine at levels below detectable levels, while control host cell fermentation without methionine feed resulted in the accumulation of high levels of ortholeucine in methionine-containing peptides. These results indicate that using the *E. coli* host cell strain of the present invention leads to a reduction or prevention of ortholeucine incorporation into heterologous (e.g., recombinant) peptides.

[0226] While the invention described above has been illustrated in detail for clarity of understanding, the illustrations and embodiments should not be construed as limiting the scope of the invention. All patent and scientific disclosures cited herein are expressly incorporated in their entirety by reference.

Claims

1. A method for preventing or reducing the erroneous incorporation of oroleucine into a protein or polypeptide, the method comprising expressing the protein or polypeptide in a microorganism, wherein the microorganism is a mutant microorganism that produces methionine to a degree or range sufficient to prevent or reduce the erroneous incorporation of oroleucine into the protein or polypeptide.

2. The method of claim 1, wherein the microorganism is bacteria.

3. The method according to claim 1, wherein the microorganism is Escherichia coli.

4. The method according to claim 1, wherein the microorganism is a feedback-inhibiting or feedback-insensitive homoserine succinyltransferase microorganism.

5. The method according to claim 1, wherein the microorganism is a microorganism that relieves inhibition of methionine production.

6. The method according to claim 1, wherein the microorganism contains a mutated metA allele, a mutated metK allele, or a mutated metA allele and a mutated metK allele.

7. The method according to claim 1, wherein the expression of proteins or polypeptides in microorganisms is carried out in the absence of exogenous addition of methionine to the culture medium or in the absence of methionine-free feed.

8. A microorganism containing a mutated metA allele, a mutated metK allele, or a mutated metA allele and a mutated metK allele.

9. The microorganism of claim 8, wherein the microorganism comprises a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence encoding an amino acid substitution in MetA, the MetA amino acid substitution being selected from the substitution of arginine to cysteine ​​at amino acid position 27, the substitution of glutamine to glutamate at amino acid position 64, the substitution of tyrosine to cysteine ​​at amino acid position 294, the substitution of isoleucine to serine at amino acid position 296, the substitution of proline to leucine at amino acid position 298, and the substitution of isoleucine to serine at amino acid position 296 and the substitution of proline to leucine at amino acid position 298.

10. The microorganism of claim 9, wherein the microorganism comprises a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:

26.

11. The microorganism of claim 8, wherein the mutated metK allele comprises a nucleic acid sequence encoding an amino acid substitution in MetK with a valine to glutamic acid substitution at amino acid position 185, or a nucleic acid sequence with a cytosine base deletion at nucleic acid residue position 1132 of the metK allele.

12. The microorganism of claim 11, wherein the microorganism comprises a mutated metK allele, wherein the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO:27 and SEQ ID NO:

28.

13. The microorganism of claim 8, wherein the microorganism comprises a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence encoding an amino acid substitution in MetA, the MetA amino acid substitution being selected from an arginine-cysteine ​​substitution at amino acid position 27, a glutamine-glutamate substitution at amino acid position 64, a tyrosine-cysteine ​​substitution at amino acid position 294, an isoleucine-serine substitution at amino acid position 296, a proline-leucine substitution at amino acid position 298, and an isoleucine-serine substitution at amino acid position 296 and a proline-leucine substitution at amino acid position 298, and further wherein the mutated metK allele comprises a nucleic acid sequence encoding an amino acid substitution in MetK containing a valine-glutamate substitution at amino acid position 185, or a nucleic acid sequence containing a cytosine base deletion at nucleic acid residue position 1132 of the metK allele.

14. The microorganism of claim 13, wherein the microorganism comprises a mutated metA allele, wherein the mutated metA allele comprises a nucleic acid sequence selected from SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, and further wherein the microorganism comprises a mutated metK allele, wherein the mutated metK allele comprises a nucleic acid sequence selected from SEQ ID NO:27 and SEQ ID NO:

28.

15. The microorganism of claim 8 further comprises nucleic acid encoding an anti-VEGF antibody or an anti-VEGF antibody fragment.

16. The microorganism according to claim 15, wherein the nucleic acid encoding the anti-VEGF antibody or the anti-VEGF antibody fragment is a nucleic acid encoding the amino acid sequence SEQ ID NO:46 and a nucleic acid encoding the amino acid sequence SEQ ID NO:

47.

17. The microorganism according to claim 15, wherein the nucleic acid encoding an anti-VEGF antibody or an anti-VEGF antibody fragment is selected from the nucleic acid sequence comprising SEQ ID NO:33 and the nucleic acid sequence comprising SEQ ID NO:

34.

18. The microorganism of claim 8, further comprising nucleic acid encoding an anti-factor D antibody or an anti-factor D antibody fragment.

19. The microorganism according to claim 18, wherein the nucleic acid encoding anti-factor D antibody or anti-factor D antibody fragment is a nucleic acid encoding the amino acid sequence SEQ ID NO:48 and a nucleic acid encoding the amino acid sequence SEQ ID NO:

49.

20. The microorganism of claim 8, further comprising nucleic acid encoding an anti-MET antibody or an anti-MET antibody fragment.

21. The microorganism according to claim 20, wherein the nucleic acid encoding the anti-MET antibody or the anti-MET antibody fragment is selected from the nucleic acid encoding the amino acid sequence SEQ ID NO:50, the nucleic acid encoding the amino acid sequence SEQ ID NO:51, and the nucleic acid encoding the amino acid sequence SEQ ID NO:

52.

22. A method for producing a protein or polypeptide in a bacterial host cell, wherein the protein or polypeptide is free from leucine misincorporation, the method comprising expressing a nucleic acid encoding the protein or polypeptide in a bacterial host cell under suitable culture conditions that allow for protein or polypeptide expression, wherein the bacterial host cell contains a mutated metA allele, a mutated metK allele, or a mutated metA allele and a mutated metK allele, thereby producing a protein or polypeptide free from leucine misincorporation.

23. The method of claim 22, wherein the bacterial host cell is selected from the microorganisms of claim 9, 10, 11, 12, 13, and 14.

24. The method of claim 22, wherein the protein or polypeptide is an antibody or an antibody fragment.

25. The method of claim 23, wherein the protein or polypeptide is an antibody or an antibody fragment.

26. The method of claim 24, wherein the antibody or antibody fragment is an anti-VEGF antibody or an anti-VEGF antibody fragment.

27. The method of claim 26, wherein the nucleic acid encoding the anti-VEGF antibody or the anti-VEGF antibody fragment is a nucleic acid encoding the amino acid sequence SEQ ID NO:46 and a nucleic acid encoding the amino acid sequence SEQ ID NO:

47.

28. The method of claim 26, wherein the nucleic acid encoding an anti-VEGF antibody or an anti-VEGF antibody fragment is selected from nucleic acid sequences comprising SEQ ID NO:33 and nucleic acid sequences comprising SEQ ID NO:

34.

29. The method of claim 24, wherein the antibody or antibody fragment is an anti-factor D antibody or an anti-factor D antibody fragment.

30. The method of claim 29, wherein the nucleic acid encoding the anti-factor D antibody or the anti-factor D antibody fragment is a nucleic acid encoding the amino acid sequence SEQ ID NO:48 and a nucleic acid encoding the amino acid sequence SEQ ID NO:

49.

31. The method of claim 24, wherein the antibody or antibody fragment is an anti-MET antibody or an anti-MET antibody fragment.

32. The method according to claim 31, wherein the nucleic acid encoding the anti-MET antibody or the anti-MET antibody fragment is selected from the nucleic acid encoding the amino acid sequence SEQ ID NO:50, the nucleic acid encoding the amino acid sequence SEQ ID NO:51, and the nucleic acid encoding the amino acid sequence SEQ ID NO:

52.

33. The method of claim 22, wherein the expression of proteins or polypeptides in bacterial host cells is carried out in the absence of exogenous addition of methionine to the culture medium or in the absence of methionine-free feed.

34. The anti-VEGF antibody or anti-VEGF antibody fragment produced by the method of claim 26, claim 27 or claim 28.

35. The anti-factor D antibody or anti-factor D antibody fragment produced by the method of claim 29 or claim 30.

36. The anti-MET antibody or anti-MET antibody fragment produced by the method of claim 31 or claim 32.

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