ENGINEERED STRAINS OF CORYNEBACTERIA

[01] Each reference, patent, and published patent application cited in this disclosure is incorporated herein by reference in its entirety.

[02] This application incorporates by reference a 163 kb text file created on December 15, 2017 and named“820000028sequencelisting.txt,” which is the sequence listing for this application.

CROSS-REFERENCE TO RELATED APPLICATIONS

[03] This application claims priority to U.S. Provisional Patent Application No. 62/599607 filed December 15, 2017, the contents of the entirety of which is incorporated by this reference.

TECHNICAL FIELD [04] This disclosure relates generally to lysine production.

BRIEF DESCRIPTION OF THE FIGURES

[05] Figure 1 A-B is a multiple alignment of SEQ ID NO: 3 with the amino acid sequences of three corynebacterial ribonuclease J (maJ) proteins identified by National Center for

Biotechnology Information (NCBI) Reference Nos. WP_0l 1014791.1 (SEQ ID NO:78), WP_096456687.1 (SEQ ID NO:79), and WP_023635875. l (SEQ ID NO:80). A box identifies the amino acid in each sequence that alignments with amino acid 448 of SEQ ID NO:3. Figure 1A, alignment with amino acids 1-560 of SEQ ID NO:3. Figure 1B, alignment with amino acids 561-718 of SEQ ID NO:3.

[06] Figure 2 is a multiple alignment of SEQ ID NO:6 with the amino acid sequences of three corynebacterial accDA proteins identified by NCBI Reference Nos. WP_075348l28 (SEQ ID NO:8l), AGT04828.1 (SEQ ID NO:82), and WP_066564978.1 (SEQ ID NO:83). A box identifies the amino acid in each sequence that alignments with amino acid 310 of SEQ ID NO:6.

[07] Figure 3 is a multiple alignment of SEQ ID NO:9 with the amino acid sequences of three corynebacterial cgl l44 proteins identified by NCBI Reference Nos. WP_0l 1897001 (SEQ ID NO: 84), WP_066565l24 (SEQ ID NO:85), and EEW49979 (SEQ ID NO:86). A box identifies the amino acid in each sequence that alignments with amino acid 66 of SEQ ID NO: 9.

DETAILED DESCRIPTION

[08] This disclosure provides engineered strains of Corynebacteria for the cost-effective production of lysine, tools and methods used to produce the engineered strains, and methods of using the engineered strains to produce lysine.

Structural Alterations

[09] This disclosure provides the following six structural alterations that can be engineered into Corynebacteria to improve lysine production: (a) alteration of the maJ coding sequence to encode a corynebacterial ribonuclease J protein comprising a G448S substitution; (b) alteration of the native accDA coding sequence to encode a corynebacterial acetyl-CoA carboxylase carboxyl transferase subunit alpha/beta protein comprising a G310E substitution; (c) alteration of the native cgl 144 coding sequence to encode a corynebacterial cgl 144 protein comprising a P66S substitution; (d) replacement of the native cg2766 promoter; (e) replacement of the native promoter of butyryl-Co A: acetate coenzyme A transferase (actA); and (f) replacement of the native opcA coding sequence by a coding sequence for an opcA protein of a different

Corynebacterium species or strain. a. Altered corynebacterial ribonuclease J proteins

[10] This disclosure provides altered corynebacterial ribonuclease J (maJ) proteins comprising a G448S substitution. One example of such a protein is shown in SEQ ID NO: 3.“A

corynebacterial maJ protein comprising a G448S substitution” as used herein means an maJ protein from a strain of Corynebacterium which, when compared with SEQ ID NO:3 using the NCBTs BLAST® alignment tool, has serine instead of glycine at the position that aligns with amino acid 448 of SEQ ID NO: 3, as illustrated in Figure 1A-B. Corynebacterial maJ proteins which can be altered to comprise the G448S substitution include, but are not limited to, proteins (regardless of how named) identified with the amino acid sequences provided by the NCBI Reference numbers in Table 1. Table 1. NCBI Reference Numbers of Corynebacterial rnaJ Proteins

b. Altered corynebacterial accDA proteins

[11] This disclosure provides altered corynebacterial accDA proteins comprising a G310E substitution. One example of such a protein is shown in SEQ ID NO:6.“A corynebacterial accDA protein comprising a G310E substitution” as used herein means an accDA protein from a strain of Corynebacterium which, when compared with SEQ ID NO:6 using the National Center for Biotechnology Information’s BLAST® alignment tool, has glutamic acid instead of glycine at the position that aligns with amino acid 310 of SEQ ID NO:6, as illustrated in Figure 2. Corynebacterial accDA proteins which can be altered to comprise the G310E substitution include, but are not limited to, proteins (regardless of how named) identified with the amino acid sequences provided by the NCBI Reference numbers in Table 2.

Table 2. NCBI Reference Numbers of Corynebacterial accDA Proteins

c. Altered corynebacterial cgll44 proteins

[12] This disclosure provides altered corynebacterial cgl 144 proteins comprising a P66S substitution. One example of such a protein is shown in SEQ ID NO:9.“A corynebacterial cgl 144 protein comprising a P66S substitution” as used herein means a cgl 144 protein from a strain of Corynebacterium which, when compared with SEQ ID NO:9 using the National Center for Biotechnology Information’s BLAST® alignment tool, has serine instead of proline at the position that aligns with amino acid 66 of SEQ ID NO:9, as illustrated in Figure 3.

Corynebacterial cgl 144 proteins which can be altered to comprise the P66S substitution include, but are not limited to, proteins (regardless of how named) identified with the amino acid sequences provided by the NCBI Reference numbers in Table 3.

Table 3. NCBI Reference Numbers of Corynebacterial cgll44 Proteins

d. Replacement of the Native cg2766 promoter

[13] In some embodiments, the native cg2766 promoter is replaced with a replacement promoter. The replacement promoter can be a promoter from a different gene of the

Corynebacterium species or strain being engineered or can be a heterologous promoter (i.e., a promoter of another Corynebacterium species or strain or an artificially constructed promoter). These promoters include, but are not limited to, promoters disclosed in Nesvera et al, 2012; Patek et al, 2003(a); Patek et al., 2003(b); Patek et al, 2013; Rytter et al, 2014; Shang et al., 2017; Yim et al., 2013; US 2017/0159045; and WO 2017/00376. In some embodiments, the replacement promoter is promoter Pcg0007_39 (SEQ ID NO:20); see US 2017/0159045 and WO 2017/00376. e. Replacement of the Native actA promoter

[14] In some embodiments, the native actA promoter is replaced with a replacement promoter. The replacement promoter can be a promoter from a different gene of the Corynebacterium species or strain being engineered or can be a heterologous promoter (i.e., a promoter of another Corynebacterium species or strain or an artificially constructed promoter). These promoters include, but are not limited to, promoters disclosed in Nesvera et al, 2012; Patek et al., 2003(a); Patek et al, 2003(b); Patek et al., 2013; Rytter et al, 2014; Shang et al, 2017; Yim et al, 2013; US 2017/0159045; and WO 2017/00376. In some embodiments, the replacement promoter is promoter Pcg0007_39 (SEQ ID NO:20). f. Replacement of the Native opcA Coding Sequence

[15] In some embodiments, the native opcA coding sequence is replaced with a coding sequence for an opcA protein of a different Corynebacterium species or strain. Suitable opcA proteins are listed in Table 4A and Table 4B. Using the NCBI’s BLAST® alignment tool, the amino acid sequences of the opcA proteins in Table 4A are less than 75% identical to the amino acid sequence of the opcA protein of NRRL B-11474 (SEQ ID NO:94), and the amino acid sequences of the opcA proteins in Table 4B are more than 75% identical to the amino acid sequence of the opcA protein of NRRL B-l 1474 (SEQ ID NO:94).

Table 4A. NCBI Reference Numbers for Corynebacterial opcA Proteins

Table 4B. NCBI Reference Numbers for Corynebacterial OpcA Proteins

Nucleic Acids and Vectors

[16] This disclosure provides nucleic acids encoding the altered corynebacterial maJ, accDA, and cgl l44 proteins described above. SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8 are examples of nucleotide sequences encoding SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:9, respectively, but any nucleotide sequence that encodes the altered corynebacterial protein can be used. The nucleotide sequences can be optimized for expression in various species or strains of Corynebacteria as is well known in the art.

[17] Nucleic acids encoding the altered corynebacterial proteins described above can be included in vectors in which a coding sequence is operably linked to a suitable regulatory sequence for expression in a Corynebacterium. The regulatory sequence includes a suitable mRNA ribosome binding site and a sequence for regulating the termination of transcription and translation and may include other elements, such as a promoter or operator. Once transformed into a host Corynebacterium, the vector may replicate or function independently of the host genome or may integrate into the genome itself. The vector that is used is not specifically limited and may be any vector known in the art, as long as it can replicate in a Corynebacterium host. See, for example, Lee, 2014; Knoppova et al., 2007; and Patek & Nesvera, 2013. [18] A vector can include at least one selectable marker, such as an antibiotic resistance gene. Suitable antibiotics include, e.g., amikacin, ampicillin, augmentin (amoxicillin plus clavulonic acid), cefazolin, cefoxitin, ceftazidime, ceftiofur, cephalothin, chloramphenicol,

enrofloxacin, florfenicol, gentamicin, imipenem, kanamycin, penicillin, sarafloxicin, spectinomycin, streptomycin, tetracycline, ticarcillin, and tilmicosin.

[19] Vectors can be used to engineer a Corynebacterium having one or more of the structural alterations described above, resulting in improved lysine production compared with the corresponding native Corynebacterium (i.e., the Corynebacterium which has not been engineered to include the structural alterations). Such corynebacteria include, but are not limited to, the Corynebacterium deposited as NRRL B-l 1474 and the corynebacterial species and strains in Tables 1, 2, 3, 4A, and 4B. Methods of delivering vectors to Corynebacteria are well known and include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, lipid-mediated transfection, electroporation, conjugation, and infection.

Engineered Strains of Corynebacteria

[20] This disclosure provides strains of Corynebacteria engineered to contain at least one of the six alterations described above; i.e., (a) alteration of the maJ coding sequence to encode a ribonuclease J protein comprising a G448S substitution; (b) alteration of the native accDA coding sequence to encode an acetyl-CoA carboxylase carboxyl transferase subunit alpha/beta protein comprising a G310E substitution; (c) alteration of the native cgl 144 coding sequence to encode a cgl 144 protein comprising a P66S substitution; (d) replacement of the native cg2766 promoter; (e) replacement of the native actA promoter; and (f) replacement of the native opcA coding sequence by coding sequence for an opcA protein of a different Corynebacterium species or strain.

[21] In some embodiments, the genome of a Corynebacterium is engineered to contain a coding sequence for an maJ protein comprising a G448S substitution. In some of these embodiments, the engineered Corynebacterium is any of the species or strains listed in Tables 1, 2, 3, 4A, and 4B. In other of these embodiments, the engineered Corynebacterium is NRLL B- 11474. In some of these embodiments, the genome of the engineered Corynebacterium consists essentially of the genome of the native Corynebacterium but for the modification of the maJ coding sequence to include the G448S substitution; i.e., this modification is the only change engineered into the native Corynebacterium. In other embodiments, the genome of the engineered Corynebacterium includes one or more of the alterations described above. [22] In some of the embodiments described in the paragraph above, the engineered

Corynebacterium comprises a coding sequence for one of the maJ proteins disclosed in Table 1, but for a codon change to include the G448S substitution. In other of these embodiments, the maJ protein comprising the G448S substitution has the amino acid sequence SEQ ID NO:3.

[23] In some embodiments, the genome of a Corynebacterium is engineered to contain a coding sequence for an accDA protein comprising a G310E substitution. In some of these embodiments, the engineered Corynebacterium is any of the species or strains listed in Tables 1, 2, 3, 4A, and 4B. In other of these embodiments, the engineered Corynebacterium is NRRL B- 11474. In some of these embodiments, the genome of the engineered Corynebacterium consists essentially of the genome of the native Corynebacterium but for the modification of the accDA coding sequence to include the G310E substitution; i.e., this modification is the only change engineered into the native Corynebacterium. In other embodiments, the genome of the engineered Corynebacterium includes one or more of the alterations described above.

[24] In some of the embodiments described in the paragraph above, the engineered

Corynebacterium comprises a coding sequence for one of the accDA proteins disclosed in Table 2, but for a codon change to include the G310E substitution. In other of these embodiments, the accDA protein comprising the G310E substitution has the amino acid sequence SEQ ID NO:6.

[25] In some embodiments, the genome of a Corynebacterium is engineered to contain a coding sequence for cgl l44 protein comprising a P66S substitution. In some of these embodiments, the engineered Corynebacterium is any of the species or strains listed in Tables 1,

2, 3, 4A, and 4B. In other of these embodiments, the engineered Corynebacterium is NRRL B- 11474. In some of these embodiments, the genome of the engineered Corynebacterium consists essentially of the genome of the native Corynebacterium but for the modification of the cgl 144 coding sequence to include the P66S substitution; i.e., this modification is the only change engineered into the native Corynebacterium. In other embodiments, the genome of the engineered Corynebacterium includes one or more of the alterations described above.

[26] In some of the embodiments described in the paragraph above, the engineered

Corynebacterium comprises a coding sequence for one of the cgl 144 proteins disclosed in Table

3, but for a codon change to include the P66S substitution. In other of these embodiments, the cgl 144 protein comprising the P66S substitution has the amino acid sequence SEQ ID NO:9.

[27] In some embodiments, the genome of a Corynebacterium is engineered to replace the native cg2766 promoter with a replacement promoter. In some of these embodiments, the engineered Corynebacterium is any of the species or strains listed in Tables 1, 2, 3, 4A, and 4B. In other of these embodiments, the engineered Corynebacterium is NRRL B-11474. In some of these embodiments, the genome of the engineered Corynebacterium consists essentially of the genome of the native Corynebacterium but for the replacement of the native cg2766 promoter; i.e., this modification is the only change engineered into the native Corynebacterium. In other embodiments, the genome of the engineered Corynebacterium includes one or more of the alterations described above. In some embodiments described in this paragraph, the replacement promoter is promoter Pcg0007_39 (SEQ ID NO: 20).

[28] In some embodiments, the genome of a Corynebacterium is engineered to replace the native actA promoter with a replacement promoter. In some of these embodiments, the engineered Corynebacterium is any of the species or strains listed in Tables 1, 2, 3, 4A, and 4B. In other of these embodiments, the engineered Corynebacterium is NRRL B-11474. In some of these embodiments, the genome of the engineered Corynebacterium consists essentially of the genome of the native Corynebacterium but for the replacement of the native actA promoter; i.e., this modification is the only change engineered into the native Corynebacterium. In other embodiments, the genome of the engineered Corynebacterium includes one or more of the alterations described above. In some embodiments described in this paragraph, the replacement promoter is promoter Pcg0007_39 (SEQ ID NO: 20).

[29] In some embodiments, the genome of a Corynebacterium is engineered to replace the native coding sequence for the opcA protein by a coding sequence for an opcA protein of a different Corynebacterium species or strain. In some of these embodiments, the engineered Corynebacterium is any of the species or strains listed in Tables 1, 2, 3, 4A, and 4B. In other of these embodiments, the engineered Corynebacterium is NRRL B-l 1474. In some of these embodiments, the genome of the engineered Corynebacterium consists essentially of the genome of the native Corynebacterium but for the replacement of the native opcA coding sequence; i.e., this modification is the only change engineered into the native Corynebacterium. In other embodiments, the genome of the engineered Corynebacterium includes one or more of the alterations described above.

[30] In some of embodiments described in the paragraph above, the genome of the engineered Corynebacterium comprises a replacement opcA coding sequence that encodes an opcA protein selected from the opcA proteins in Table 4A and Table 4B, provided that, if the opcA replacement is the only change made to the bacterium, the replacement opcA coding sequence does not encode an opcA protein selected from the opcA proteins in Table 4B. In other embodiments, the replacement opcA coding sequence encodes an opcA protein selected from C. casei UCMA 3821 (SEQ ID NO:92), C. humireducens (SEQ ID NO: 25), C. falsenii (SEQ ID NO:89), C. vitaeruminis (SEQ ID NO:87), C. terpenotabidum (SEQ ID NO:93), C.

pyruviciproducens (SEQ ID NO:9l), C. matruchotii (SEQ ID NO:88), and C. halotolerans (SEQ ID NO: 90). In other embodiments, the replacement opcA coding sequence encodes the opcA protein of C. humireducens (SEQ ID NO:25).

[31] In some of the embodiments described in the paragraph above, the replacement opcA coding sequence is under the control of its native promoter. In other embodiments, the replacement opcA coding sequence is under the control of a promoter from a different gene of the Corynebacterium species or strain being engineered or a heterologous promoter (i.e., a promoter of another Corynebacterium species or strain or an artificially constructed promoter). These promoters include, but are not limited to, promoters disclosed in Nesvera et al, 2012; Patek et al, 2003(a); Patek et al., 2003(b); Patek et al, 2013; Rytter et al, 2014; Shang et al., 2017; Yim et al., 2013; US 2017/0159045; and WO 2017/00376. In some embodiments, the replacement opcA coding sequence is under the control of promoter Pcg0007_39 (SEQ ID NO:20).

Optional Alterations

[32] Any of the engineered Corynebacteria described above optionally can include one or more additional structural alterations for improved lysine production. These alterations include, but are not limited to, one or more of the following structural alterations: (f) insertion of at least one additional copy of at least one gene selected from the group consisting ask. asd, ddh, and dapB (g) replacement of the start codon of the native aceE gene; (h) replacement of the native pyc gene promoter; (i) insertion of an additional promoter in front of the zwf open reading frame; and (k) insertion of a codon-optimized lysA coding sequence.

[33] Nucleic acids that can be used to engineer these optional structural alterations described above are well known and are described, for example, in U.S. Patent 7,368,276, U.S. Patent 6,927,046, US 2017/0159045, and WO 2017/00376 and in the Examples, below. g. Insertion of at least one additional copy of lysA , ask, asd , ddh, and/or dapB

[34] In any of the embodiments described above, the engineered Corynebacterium includes at least one additional copy of at least one gene selected from the group consisting of lysA, ask, asd, ddh, and dapB. In some of these embodiments, this is the only optional alteration present in the engineered Corynebacterium genome. In other embodiments, one or more other optional alterations are present. The additional copies can be provided using one or more vectors.

Example 1 describes two integration vectors that can be used to insert copies of ask. asd. ddh, and dapB.

[35] In some embodiments, the engineered Corynebacterium contains at least one additional copy of lysA . ask, asd, ddh, and dapB.

[36] In some embodiments, the engineered Corynebacterium contains at least one additional copy of ask, asd, ddh, and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA, asd, ddh, and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA, ask, ddh, and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA, ask, asd, and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA, ask, asd, and ddh.

[37] In some embodiments, the engineered Corynebacterium contains at least one additional copy of asd, ddh, and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of ask, ddh, and dapB. In other embodiments, the engineered

Corynebacterium contains at least one additional copy of ask, asd, and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of ask, asd, and ddh. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA, ddh, and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA, asd, and dapB. In other embodiments, the engineered

Corynebacterium contains at least one additional copy of lysA, asd, and ddh. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA, ask, and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA, ask, and ddh.

[38] In some embodiments, the engineered Corynebacterium contains at least one additional copy of ddh and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of asd and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of asd and ddh. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA and dapB. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA and ddh. In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA . and ask.

[39] In other embodiments, the engineered Corynebacterium contains at least one additional copy of lysA. In other embodiments, the engineered Corynebacterium contains at least one additional copy of ask. In other embodiments, the engineered Corynebacterium contains at least one additional copy of asd. In other embodiments, the engineered Corynebacterium contains at least one additional copy of ddh. In other embodiments, the engineered Corynebacterium contains at least one additional copy of dapB.

[40] In any of the embodiments described above in which the engineered Corynebacterium contains at least one additional copy of lysA. the additional copy of lysA is a codon-optimized version described below. h. Replacement of the start codon of aceE

[41] In any of the embodiments described above, the start codon of the native aceE gene in the genome of the engineered Corynebacterium is replaced. In some of these embodiments, this is the only optional alteration present in the engineered Corynebacterium genome. In other embodiments, one or more other optional alterations are present.

[42] In some of these embodiments, the start codon is replaced with GTG, CTG, or TTG. In some embodiments, the start codon is replaced with TTG. i. Replacement of the native pyc gene promoter

[43] In any of the embodiments described above, the native pyc gene promoter is replaced with a replacement promoter. The replacement promoter can be a promoter from a different gene of the Corynebacterium species or strain being engineered or can be a heterologous promoter (i.e., a promoter of another Corynebacterium species or strain or an artificially constructed promoter). These promoters include, but are not limited to, promoters disclosed in Nesvera et al., 2012; Patek et al, 2003(a); Patek et al., 2003(b); Patek et al, 2013; Rytter et al, 2014; Shang et al, 2017; Yim et al., 2013; US 2017/0159045; and WO 2017/00376. In some embodiments, the replacement promoter is promoter Pcgl860 (SEQ ID NO: l l; see US 2017/0159045). In some of these embodiments, this is the only optional alteration present in the engineered

Corynebacterium genome. In other embodiments, one or more other optional alterations are present. j. Insertion of a promoter in front of the zwf open reading frame

[44] In any of the embodiments described above, the genome of the engineered

Corynebacterium contains a promoter inserted in front of the zwf open reading frame. The inserted promoter can be a promoter from a different gene of the Corynebacterium species or strain being engineered or can be a heterologous promoter (i.e., a promoter of another

Corynebacterium species or strain or an artificially constructed promoter). These promoters include, but are not limited to, promoters disclosed in Nesvera et al, 2012; Patek et al., 2003(a); Patek et al, 2003(b); Patek et al., 2013; Rytter et al, 2014; Shang et al., 2017; Yim et al, 2013; US 2017/0159045; and WO 2017/00376. In some embodiments, the additional promoter is promoter Pcg0007_39 (SEQ ID NO:20). In some of these embodiments, this is the only optional alteration present in the engineered Corynebacterium genome. In other embodiments, one or more other optional alterations are present. k. Insertion of a codon-optimized lysA coding sequence

[45] In any of the embodiments described above, the genome of the engineered

Corynebacterium contains a codon-optimized lysA coding sequence under the control of a promoter and including terminator sequence. The promoter can be a promoter from a different gene of the Corynebacterium species or strain being engineered or can be a heterologous promoter (i.e., a promoter of another Corynebacterium species or strain or an artificially constructed promoter). These promoters include, but are not limited to, promoters disclosed in Nesvera et al, 2012; Patek et al, 2003(a); Patek et al, 2003(b); Patek et al, 2013; Rytter et al., 2014; Shang et al., 2017; Yim et al., 2013; US 2017/0159045; and WO 2017/00376. Terminator sequences include, but are not limited to, those disclosed in Pfeifer-Sancar et al, 2013. In some of these embodiments, this is the only optional alteration present in the engineered

Corynebacterium genome. In other embodiments, one or more other optional alterations are present.

[46] In some embodiments, the promoter is Pcg0007_39 (SEQ ID NO:20). In some of these embodiments, the codon-optimized sequence is SEQ ID NO: 16. In some of these embodiments, the terminator is the sod terminator (nucleotides 1436-1516 of SEQ ID NO: 17). In some embodiments, the genome of the engineered Corynebacterium contains the nucleotide sequence SEQ ID NO: 17, which is codon-optimized lysA sequence SEQ ID NO: 16 under the control of promoter Pcg0007_39 (SEQ ID NO:20) and having a sod terminator (nucleotides 1436-1516 of SEQ ID NO: 17). [47] The following non-limiting embodiments of engineered Corynebacteria fall within the description above.

Embodiment 1. The Corynebacterium deposited as NRRL B-67439.

Embodiment 2. A Corynebacterium having a bacterial genome consisting essentially of the genome of the bacterium deposited as NRRL B-l 1474 but for

(i) up to 6 structural alterations selected from the group consisting of:

(a) alteration of the maJ coding sequence to encode a

corynebacterial maJ protein comprising a G448S substitution;

(b) alteration of the native accDA coding sequence to encode a corynebacterial accDA protein comprising a G310E substitution;

(c) alteration of the native cgl l44 coding sequence to encode a corynebacterial cgl l44 protein comprising a P66S substitution;

(d) replacement of the native cg2766 promoter;

(e) replacement of the native actA promoter; and

(f) replacement of the native opcA coding sequence by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium, and, optionally,

(ii) up to 5 structural alterations selected from the group consisting of:

(g) insertion of at least one additional copy of at least one gene selected from the group consisting of lysA, ask, asd, ddh, and dapB,

(h) replacement of the native start codon of aceE by GTG, CTG, or

TTG;

(i) replacement of the native pyc gene promoter by promoter Pcgl860 (SEQ ID NO: 11);

(j) insertion of promoter Pcg0007_39 (SEQ ID NO:20) in front of the zwf open reading frame;

(k) insertion of a codon-optimized lysA coding sequence.

Embodiment 3. The Corynebacterium of Embodiment 2, in which (a) the native maJ coding sequence is altered to encode the corynebacterial maJ protein comprising a G448S substitution.

Embodiment 4. The Corynebacterium of Embodiment 2 or 3, in which (a) the native maJ coding sequence is altered to encode the amino acid sequence SEQ ID NO:3. Embodiment 5. The Corynebacterium of any of Embodiments 2-4, in which (b) the native accDA coding sequence is altered to encode the corynebacterial accDA protein comprising the G310E substitution.

Embodiment 6. The Corynebacterium bacterium of any of Embodiments 2-5, in which

(b) the native accDA coding sequence is altered to encode the amino acid sequence SEQ ID NO:6.

Embodiment 7. The Corynebacterium of any of Embodiments 2-6, in which (c) the native cgl l44 coding sequence is altered to encode the corynebacterial cgl l44 protein comprising the P66S substitution.

Embodiment 8. The Corynebacterium bacterium of any of Embodiments 2-7, in which

(c) the native cgl l44 coding sequence is altered to encode the amino acid sequence SEQ ID NO:9.

Embodiment 9. The Corynebacterium bacterium of any of Embodiments 2-8, in which

(d) the native cg2766 promoter is replaced.

Embodiment 10. The Corynebacterium of any of Embodiments 2-9, in which (d) the native cg2766 promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 11. The Corynebacterium bacterium of any of Embodiments 2-10, in which

(e) the native aclA promoter is replaced.

Embodiment 12. The Corynebacterium of any of Embodiments 2-11 in which (e) the native actA promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 13. The Corynebacterium bacterium of any of Embodiments 2-12, in which

(f) the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium.

Embodiment 14. The Corynebacterium of any of Embodiments 2-13, in which the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a Corynebacterium selected from the group consisting of Corynebacterium vitaeruminis DSM 20294, Corynebacterium matruchotii ATCC 33806, Corynebacterium falsenii DSM 44353, Corynebacterium halotolerans YIM 70093, Corynebacterium pyruviciproducens ATCC BAA- 1742, Corynebacterium casei UCMA 3821, Corynebacterium terpenotabidum Y-l l, and C. humireducens NBRC 106098.

Embodiment 15. The Corynebacterium of any of Embodiments 2-14, in which the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of C. humireducens NBRC 106098.

Embodiment 16. The Corynebacterium of any of Embodiments 2-15, in which the replacement opcA coding sequence is under control of a replacement promoter. Embodiment 17. The Corynebacterium of Embodiment 16, in which the replacement promoter is promoter Pcg0007_39 (SEQ ID NO: 20).

Embodiment 18. The Corynebacterium of Embodiment 2, in which (b) the native accDA coding sequence is altered to encode the corynebacterial accDA protein comprising the G310E substitution.

Embodiment 19. The Corynebacterium bacterium of Embodiment 18, in which (b) the native accDA coding sequence is altered to encode the amino acid sequence SEQ ID NO:6.

Embodiment 20. The Corynebacterium bacterium of Embodiment 18 or 19, in which (c) the native cgl 144 coding sequence is altered to encode the corynebacterial cgl 144 protein comprising the P66S substitution.

Embodiment 21. The Corynebacterium of any of Embodiments 18-20, in which (c) the native cgl 144 coding sequence is altered to encode the amino acid sequence SEQ ID NO:9.

Embodiment 22. The Corynebacterium bacterium of any of Embodiments 18-21, in which (d) the native cg2766 promoter is replaced.

Embodiment 23. The Corynebacterium of any of Embodiments 18-22, in which (d) the native cg2766 promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 24. The Corynebacterium of any of Embodiments 18-23, in which (e) the native actA promoter is replaced.

Embodiment 25. The Corynebacterium bacterium of any of Embodiments 18-23, in which (e) the native actA promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 26. The Corynebacterium bacterium of any of Embodiments 18-25, in which in which (f) the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium.

Embodiment 27. The Corynebacterium bacterium of any of Embodiments 18-26, in which the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a Corynebacterium selected from the group consisting of Corynebacterium vitaeruminis DSM 20294, Corynebacterium matruchotii ATCC 33806, Corynebacterium falsenii DSM 44353, Corynebacterium halotolerans YIM 70093, Corynebacterium pyruviciproducens ATCC BAA-1742, Corynebacterium casei UCMA 3821, Corynebacterium terpenotabidum Y- 11, and C. humireducens NBRC 106098.

Embodiment 28. The Corynebacterium of any of Embodiments 18-27, in which the native opcA coding sequence is replaced by a replacement coding sequence for the opcA protein of C. humireducens NBRC 106098.

Embodiment 29. The Corynebacterium of any of Embodiments 26-28, in which the replacement opcA coding sequence is under control of a replacement promoter. Embodiment 30. The Corynebacterium of Embodiment 29, in which the replacement promoter is promoter Pcg0007_39 (SEQ ID NO: 20).

Embodiment 31. The Corynebacterium of Embodiment 2, in which the native cgl 144 coding sequence is altered to encode the corynebacterial cgl 144 protein comprising the P66S substitution.

Embodiment 32. The Corynebacterium bacterium of Embodiment 31, in which (c) the native cgl 144 coding sequence is altered to encode the amino acid sequence SEQ ID NO:9.

Embodiment 33. The Corynebacterium of Embodiment 31 or 32, in which (d) the native cg2766 promoter is replaced.

Embodiment 34. The Corynebacterium bacterium of any of embodiments 31-33, in which (d) the native cg2766 promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 35. The Corynebacterium bacterium of any of Embodiments 31-34, in which (e) the native actA promoter is replaced.

Embodiment 36. The Corynebacterium bacterium of any of Embodiments 31-35, in which (e) the native actA promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 37. The Corynebacterium bacterium of any of Embodiments 31-36, in which in which (f) the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium.

Embodiment 38. The Corynebacterium of any of Embodiments 31-37, in which the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a Corynebacterium selected from the group consisting of Corynebacterium

vitaeruminis DSM 20294, Corynebacterium matruchotii ATCC 33806, Corynebacterium falsenii DSM 44353, Corynebacterium halotolerans YIM 70093, Corynebacterium pyruviciproducens ATCC BAA-1742, Corynebacterium casei UCMA 3821, Corynebacterium terpenotabidum Y- 11, and C. humireducens NBRC 106098.

Embodiment 39. The Corynebacterium of any of Embodiments 31-38, in which the native opcA coding sequence is replaced by a replacement coding sequence for an opcA protein of C. humireducens NBRC 106098.

Embodiment 40. The Corynebacterium of any of Embodiments 37-39, in which the replacement opcA coding sequence is under control of a replacement promoter.

Embodiment 41. The Corynebacterium of Embodiment 40, in which the replacement promoter is promoter Pcg0007_39 (SEQ ID NO: 20).

Embodiment 42. The Corynebacterium bacterium of any of Embodiments 31-41, in which (f) the native opcA coding sequence is replaced by a coding sequence for the opcA protein of C. humireducens. Embodiment 43. The Corynebacterium bacterium of Embodiment 2, in which (d) the native cg2766 promoter is replaced.

Embodiment 44. The Corynebacterium of Embodiment 43, in which (d) the native cg2766 promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 45. The Corynebacterium of Embodiment 43 or 44, in which (e) the native actA promoter is replaced.

Embodiment 46. The Corynebacterium bacterium of any of Embodiments 43-46, in which (e) the native actA promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 47. The Corynebacterium bacterium of any of Embodiments 43-46, in which in which (f) the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium.

Embodiment 48. The Corynebacterium of any of Embodiments 43-47, in which the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a Corynebacterium selected from the group consisting of Corynebacterium

vitaeruminis DSM 20294, Corynebacterium matruchotii ATCC 33806, Corynebacterium falsenii DSM 44353, Corynebacterium halotolerans YIM 70093, Corynebacterium pyruviciproducens ATCC BAA-1742, Corynebacterium casei UCMA 3821, Corynebacterium terpenotabidum Y- 11, and C. humireducens NBRC 106098.

Embodiment 49. The Corynebacterium of any of Embodiments 43-48, in which the replacement coding sequence encodes an opcA protein of C. humireducens NBRC 106098.

Embodiment 50. The Corynebacterium of any of Embodiments 43-49, in which the replacement opcA coding sequence is under control of a replacement promoter.

Embodiment 51. The Corynebacterium of Embodiment 50, in which the replacement promoter is promoter Pcg0007_39 (SEQ ID NO: 20).

Embodiment 52. The Corynebacterium bacterium of Embodiment 2, in which (e) the native actA promoter is replaced.

Embodiment 53 The Corynebacterium bacterium of Embodiment 52, in which (e) the native actA promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 54. The Corynebacterium of Embodiment 2, in which in which (f) the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium.

Embodiment 55. The Corynebacterium of Embodiment 54, in which the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a Corynebacterium selected from the group consisting of Corynebacterium vitaeruminis DSM 20294, Corynebacterium matruchotii ATCC 33806, Corynebacterium falsenii DSM 44353, Corynebacterium halotolerans YIM 70093, Corynebacterium pyruviciproducens ATCC BAA- 1742, Corynebacterium casei UCMA 3821, Corynebacterium terpenotabidum Y-l l, and C. humireducem NBRC 106098.

Embodiment 56. The Corynebacterium bacterium of Embodiment 54 or 55, in which the replacement coding sequence encodes an opcA protein of C. humireducens NBRC 106098.

Embodiment 57. The Corynebacterium of any of Embodiments 54-56, in which the replacement opcA coding sequence is under control of a replacement promoter.

Embodiment 58. The Corynebacterium of Embodiment 57, in which the replacement promoter is promoter Pcg0007_39 (SEQ ID NO: 20).

Embodiment 59. The Corynebacterium bacterium of Embodiment 2, in which:

(a) the native maJ coding sequence is altered to encode the amino acid sequence SEQ ID NO:3;

(b the native accDA coding sequence is altered to encode the amino acid sequence SEQ ID NO:6;

(c) the native cgl l44 coding sequence is altered to encode the amino acid sequence SEQ ID NO:9;

(d) the native cg2766 promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20);

(e) the native actA promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20); and

(f) the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of C. humireducens NBRC 106098.

Embodiment 60. The Corynebacterium bacterium of any of Embodiments 2-59, in which the genome of the bacterium contains the at least one additional copy of the at least one gene selected from the group consisting of lysA. ask, asd, ddh, and dapB.

Embodiment 61. The Corynebacterium bacterium of Embodiment 60, in which the genome of the bacterium contains (a) at least one additional copy of lysA . ask, asd, ddh, and dapB.

Embodiment 61. The Corynebacterium bacterium of Embodiment 60, in which the genome of the bacterium contains:

(b) at least one additional copy of ask, asd, ddh, and dapB,'

(c) at least one additional copy of lysA, asd, ddh, and dapB,'

(d) at least one additional copy of lysA, ask, ddh, and dapB,'

(e) at least one additional copy of lysA, ask, asd, and dapB,' or

(f) at least one additional copy of lysA, ask, asd, and ddh. Embodiment 62. The Corynebacterium bacterium of Embodiment 60, in which the genome of the bacterium contains:

(g) at least one additional copy of asd, ddh, and dapB;

(h) at least one additional copy of ask. ddh, and dapB;

(i) at least one additional copy of ask, asd, and dapB,'

(j) at least one additional copy of ask, asd, and ddh:

(k) at least one additional copy of lysA . ddh, and dapB,'

(l) at least one additional copy of lysA . asd, and dapB,'

(m) at least one additional copy of lysA . asd, and ddh;

(n) at least one additional copy of lysA . ask, and dapB; or

(o) at least one additional copy of lysA . ask, and ddh.

Embodiment 63. The Corynebacterium bacterium of Embodiment 60, in which the genome of the bacterium contains:

(p) at least one additional copy of ddh and dapB;

(q) at least one additional copy of asd and dapB;

(r) at least one additional copy of asd and ddh;

(s) at least one additional copy of lysA and dapB;

(t) at least one additional copy of lysA and ddh;

(u) at least one additional copy of lysA . and ask.

Embodiment 64. The Corynebacterium bacterium of Embodiment 60, in which the genome of the bacterium contains:

(v) at least one additional copy of lysA;

(w) at least one additional copy of ask;

(x) at least one additional copy of asd;

(y) at least one additional copy of ddh; or

(z) at least one additional copy of dapB.

Embodiment 65. The Corynebacterium bacterium of any of Embodiments 2-64, in which the start codon of aceE is replaced by TTG, GTG, or CTG.

Embodiment 66. The Corynebacterium bacterium of any of Embodiments 2-65, in which the start codon of aceE is replaced by TTG.

Embodiment 67. The Corynebacterium bacterium of any of Embodiments 2-66, in which the native pyc gene promoter is replaced.

Embodiment 68. The Corynebacterium bacterium of any of Embodiments 2-67, in which the native pyc gene promoter is replaced by promoter Pcgl 860 (SEQ ID NO: 11). Embodiment 69. The Corynebacterium bacterium of any of Embodiments 2-68, in which a promoter is inserted in front of the zwf open reading frame.

Embodiment 70. The Corynebacterium bacterium of any of Embodiments 2-69, in which promoter Pcg0007_39 (SEQ ID NO:20) is inserted in front of the zwf open reading frame.

Embodiment 71. The Corynebacterium of any of Embodiments 2-70, which contains a codon-optimized lysA coding sequence.

Embodiment 72. The Corynebacterium of Embodiment 71, in which the codon- optimized lysA coding sequence is under control of Pcg0007_39 (SEQ ID NO:20).

Embodiment 73. The Corynebacterium of Embodiment 71 or 72, which comprises a heterologous terminator for the codon-optimized lysA coding sequence.

Embodiment 74. The Corynebacterium bacterium of any of Embodiments 71-73, which comprises the nucleotide sequence SEQ ID NO: 17.

Embodiment 75. A Corynebacterium comprising a structural alteration selected from the group consisting of:

(a) alteration of the maJ coding sequence to encode a corynebacterial maJ protein comprising a G448S substitution;

(b) alteration of the native accDA coding sequence to encode a corynebacterial accDA protein comprising a G310E substitution;

(c) replacement of the native cg2766 promoter;

(d) replacement of the native actA promoter; and

(e) replacement of the native opcA coding sequence by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium.

Embodiment 76. The Corynebacterium of Embodiment 77further comprising a structural alteration selected from the group consisting of:

(f) alteration of the native cgl l44 coding sequence to encode a corynebacterial cgl l44 protein comprising a P66S substitution;

(g) insertion of at least one additional copy of at least one gene selected from the group consisting of lysA, ask, asd, ddh, and dapB,'

(h) replacement of the start codon of aceE by TTG, GTG, or CTG;

(i) replacement of the native pyc gene promoter;

(j) insertion of a promoter in front of the zwf open reading frame; and

(k) insertion of a codon-optimized lysA coding sequence. Production of Lysine

[48] Methods of using Corynebacteria to produce lysine are well known in the art, and the engineered Corynebacteria provided in this disclosure can be used with any of these methods. See, for example, U.S. Patent 8,048,649, U.S. Patent 7,635,579, U.S. Patent 7,300,777, U.S. Patent 7,141,388, U.S. Patent 7,122,369, U.S. Patent 6,927,046, and U.S. Patent 6,830,903.

[49] Those skilled in the art will appreciate that there are numerous variations and

permutations of the above described embodiments that fall within the scope of the appended claims.

EXAMPLE 1. Preparation of L-Lysine Pathway Four-Gene Constructs

Example 1A. Construction of an integration vector containing lysine biosynthesis genes ask-asd-dapB-ddh in the bioD region.

[50] pBKMS vector is a pBR322 derivative unable to replicate in C. glutamicum and which contains a kanamycin resistance gene marker and a levansucrase sacB gene from Bacillus subtilis under the control of a strong synthetic promoter for sucrose counter-selection. The 5.4 kb ask-asd-ddh-dapB-orf2’ cassette (4Go) was digested from p F C 3 -ask-asd-dapB-ddh plasmid (U.S. Patent 7,368,276) with Pmel and Xmal and ligated into pDElial 1 (U.S. Patent 6,927,046) linearized with Hindi and Xmal to construct pDl l-KBDH.

[51] To generate a 9.4 kb homology region containing a Spel site, a DNA fragment was amplified from NRRL B-11474 genomic DNA using primers l84f7 (SEQ ID NO: 70 ) and 184F8 (SEQ ID NO:7l) and cloned into the pBKMS vector Ndel/Pstl sites by IN-FUSION ^® (Clontech). The resulting plasmid pBKMS 184 3p was digested with Spel and a second PCR product similarly obtained from NRRL B-11474 genomic DNA using primers 161-184E5 and l62-l84r6 was inserted by IN-FUSION ^® reaction to generate pBKMSl84. The 5.4 kb 4Go cassette was amplified from pDl l-KDBH with primers 244-4Go F2 Inf Spel (SEQ ID NO:72) and 253-4Go R3 (SEQ ID NO:), digested with Spel and cloned by ligation into pBKMS 184 Spel site.

Example IB. Construction of a vector containing the lysine biosynthesis genes ask-asd- dapB-ddh and a Farl homology region for targeted integration.

[52] The 3' Farl homology region from a NRRL B-11474 derived strain was amplified by PCR using primers Farl 3P InfusF (SEQ ID NO:30) and Farl 3P InfusR AvrII (SEQ ID NO:3l). The purified PCR fragment was cloned by IN-FUSION ^® reaction (Clontech) into pBKMS digested with Ndel and Pstl to generate pBKMS Farl 3p. Similarly, the 5' Farl homology region from aNRRL B-l l474-derived strain was PCR amplified with primers Farl 5p InfusF (SEQ ID NO:32) and Farl 5p InfusR (SEQ ID NO:33) and cloned into the Xbal/AvrII sites of pBKMS Farl 3p to generate pBKMS Farl.

[53] The 5.4 kb ask-asd-ddh-dapB-orf2’ cassette (4Go) was digested from pFC 3-ask-asd- dapB-ddh plasmid (US7368276) with Pmel and Xmal and ligated into pDElial 1 (U.S. Patent 6,927,046) linearized with Hindi and Xmal. The resulting vector pDl l-KBDH was digested with Nrul and Swal to eliminate a ddh-dapB 2.9-kb fragment to generate pDl 1KD. pDl 1KD was then digested with Smal and a 1.36 kb PCR fragment amplified from pDl 1KDBH using primers 685 (SEQ ID NO:74) and 686 (SEQ ID NO:75) was inserted using IN-FUSION ^® (Clontech) to generate pDl 1KDH. A 986-bp dapB fragment was then amplified from pDl 1KDBH with primers 687 (SEQ ID NO:76) and 693 (SEQ ID NO:77) and cloned into the pDl 1KDH Sbfl site by IN-FUSION ^® (Clontech) reaction. The KBDH fragment was excised from the resulting pDl 1KBDH R plasmid by restriction with Spel and cloned into the AvrII site of pBKMS Farl to generate pBKMSFarI4GRA.

EXAMPLE 2. Allelic Replacement in C. glutamicum

[54] This examples describes the methods used for allelic replacement in Example 4, below. Strains were cultured in Medium B (Table 5) or BHI broth (BD Biosciences) at 30°C until OD 660 nm reached 0.5. Cells were harvested at 4°C by centrifugation, washed twice in ice-cold deionized water, and resuspended in ice-cold 10% glycerol to generate electrocompetent C. glutamicum cells. Plasmid and cells were mixed together, transformed by electroporation, plated on BHI agar plates with 10 pg/ml kanamycin, and incubated until transformants (kanamycin resistant, sucrose sensitive) which have integrated the plasmid appeared. Transformants were further cultivated overnight at 30°C in Medium B (Table 1) supplemented with an additional 5% sucrose (final concentration 10% sucrose) and plated on Medium B to select for strains that excised the plasmid through a second recombination event and thus were sucrose resistant and kanamycin sensitive. Clones were further screened by PCR and sequencing to verify the presence of the desired mutation.

EXAMPLE 3. Genomic DNA Extraction

[55] For the extraction of genomic DNA, the selected strains were grown overnight in Medium B (Table 5) at 30°C. Cultures were precipitated by centrifugation at 5000 x g (4°C) for 10 minutes. The pellets were suspended in 10 ml of a solution containing 25mM Tris-HCl pH 8.0, 10 mM EDTA, 50mM glucose, and 20 mg/ml lysozyme and incubated for 2 hours at 37°C. The incubation was extended for an additional 2 hours following addition of 1.3 ml 10% SDS,

67 pl lOmg/ml RNAse A, and 167 mΐ of Proteinase K (20 mg/ml stock). Genomic DNA was further purified by phenol-chloroform extraction and precipitation by addition of two volumes of ice cold ethanol and 0.1 volume of 3M sodium acetate (pH 5.2). After incubation at -80°C for 1 hour, the DNA pellet was separated by centrifugation at 14,000 rpm (4°C) for 1 hour, washed with 70% ethanol, air dried, and dissolved in nuclease-free water.

EXAMPLE 4. Assembly of Insertion Fragments into a pK18mobsacB-Derived

Vector and Transformation into C. glutamicum Strains

Example 4A. Assembly of plasmids for integration and allelic replacement [56] All DNA fragments used in the generation of upstream or downstream homologous recombination regions, as well as inserts, were either amplified from purified genomic DNA extracted from strain NRRL B-l 1474 or from plasmids containing sequences derived from this strain as described in Example 2 using the polymerase chain reaction (PCR), or were chemically synthesized (DNA 2.0). A pZ vector derived from pKl8mobsacB (Schaffer et al, Gene 145: 69- 73, 1994; Accession FJ437239) containing an URA3 gene for selection in yeast was used as the vector backbone for introducing the changes into C. glutamicum. Homology arms and inserts were assembled into the vector backbone by homologous recombination in yeast (Ma et al, Gene (58): 201-16, 1987). Each DNA fragment contained a 50-bp overlap on each side to ensure correct assembly with its adjacent parts. For assembly, the linearized vector backbone, the two homology arms, and optionally the inserted DNA fragment were simultaneously transformed into Saccharomyces cerevisiae CEN.PK (Entian & Kotter, Methods in Microbiology 36: 629-66, 2007) and plated on synthetic complete agar plates without uracil (Sigma). The assembled plasmids were extracted from yeast using a ZYMOPREP™ I yeast plasmid miniprep kit (Zymo Research) and propagated in E. coli lO-Beta cells (New England Biolabs) with 50 pg/ml kanamycin selection before transformation into C. glutamicum as described in Example 2.

Example 4B. Construction of transformation vector to introduce rna./ciiss allele

[57] Two homology arms were amplified using PCR from NRRL B-l 1474 genomic DNA obtained as described in Example 3.

[58] The approximately 2.1 kb downstream homology arm (nucleotides 2026 to 4101 from SEQ ID NO: l) was amplified using primers SNP_084_Pl (SEQ ID NO:38) and SNP_084_P2 (SEQ ID NO:39). Primer SNP 084 P1 includes a 50-nucleotide 5' extension that overlaps with the pZ backbone described in Example 4A. Primer SNP 084 P2 contains a G to A substitution at position 25 to introduce the G448S substitution. Similarly, the approximately 2.1 kb upstream homology arm (nucleotides 1 to 2075 from SEQ ID NO: l) was amplified using primers

SNP_084_P3 (SEQ ID NO:40) and SNP_084_P4 (SEQ ID NO:4l). Primer SNP_084_P3 contains a C to T substitution at position 20 to introduce the G448S substitution. SNP_084_P4 includes a 50-base 5' extension that overlaps with the pZ described in Example 4A. Alignment of the two homology arms results in a 50-bp overlap defined by primers SNP_084_P2 and SNP_084_P3. The two homology arms were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-l 1474 and its derived strains as described in Example 2. Resulting strains carry the altered maJ coding sequence (SEQ ID NO:2), encoding the maJ amino acid sequence with the G448S substitution (SEQ ID NO:3). Example 4C. Construction of transformation vector to introduce accDAGum allele

[59] Two homology arms were amplified from NRRL B-11474 genomic DNA by PCR. The 2070 bp downstream homology arm (nucleotides 2032 to 4101 from SEQ ID NO:4) was amplified using primers SNP_033_Pl (SEQ ID NO:46) and SNP_033_P2 (SEQ ID NO:47). Primer SNP_033_Pl includes a 50-nucleotide 5' extension that overlaps with the pZ vector backbone described in Example 4A. Primers SNP 033 P2 contains a G to A substitution at position 20 to introduce the G310E mutation. Similarly, the approximately 2.1 kb upstream homology arm (nucleotides 1 to 2071 from SEQ ID NO:4) was amplified using primers SNP_084_P3 (SEQ ID NO:48) and SNP_084_P4 (SEQ ID NO:49). Primer SNP_033_P3 contains a C to T substitution at position 21 to introduce the G310E mutation. SNP_033_P4 includes a 50-base 5' extension that overlaps with the pZ backbone described in Example 4A. Alignment of the two homology arms results in a 40-bp overlap defined by primers SNP_033_P2 and SNP_033_P3. The two homology arms were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-l 1474 and its derived strains as described in Example 2. Resulting strains carry the altered accDA coding sequence (SEQ ID NO:5), encoding the accDA amino acid sequence with the G310E substitution (SEQ ID NO:6).

Example 4D. Construction of transformation vector to introduce cgll44p66S allele

[60] Two homology arms were amplified from NRRL B-l 1474 genomic DNA by PCR. The 2037 bp upstream homology arm (nucleotides 1 to 2037 from SEQ ID NO:7) was amplified using primers SNP 316 P1 (SEQ ID NO:50) and SNP 316 P2 (SEQ ID NO:5l). Primer SNP_3l6_Pl includes a 50-nucleotide 5' extension that overlaps with the pZ vector backbone described in Example 4A. Primer SNP 316 P2 contains a G to A substitution at position 21 to introduce the P66S mutation. Similarly, the 2070 bp downstream homology arm (nucleotides 1998 to 4067 from SEQ ID NO:7) was amplified using primers SNP_3l6_P3 (SEQ ID NO:52) and SNP 316 P4 (SEQ ID NO: 53). Primer SNP 316 P3 contains a C to T substitution at position 20 to introduce the P66S mutation. SNP_3l6_P4 includes a 50-base 5' extension that overlaps with the pZ backbone described in Example 4A. Alignment of the two homology arms results in a 40-bp overlap defined by primers SNP 316 P2 and SNP 316 P3. The two homology arms were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-l 1474 and its derived strains as described in Example 2. Resulting strains carry the altered cgl 144 coding sequence (SEQ ID NO:8), encoding the cgl 144 amino acid sequence with the P66S substitution (SEQ ID NO:9). Example 4E. Construction of transformation vector to replace the native pyc promoter by promoter Pcgl860

[61] Two homology arms were amplified from NRRL B-11474 genomic DNA by PCR. The 2043 bp upstream homology arm (nucleotides 1 to 2043 from SEQ ID NO: 10) was amplified using primers Pcgl860_pyc_Pl (SEQ ID NO:42) and Pcgl860_pyc_P2 (SEQ ID NO:43). Similarly, the 2050 bp downstream homology arm (nucleotides 2161 to 4210 from SEQ ID NO: lO) was amplified using primers Pcgl860_pyc _P3 (SEQ ID NO:44) and Pcgl860_pyc_P4 (SEQ ID NO:45). The 93-bp Pcgl860 nucleotide sequence (SEQ ID NO: 11) was obtained by overlap of the 5' extensions of primers Pcgl860_pyc_P2 and Pcgl860-pyc_P3. The two homology arms were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-11474 and its derived strains as described in Example 2. Resulting strains carry the promoter Pcgl 860 (SEQ ID NO: 11) instead of the pyc promoter (SEQ ID NO: 12).

Example 4F. Construction of transformation vector to introduce aceE _atg>ttg allele

[62] Two homology arms were amplified from NRRL B-11474 genomic DNA by PCR. The 2072 bp upstream homology arm (nucleotides 1 to 2072 from SEQ ID NO: 13) was amplified using primers SNP aceE Pl (SEQ ID NO:54) and SNP_aceE_P2 (SEQ ID NO:55). Primer SNP aceE Pl includes a 50-nucleotide 5' extension that overlaps with the pZ vector backbone described in Example 4A. Primer SNP_aceE_P2 contains a T to A substitution at position 22 to introduce the A mutation. Similarly, the 2073 bp downstream homology arm (nucleotides 2029 to 4101 from SEQ ID NO: 13) was amplified using primers SNP_aceE_P3 (SEQ ID NO:56) and SNP_aceE_P4 (SEQ ID NO:57). Primer SNP_aceE_P3 contains an A to T substitution at position 23 to introduce the T mutation. SNP_aceE_P4 includes a 50-base 5' extension that overlaps with the pZ backbone described in Example 4. Alignment of the two homology arms results in a 44-bp overlap defined by primers SNP_aceE_P2 and SNP_aceE_P3. The two homology arms were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-11474 and its derived strains as described in Example 2. Resulting strains carry the TTG start codon in the aceE open reading frame (SEQ ID NO: 14).

Example 4G. Insertion of a codon-optimized lysA coding sequence

[63] A cassette containing the codon optimized lysA fragment of SEQ ID NO: 16 was inserted between nucleotides 2048 and 2049 of SEQ ID NO: 15 and synthetically assembled (DNA 2.0) into the pZ vector described in Example 4A. The final plasmid contains two ~2kb homology arms (nucleotides 1 to 2048, and nucleotides 2049 to 4099 of SEQ ID NO: 15) flanking a Pcg0007_39-lysA ^co -sodT (SEQ ID NO: 17). The lysA ^C0 containing plasmid was transformed into C. glutamicum NRRL B-11474 and its derived strains as described in Example 2. Resulting strains carry an additional copy of the codon-optimized lysA coding sequence under the control of promoter Pcg0007_39.

Example 4H. Insertion of a promoter upstream of the zwf coding sequence

[64] Two homology arms were amplified from a NRRL B-l 1474 genomic DNA by PCR. The 2050 bp upstream homology arm (nucleotides 1 to 2050 from SEQ ID NO: 19) was amplified using primers Pcg0007_39-zwf_Pl (SEQ ID NO:34) and Pcg0007_39-zwf_P2 (SEQ ID NO:35) Similarly, the 2050 bp downstream homology arm (nucleotides 2052 to 4101 from SEQ ID NO: 19) was amplified using primers Pcg0007_39-zwf_P3 (SEQ ID NO:36) and Pcg0007_39- zwf_P4 (SEQ ID NO:37). The 93-bp Pcg0007_39 nucleotide sequence was obtained by overlap of the 5' extensions of primers Pcg0007_39-zwf_P2 and Pcg0007_39-pyc_P3. The two homology arms were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-l 1474 and its derived strains as described in Example 2. Resulting strains result in the replacement of nucleotide 2051 of SEQ ID NO: 19 by the promoter

Pcg0007_39 (SEQ ID NO:20). Resulting strains carry the promoter Pcg0007_39 upstream of the zwfORF (SEQ ID NO:2l).

Example 41. Replacement of native opcA coding sequence by the opcA coding sequence of C. humireducens NBRC 106098

[65] Two homology arms were amplified from NRRL B-l l474-derived strain BS2CZ genomic DNA (Example 3) by PCR. The 2039 bp upstream homology arm (nucleotides 1 to 2039 from SEQ ID NO:22) was amplified using primers opcA Pl (SEQ ID NO:58) and opcA_P2 (SEQ ID NO:59). Similarly, the 2083 bp downstream homology arm (nucleotides 3000 to 5082 from SEQ ID NO:22) was amplified using primers opcA_P3 (SEQ ID NO:60) and opcA_P4 (SEQ ID NO:6l). A gene cassette consisting of the 93-bp Pcg0007_39 nucleotide sequence (SEQ ID NO:20) and C. humireducens opcA (SEQ ID NO:23) carries sequence overlaps of the 5' extensions of primers opcA_P2 and opcA_P3. The two homology arms and the gene cassette were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-l 1474 and its derived strains as described in Example 2. Resulting strains result in the replacement of nucleotide 2040 to 2999 of SEQ ID NO:22 by the Pcg0007_39-opcA (SEQ ID NO: 24). Example 4 J. Construction of transformation vector to replace the cg2766 promoter with promoter Pcg0007_39

[66] Two homology arms were amplified from NRRL B-11474 genomic DNA by PCR. The

2050 bp downstream homology arm (nucleotides 2051 to 4100 from SEQ ID NO:26) was amplified using primers Pcg0007_39-cg2766_Pl (SEQ ID NO:62) and Pcg0007_39-cg2766_P2 (SEQ ID NO: 63). Similarly, the 2050 bp upstream homology arm (nucleotides 1 to 2050 from SEQ ID NO:26) was amplified using primers Pcg0007_39-cg2766_P3 (SEQ ID NO:64) and Pcg0007_39-cg2766 _P4 (SEQ ID NO:65). The 93-bp Pcg0007_39 nucleotide sequence (SEQ ID NO:20) was obtained by overlap of the 5' extensions of primers Pcg0007_39-cg2766_P2 and Pcg0007_39-cg2766_P3. The two homology arms were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-l 1474 and its derived strains as described in Example 2. Resulting strains carry the promoter Pcg0007_39 inserted in front of cg2766 (SEQ ID NO:27).

Example 4K. Construction of transformation vector to replace the actA promoter with promoter Pcg0007_39.

[67] Two homology arms were amplified from NRRL B-l 1474 genomic DNA (Example 3) by PCR.

[68] The 2021 bp downstream homology arm (nucleotides 2263 to 4283 from SEQ ID NO:28) was amplified using primers Pcg0007_39-actA_Pl (SEQ ID NO:66) and Pcg0007_39-actA_P2 (SEQ ID NO: 67). Similarly, the 2050 bp upstream homology arm (nucleotides 1 to 2050 from SEQ ID NO:28) was amplified using primers Pcg0007_39-act A_P3 (SEQ ID NO:68) and Pcg0007_39-actA_P4 (SEQ ID NO: 69). The 93-bp Pcg0007_39 nucleotide sequence (SEQ ID NO:20) was obtained by overlap of the 5' extensions of primers Pcg0007_39-actA_P2 and Pcg0007_39-actA_P3. The two homology arms were then assembled into pZ as described in Example 4A and transformed into C. glutamicum NRRL B-l 1474 and its derived strains as described in Example 2. Resulting strains carry the promoter Pcg0007_39 in place of nucleotides

2051 to 2262 of SEQ ID NO:28 and inserted in front of actA (SEQ ID NO:29).

EXAMPLE 5. Evaluation of altered C. glutamicum strains for

lysine production in microtiter plates

Example 5A. Lysine production by altered C. glutamicum strains

[69] Each of the altered strains described in the previous examples as well as strains resulting from combining the different alterations were evaluated for lysine production by a three-stage fermentation in microtiter plates. Cells were grown in Medium C (Table 5). After 48 hours a 10% inoculum was transferred to Medium D (Table 5). Ten percent of the cell culture was transferred after 19 hours to Medium E (Table 5) and grown for an additional 24 hours. Culture conditions were 32°C, 1000 rpm (Infors HT).

[70] The amount of lysine produced was determined using a coupled lysine oxidase assay. Culture supernatants were added to a lysine oxidase reaction solution (250 mM potassium phosphate buffer, pH 7.5, 824 mg/ml phenol, 76 mg/ml 4-amino antipyrene (Sigma), 0.03 mg/ml peroxidase (Sigma), 0.015 units/ml Lysine oxidase (Sigma). Samples were incubated at 25°C for 40 min and absorbance was read at 490 nm. The amount of dextrose in the microtiter plates was determined by a couple glucose oxidase assay. Culture supernatants were added to a glucose oxidase reaction solution (275 mM sodium maleate buffer pH 5.5, 730 mg/ml phenol, 680 mg/ml 4-amino antipyrene (Sigma), 0.027 mg/ml peroxidase (Sigma), 56 mg/L glucose oxidase (Sigma). Samples were incubated at 25°C for 40 min and absorbance was read at 490 nm.

[71] The amount of lysine (g/L) produced by each recombinant strain and its immediate parent strain is shown in the Table 6. Genomic alterations in the tested strains are with respect to the native genome of NRRL B-l 1474. Table 7A is a list of genomic alterations, and Table 7B identifies which alterations are present in the tested strains.

Table 6. Lysine Production by Engineered C. glutamicum Strains and their Immediate Parents

Table 7A. Genomic Alterations in Table 7B

ADM Ref. No. BI0.0065.PC01

ADM Ref. No. BI0.0065.PC01

3

O

O

C/I

h3 n H in bo o

Example 5B. Comparison of lysine production by strains 12145 and 9659.

[72] The strain carrying accDA _G3i oE allele (strain 12145) described in Example 4C and its immediate parent strain (strain 9659; see Tables 7A and 7B) were grown in 3ml Medium C (Table 5) supplemented with 20 g/L soytone. After 7 hours a 10% inoculum was transferred to the same media in microplates for 24 hours incubation. A 10% inoculum was transferred to Medium D (Table 5) supplemented with 5 g/L (NEE^SCri for growth. Ten percent of the cell culture was transferred after 19 hours to Medium F (Table 5) and grown for an additional 24 hours. Culture conditions were 32°C, 900 rpm (Infors HT).

[73] The amount of lysine produced was determined by HPLC. The amount of dextrose in the microtiter plates was determined by a glucose oxidase assay. The growth optical density (OD) of 1:40 diluted sample was measured at 600nm using a BIOTEK ^® SYNERGY™ plate reader.

[74] Lysine titer, OD, and dextrose consumption for strains 9659 and 12145 after 22 hours fermentation are shown in Table 8.

Table 8. Comparison of Lysine Production by Strains 9659 and 12145

Example 5C. Lysine production by strains 12145 and 20478

[75] Strain 20478 carrying cgl l44p _66S allele, described in Example 4D, and its parent strain 12145 (see Tables 7A and 7B) were grown in 3ml Medium C (Table 5) supplemented with 20 g/L soytone. After 7 hours a 10% inoculum was transferred to the same media in microplates for 24 hours incubation. A 10% inoculum was transferred to Medium D (Table 5) supplemented with 5 g/L (NEE^SCri for growth. Ten percent of the cell culture was transferred after 19 hours to Medium F (3.5% dextrose, Table 5), and modified Medium F containing 5%, 10% or 20% dextrose. The microtiter plate cultures were incubated at 32°C, 900rpm (6mm throw) for 24 hours and then removed for analysis. The growth optical density (OD) of 1 :40 diluted sample was measured at 600nm through a BIOTEK ^® SYNERGY™ plate reader. Result shows at 24 hours, strain 20478 statistically grew more efficiently in the presence of increased background dextrose (concentrations 5%, 10%, and 20%) over strain 12145.

[76] The results are shown in Table 9.

EXAMPLE 6. Evaluation of C. glutamicum strains for

lysine production in microtiter plates

[77] Strains carrying opcA coding sequences from the following Corynebacteria were tested as described in Example 5A: C. vitaeruminis DSM 20294 (SEQ ID NO:87; opcA_l), C.

matruchotii ATCC 33806 (SEQ ID NO:88, opA_2), C. falsenii DSM 44353 (SEQ ID NO:89, opcA_3), C. humireducens NBRC 106098 (SEQ ID NO:25, opcA_4), and C. halotolerans YIM 70093 (SEQ ID NO:90, opcA_5). Inclusion of“P” in the name of the opcA coding sequence indicates that the sequence was placed under the control of promoter Pcg0007_39. See Tables 7A and 7B for the genomic changes in the strains. The results are shown in Table 10.

Table 10. Lysine Production by Modified Strains of NRRL B-11474 Having Different opcA Coding Sequences

Lysine concentration (mM)

opcA in plates

parent id strain id

change Upper

Average Lower 95%

95%

( ) i ( ( > ( ¹ (

EXAMPLE 7. Lysine Production by Strains NRRL B-11474 and NRRL B-67439

[78] Strain NRRL B-l 1474 was modified to generate an engineered strain which has the following 12 changes, each described above, with respect to the NRRL B-l 1474 genome:

1. alteration of the native maJ coding sequence to encode the amino acid

sequence SEQ ID NO:3;

2. alteration of the native accDA coding sequence to encode the amino acid sequence SEQ ID NO:6;

3. alteration of the native cgl 144 coding sequence to encode the amino acid sequence SEQ ID NO:9;

4. replacement of the native cg2766 promoter by promoter Pcg0007_39 (SEQ ID NO:20);

5. replacement of the native actA promoter by promoter Pcg0007_39 (SEQ ID NO:20);

6. replacement of the native opcA coding sequence by the opcA coding sequence of C. humireducens under control of promoter Pcg0007_39 (SEQ ID NO:20);

7. insertion of lysine biosynthesis genes ask-asd-dapB-ddh in the bioD region;

8. insertion of biosynthesis genes ask-asd-dapB-ddh in a Farl homology region;

9. replacement of aceE start codon with TTG;

10. replacement of native pyc promoter by promoter Pcgl860;

11. insertion of promoter Pcg0007_39 (SEQ ID NO:20) in front of the zwf open reading frame; and

12. insertion of codon-optimized lysA sequence under the control of promoter Pcg0007_39 and having a sod terminator (SEQ ID NO: 17).

[79] The engineered strain was deposited with the Agriculture Research Culture Collection (NRRL) International Depositary Authority, 1815 N. University Street, Peoria, IL 61604 on April 12, 2017 under the provisions of the Budapest Treaty and assigned Accession No. NRRL B-67439.

[80] Both strains were streaked onto Medium B (Table 1) agar plates for well-isolated colonies. Five individual colonies of each respective strain were inoculated into 3 ml Medium B broth. After 24 hours, a 10% inoculum (2 ml) was transferred to Medium G (Table 1) in flasks and grown for an additional 48 hours of lysine production. Lysine production by each strain is shown in Table 11.

Table 11. Lysine Production by Strains NRRL B-11474 and NRRL B-67439

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