ENGINEERED STRAINS OF CORYNEBACTERIA

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

This application incorporates by reference a 195 kb text file created on Sep. 7, 2018 and named “BIO066US00sequencelisting.txt,” which is the sequence listing for this application.

TECHNICAL FIELD

This disclosure relates generally to lysine production.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B is a multiple alignment of SEQ ID NO:3 with the amino acid sequences of three corynebacterial ribonuclease J (rnaJ) proteins identified by National Center for Biotechnology Information (NCBI) Reference Nos. WP_011014791.1 (SEQ ID NO:78), WP_096456687.1 (SEQ ID NO:79), and WP_023635875.1 (SEQ ID NO:80). A box identifies the amino acid in each sequence that alignments with amino acid 448 of SEQ ID NO:3. FIG. 1A, alignment with amino acids 1-560 of SEQ ID NO:3. FIG. 1B, alignment with amino acids 561-718 of SEQ ID NO:3.

FIG. 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_075348128 (SEQ ID NO:81), 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.

FIG. 3 is a multiple alignment of SEQ ID NO:9 with the amino acid sequences of three corynebacterial cg1144 proteins identified by NCBI Reference Nos. WP_011897001 (SEQ ID NO:84), WP_066565124 (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

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

This disclosure provides the following four structural alterations that can be engineered into Corynebacteria to improve lysine production: (a) insertion of a promoter in front of the cg1383 start codon, (b) replacement of the native phoU promoter; (c) replacement of the native cg3210 promoter; and (d) replacement of the native cg0800 promoter.

a. Insertion of a Promoter in Front of the cg1383 Start Codon

cg1383 is also known as NCg11179 and encodes an ATPase component of the ABC-type molybdenum transport system (e.g., WP_011014216.1). In some embodiments, a promoter is inserted in front of the cg1383 start codon. 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 inserted promoter is promoter Pcg0007_39 (SEQ ID NO:20); see US 2017/0159045 and WO 2017/00376.

b. Replacement of the Native phoU Promoter

phoU encodes a phosphate uptake regulator (e.g., BAB99964.1) and is also known as cgl2571 and NCgl2482. In some embodiments, the native phoU promoter is replaced with a phoU replacement promoter. The phoU 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 phoU replacement promoter is promoter Pcg0007_39 (SEQ ID NO:20); see US 2017/0159045 and WO 2017/00376.

c. Replacement of the Native Cg3210 Promoter

cg3210 is also known as NCgl2802 and encodes a cell envelope-related transcriptional regulator (e.g., WP_011266029.1). In some embodiments, the native cg3210 promoter is replaced with a cg3210 replacement promoter. The cg3210 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 cg3210 replacement promoter is promoter Pcg0007_39 (SEQ ID NO:20); see US 2017/0159045 and WO 2017/00376.

d. Replacement of the Native Cg0800 Promoter

cg0800 is also known as NCg10668 or prpR and encodes a transcriptional regulatory of the MerR family (e.g., WP_011013825.1). In some embodiments, the native cg0800 promoter is replaced with a cg0800 replacement promoter. The cg0800 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 cg0800 replacement promoter is promoter Pcg0007_39 (SEQ ID NO:20); see US 2017/0159045 and WO 2017/00376.

Additional Structural Alterations

In some embodiments, engineered strains of Corynebacteria comprising one or more of the structural alterations described above may also comprise one or more of the additional structural alterations (e)-(o) described below.

e. Altered Corynebacterial Ribonuclease J Proteins

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise an altered corynebacterial ribonuclease (rnaJ) protein. Altered corynebacterial rnaJ proteins comprise a G448S substitution. One example of such a protein is shown in SEQ ID NO:3. “A corynebacterial rnaJ protein comprising a G448S substitution” as used herein means an rnaJ protein from a strain of Corynebacterium which, when compared with SEQ ID NO:3 using the NCBI's 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 FIG. 1A-B. Corynebacterial rnaJ 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

Reference number
Species and/or Strain(s)

WP_011014791.1

C. glutamicum 617, 1006, AJ1511, AS 1.299,

ATCC 13032 (K051) and DSM 20300, ATCC

13869, ATCC 21831, C1, CP, MB001, TQ2223,

XV, and ZL-; B. flavum ZL-1

WP_003857476.1

C. glutamicum AS 1.542, ATCC 13870,

ATCC 14067, ATCC 21493, MT, S9114, SCgG1,

SCgG2, SYPA 5-5, SYPS-062, SYPS-062-33a,

T6-13, and Z188; B. flavum ATCC 15168

WP_077312485.1

C. glutamicum

WP_089158555.1

C. glutamicum

WP_044030039.1

C. glutamicum

WP_040967540.1

C. glutamicum

WP_059289432.1

C. glutamicum

WP_063967576.1

C. glutamicum

WP_011897391.1

C. glutamicum

WP_096456687.1

C. glutamicum

WP_080794542.1

Corynebacterium sp. Marseille-P2417

WP_015651527.1

C. callunae

WP_011075647.1

C. efficiens

WP_095075511.1

C. ulcerans

WP_014836513.1

C. ulcerans

WP_087454146.1

C. ulcerans

WP_013911733.1

C. ulcerans

WP_014525921.1

C. ulcerans

WP_023635875.1

C. ulcerans

WP_014300762.1

C. pseudotuberculosis

WP_013242122.1

C. pseudotuberculosis

AJC14005.1

C. pseudotuberculosis

WP_058832312.1

C. pseudotuberculosis

WP_014523406.1

C. pseudotuberculosis

WP_014558731.1

C. pseudotuberculosis

OMH75460.1

C. pseudotuberculosis

WP_014367221.1

C. pseudotuberculosis

WP_092284332.1

C. spheniscorum

WP_020976441.1

C. argentoratense

WP_075726535.1

C. aquilae

WP_027012113.1

C. freiburgense

WP_034995241.1

C. matruchotii

WP_005526573.1

C. matruchotii

WP_018339963.1

C. caspium

WP_018020369.1

C. ciconiae

WP_023019341.1

Corynebacterium sp. KPL1995 and KPL1989

OUJ24662.1

C. kefirresidentii

WP_018119731.1

C. propinquum

ART21077.1

C. striatum

ERS47112.1

Corynebacterium sp. KPL1856

WP_021352644.1

C. pseudodiphtheriticum

WP_027017879.1

C. pseudodiphtheriticum

WP_075691844.1

C. sphenisci

WP_046202207.1

C. kroppenstedtii

WP_012731758.1

C. kroppenstedtii

WP_064833244.1

Corynebacterium sp.

EPI-003-04-2554_SCH2473622

WP_070446719.1

Corynebacterium sp. HMSC05D03,

HMSC066C02, and HMSC072D12

WP_070527124.1

Corynebacterium sp. HMSC074E01

and HMSC074C01

WP_010190304.1

C. aurimucosum

WP_070719120.1

Corynebacterium sp. HMSC069E04

WP_070644981.1

Corynebacterium sp. HMSC062A03

WP_083303801.1

Corynebacterium sp. HMSC072A02

WP_070683378.1

Corynebacterium sp. HMSC056E09

WP_046649347.1

C. minutissimum

WP_083299156.1

Corynebacterium sp. HMSC055A01,

HMSC065D07, and HMSC078C09

WP_016829761.1

C. diphtheriae

WP_010935082.1

C. diphtheriae

WP_045143380.1

C. diphtheriae

WP_003851901.1

C. diphtheriae

WP_072574180.1

C. diphtheriae

WP_071571681.1

C. diphtheriae

OLN14990.1

C. diphtheriae

WP_014319143.1

C. diphtheriae

WP_071572121.1

C. diphtheriae

WP_088246382.1

C. diphtheriae

WP_088266276.1

C. diphtheriae

WP_014302046.1

C. diphtheriae

WP_071575574.1

C. diphtheriae

WP_047262243.1

C. mustelae

WP_048379222.1

C. renale

WP_098389157.1

C. renale

WP_079005470.1

C. stationis

WP_083640700.1

C. stationis

WP_082869553.1

C. stationis

WP_054468244.1

C. ulcerans

WP_015401129.1

C. halotolerans

WP_014010148.1

C. variable

WP_085548717.1

C. pollutisoli

WP_047253276.1

C. testudinoris

WP_040086051.1

C. humireducens

EPD69865.1

C. pyruviciproducens ATCC BAA-1742

OFO43484.1

Corynebacterium sp. HMSC073D01

WP_046439708.1

C. kutscheri

WP_006063636.1

C. durum

WP_070434230.1

Corynebacterium sp. HMSC28B08

WP_025253035.1

C. vitaeruminis

WP_035002300.1

C. jeikeium

WP_025402845.1

C. falsenii

WP_066566197.1

C. crudilactis

WP_053545137.1

C. deserti

WP_042621626.1

C. marinum

WP_055121720.1

C. oculi

WP_026165808.1

C. mastitidis

WP_047240411.1

C. epidermidicanis

WP_082099190.1

C. camporealensis

EEW49517.1

C. efficiens YS-314

EEG27126.1

C. matruchotii ATCC 33806

WP_013888590.1

C. resistens

WP_099298604.1

Corynebacterium sp. Marseille-P4122

WP_055176585.1

C. lowii

f. Altered Corynebacterial accDA Proteins

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise an altered corynebacterial accDA protein. Altered corynebacterial accDA proteins comprise 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 FIG. 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

Reference No.
Species and/or Strain(s)

WP_075348128.1

C. glutamicum AJ1511, ZL-6, ATCC 13869, XV, CP,

and WM001; B. flavum ZL-1

WP_081099520.1

C. glutamicum

WP_077311448.1

C. glutamicum

WP_087061768.1

C. glutamicum

ALP51538.1

C. glutamicum

WP_074508465.1

C. glutamicum

WP_080724089.1

C. glutamicum

WP_003862368.1

C. glutamicum ATCC 14067, SYPS-062, SYPS-062-33a,

ATCC 21493; B. flavum ATCC 15168

WP_074491484.1

C. glutamicum

WP_011013915.1

C. glutamicum

CAC42827.1

C. glutamicum

WP_081253003.1

C. glutamicum

WP_080506411.1

C. glutamicum

BAF53919.1

C. glutamicum

AMA01529.1

C. glutamicum

SJM44016.1

C. glutamicum

AIK87334.1

C. glutamicum

KEI22570.1

C. glutamicum ATCC 14067

WP_074508304.1

C. glutamicum

WP_003858326.1

C. glutamicum SCgG1, SCgG2, S9114, Z188, AS 1.542,

and T6; C. crenatum MT, and SYPA 5-5

OKX89395.1

C. glutamicum

AGT04828.1

C. glutamicum MB001

ANE09779.1

C. glutamicum

OKX77810.1

C. glutamicum

OKX76530.1

C. glutamicum

WP_066564978.1

C. crudilactis

BAU95211.1

C. glutamicum

WP_096454843.1

C. glutamicum

WP_006770129.1

C. efficiens

WP_053544383.1

C. deserti

WP_047261525.1

C. mustelae

WP_027013104.1

C. freiburgense

WP_075725271.1

C. aquilae

WP_005527314.1

C. matruchotii

WP_080796909.1

Corynebacterium sp. Marseille-P2417

WP_005523810.1

C. matruchotii

WP_034651919.1

C. vitaeruminis

WP_047239789.1

C. epidermidicanis DSM 45586

WP_020975778.1

C. argentoratense

WP_084035934.1

C. glucuronolyticum

WP_025252271.1

C. vitaeruminis

WP_005395753.1

Corynebacterium sp. HMSC073D01; C. glucuronolyticum

ATCC 51866

WP_005391688.1

C. glucuronolyticum

WP_046095662.1

C. ulcerans

WP_046693956.1

C. ulcerans

WP_023635342.1

C. ulcerans

WP_029975236.1

C. ulcerans

WP_044032730.1

C. ulcerans

WP_014525474.1

C. ulcerans

WP_013911032.1

C. ulcerans

WP_046649933.1

C. xerosis

WP_095076119.1

C. ulcerans

WP_038617892.1

C. ulcerans

WP_087453611.1

C. ulcerans

AKN76575.1

C. ulcerans FRC58

WP_014366686.1

C. pseudotuberculosis

WP_048740779.1

C. halotolerans

WP_014523140.1

C. pseudotuberculosis

WP_032802070.1

C. pseudotuberculosis

WP_071307711.1

C. pseudotuberculosis

WP_013241466.1

C. pseudotuberculosis

WP_014558622.1

C. pseudotuberculosis

WP_048588670.1

C. pseudotuberculosis

WP_014800179.1

C. pseudotuberculosis

WP_070834218.1

Corynebacterium sp. HMSC073H12

WP_070595314.1

Corynebacterium sp. HMSC063G05 and HMSC064E08;

C. amycolatum ICIS 9

WP_040426984.1

C. pyruviciproducens

EPD71091.1

C. pyruviciproducens ATCC BAA-1742

WP_070522630.1

Corynebacterium sp. HMSC11E11

WP_035120392.1

C. freneyi

WP_070605452.1

C. sp. HMSC055G02

WP_070565960.1

Corynebacterium sp. HMSC077G01 and HMSC072D01

WP_070653141.1

Corynebacterium sp. HMSC065H09 and HMSC072B08

WP_049189720.1

C. jeikieum 212_CJEI; Corynebacterium

sp. HMSC064E07

WP_070545912.1

Corynebacterium sp. HMSC064H12, HMSC077C02,

and HMSC070B05

WP_070437593.1

Corynebacterium sp. HMSC14H10

WP_069359660.1

C. amycolatum

WP_070601703.1

Corynebacterium sp. HMSC063F04 and HMSC075F02

WP_070642800.1

Corynebacterium sp. HMSC076C10

WP_070429963.1

Corynebacterium sp. HMSC14B06 and HMSC077G07

WP_018019334.1

C. ciconiae

WP_016423306.1

Corynebacterium sp. HFH0082

AFM06966.2

C. pseudotuberculosis Cp162

WP_038626403.1

Corynebacterium sp. ATCC 6931 and HMSC11H10

WP_070862586.1

Corynebacterium sp. HMSC072B09 and HMSC073B01

WP_049171363.1

C. jeikeium

WP_070851988.1

Corynebacterium sp. HMSC074C03

WP_049180908.1

C. jeikeium 805_CJEI; Corynebacterium sp.

HMSC074C11, HMSC063A05, and HMSC064E10

WP_049193043.1

C. jeikeium

WP_070855978.1

Corynebacterium sp. HMSC074C05

WP_088611726.1

C. jeikeium

WP_005511812.1

C. amycolatum

WP_088266872.1

C. diphtheriae

WP_070858287.1

Corynebacterium sp. HMSC061H03

WP_048758709.1

C. vitaeruminis

WP_072564663.1

C. diphtheriae

WP_014303117.1

C. diphtheriae

WP_014306652.1

C. diphtheriae

WP_070794870.1

C. diphtheriae

g. Altered Corynebacterial cg1144 Proteins

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise an altered corynebacterial cg1144 protein. Altered corynebacterial cg1144 proteins comprise a P66S substitution. One example of such a protein is shown in SEQ ID NO:9. “A corynebacterial cg1144 protein comprising a P66S substitution” as used herein means a cg1144 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 FIG. 3. Corynebacterial cg1144 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 cg1144 Proteins

Reference No.
Species and/or Strains(s)

BAB98402.1

C. glutamicum ATCC 13032

WP_004568112.1

C. glutamicum 617, ATCC 21493, B Co 03.31, ATCC

13032 (K051, DSM 20300), MB001, ATCC 14067, AJ1511, CS176,

ZL-6, ATCC 21831, B253, CP, Y1, USDA-ARS-USMARC-56828, C1,

XV, TQ2223, WM001, ATCC 14067, 1006, SYPS-062, SYPS-062-33a,

and AS 1.299; B. flavum 15168 and ZL-1

WP_011897001.1

C. glutamicum

WP_044029870.1

C. glutamicum

WP_074506400.1

C. glutamicum

WP_003856809.1

C. glutamicum AS 1.542, MT, S9114, SCgG1, SCgG2,

SYPA 5-5, T6-13, and Z188

WP_096455129.1

C. glutamicum

WP_053544501.1

C. deserti

WP_066565124.1

C. crudilactis

WP_011075272.1

C. efficiens

WP_015650827.1

C. callunae

EEW49979.1

C. efficiens YS-314

WP_080795733.1

Corynebacterium sp. Marseille-P2417

WP_015400428.1

C. halotolerans

WP_042621053.1

C. marinum

AJK68412.1

C. marinum DSM 44953

WP_049168821.1

C. propinquum

WP_027017701.1

C. pseudodiphtheriticum

WP_049148216.1

C. propinquum

WP_018119926.1

C. propinquum

WP_023019702.1

Corynebacterium sp. KPL1995, KPL1989, and

EPI-003-04-2554_SCH2473622

WP_085550290.1

C. pollutisoli

WP_040085436.1

C. humireducens

EKX88113.1

C. durum F0235

WP_051033899.1

C. durum

WP_020934330.1

C. maris

WP_075814724.1

Corynebacterium sp. CNJ-954

WP_021353009.1

C. pseudodiphtheriticum

WP_014008877.1

C. variable

WP_052540339.1

C. glyciniphilum

AHW65364.1

C. glyciniphilum AJ 3170

WP_015400429.1

C. halotolerans

WP_075814720.1

Corynebacterium sp. CNJ-954

SNV91221.1

C. urealyticum

CUU66362.1

C. variable

WP_020934331.1

C. maris

WP_012361047.1

C. urealyticum

WP_015381984.1

C. urealyticum

WP_071055926.1

C. jeikeium

SCX01561.1

C. jeikeium

WP_049050177.1

C. jeikeium 931_CJEI; Corynebacterium

sp. HMSC08A12

EEW16595.1

C. jeikeium ATCC 43734

WP_034965445.1

C. jeikeium

WP_034986706.1

C. jeikeium

WP_011272910.1

C. jeikeium

WP_038550379.

C. glyciniphilum

WP_070760374.1

Corynebacterium sp. HMSC27B11

WP_035004904.1

C. jeikeium

WP_070759388.1

Corynebacterium sp. HMSC22B11

WP_020440292.1

C. terpenotabidum

WP_066586577.1

C. provencense

WP_013887703.1

C. resistens

WP_010120483.1

C. nuruki

WP_034981842.1

C. jeikeium

WP_034971855.1

C. jeikeium

WP_043012061.1

C. jeikeium Cj19409; Corynebacterium

sp. HMSC058E07

WP_034969387.1

C. jeikeium

WP_025401902.1

C. falsenii

WP_034974957.1

C. jeikeium

WP_034992651.1

C. jeikeium

WP_035007616.1

C. jeikeium

WP_035002809.1

C. jeikeium

WP_048737023.1

C. falsenii

WP_099298200.1

Corynebacterium sp. Marseille-P4122

WP_018021816.1

C. doosanense

WP_049168823.1

C. propinquum

WP_049148215.1

C. propinquum

WP_018119927.1

C. propinquum

WP_029157673.1

C. bovis

WP_021353010.1

C. pseudodiphtheriticum 090104 and DSM 44287;

Corynebacterium sp. KPL1995,

KPL1989, and EPI-003-04-2554_SCH2473622

WP_048737018.1

C. falsenii

WP_047262329.1

C. mustelae

h. Insertion of a Promoter in Front of the cg2766 Start Codon

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise a promoter inserted in front of the cg2766 start codon. 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 inserted promoter is promoter Pcg0007_39 (SEQ ID NO:20); see US 2017/0159045 and WO 2017/00376.

i. Replacement of the Native actA Promoter

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise a replacement actA promoter in place of the native actA promoter. The actA 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 actA replacement promoter is promoter Pcg0007_39 (SEQ ID NO:20).

j. Replacement of the Native opcA Coding Sequence

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise a coding sequence for an opcA protein of a different Corynebacterium species or strain in place of the native opcA coding sequence. 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-11474 (SEQ ID NO:94).

In some embodiments the replacement opcA coding sequence is under the control of a promoter that drives expression of both zwf and opcA. In some embodiments, the replacement opcA coding sequence is under the control of an inserted opcA promoter. The inserted opcA 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 inserted opcA promoter is promoter Pcg0007_39 (SEQ ID NO:20).

TABLE 4A

NCBI Reference Numbers for Corynebacterial opcA Proteins

Reference Number
Species and/or Strain(s)

WP_046439480.1

C. kutscheri

WP_099298719.1

Corynebacterium sp. Marseille-P22

WP_006823740.1

C. casei UCMA 3821 (GenBank Accession No.

CCE56308.1), LMG S-19264; Corynebacterium

sp. JB4; SEQ ID NO: 92

WP_095661089.1

C. glaucum

WP_095276461.1

Corynebacterium sp. NBT06-6, NML99-0020,

and NML92-0415

WP_095553723.1

Corynebacterium sp. NML 150383

WP_095548171.1

Corynebacterium sp. NML00-0156

WP_095545764.1

Corynebacterium sp. NML 120412

WP_095536183.1

Corynebacterium sp. NML93-0607

SNV73072.1

C. imitans

WP_092286146.1

C. spheniscorum

WP_092255258.1

C. cystitidis

WP_092148525.1

C. mycetoides

WP_088266936.1

C. diphtheriae

WP_088265045.1

C. diphtheriae

WP_088246432.1

C. diphtheriae

WP_087454020.1

C. ulcerans

WP_087117967.1

C. urinapleomorphum

WP_049360793.1

C. aurimucosum 1237_CAUR; C. kefirresidentii SB

WP_086891441.1

C. striatum

WP_085958015.1

C. fournierii

WP_085713610.1

C. diphtheriae

WP_085549995.1

C. pollutisoli

SLM96782.1

C. xerosis

WP_084759291.1

C. phoceense

WP_084168239.1

C. atypicum

WP_084082961.1

C. freneyi

WP_082796495.1

C. variabile

WP_081920634.1

C. variabile

WP_081803842.1

C. glyciniphilum

WP_078057488.1

C. provencense

WP_076598597.1

C. appendicis

WP_075816106.1

Corynebacterium sp. CNJ-954

WP_075734014.1

C. phocae

WP_075729852.1

C. flavescens

WP_075726270.1

C. aquilae

WP_075722895.1

C. stationis

WP_075691979.1

C. sphenisci

WP_075663775.1

C. frankenforstense

WP_072590090.1

C. diphtheriae

WP_071575625.1

C. diphtheriae

WP_071572602.1

Corynebacterium sp. NML120713

WP_071571645.1

C. diphtheriae

WP_071570066.1

C. diphtheriae

WP_071568288.1

Corynebacterium sp. NML140438

WP_071566266.1

Corynebacterium sp. NML130628

WP_071056558.1

C. jeikeium

WP_070976878.1

Corynebacterium sp. MC3

WP_070832090.1

Corynebacterium sp. HMSC073H12 and HMSC074C03

WP_070681225.1

Corynebacterium sp. HMSC072A04, HMSC074H12,

and HMSC035E02

WP_070653088.1

Corynebacterium sp. HMSC065H09 and HMSC072B08

WP_070451416.1

C. coyleae DSM 44184; Corynebacterium

sp. HMSC11D10 and HMSC034E211

WP_070866373.1

Corynebacterium sp. HMSC072D01

WP_070856734.1

Corynebacterium sp. HMSC074C05

WP_070843904.1

Corynebacterium sp. HMSC034A01

WP_070839048.1

Corynebacterium sp. HMSC070H05

WP_070828994.1

Corynebacterium sp. HMSC036E10

WP_070822776.1

Corynebacterium sp. HMSC036D03

WP_070816208.1

Corynebacterium sp. HMSC034B08

WP_070799051.1

C. diphtheriae

WP_070770986.1

Corynebacterium sp. HMSC075D04

WP_070768006.1

Corynebacterium sp. HMSC04H06

WP_070761406.1

Corynebacterium sp. HMSC27B11

WP_070758918.1

Corynebacterium sp. HMSC22B11

WP_070738933.1

Corynebacterium sp. HMSC073D01

WP_070732205.1

Corynebacterium sp. HMSC076D02

WP_070683023.1

Corynebacterium sp. HMSC056E09

WP_070671164.1

Corynebacterium sp. HMSC078H07

WP_070643476.1

Corynebacterium sp. HMSC062A03 and HMSC072D12

WP_070642957.1

Corynebacterium sp. HMSC076C10

WP_070615011.1

Corynebacterium sp. HMSC067D03

WP_070570372.1

Corynebacterium sp. HMSC055D05

WP_070567126.1

C. jeikeium FDAARGOS_328; Corynebacterium

sp. HMSC077G01, HMSC063F04, HMSC064E07, HMSC072B09, and HMSC073B01

WP_070564456.1

Corynebacterium sp. HMSC072A02 and HMSC078A10

WP_070549602.1

Corynebacterium sp. HMSC077G07

WP_070547341.1

Corynebacterium sp. HMSC070E08

WP_070536503.1

Corynebacterium sp. HMSC05E07

WP_070528425.1

Corynebacterium sp. HMSC074E01, HMSC074C01,

and HMSC036D02

WP_070524283.1

Corynebacterium sp. HMSC055A01, HMSC065D07,

and HMSC078C09

WP_070522356.1

Corynebacterium sp. HMSC11E11

WP_070494917.1

Corynebacterium sp. HMSC30G07

WP_070487506.1

Corynebacterium sp. HMSC08A12

WP_070484826.1

Corynebacterium sp. HMSC08F01

WP_070473987.1

Corynebacterium sp. HMSC05H05

WP_070462224.1

Corynebacterium sp. HMSC29G08

WP_070444875.1

Corynebacterium sp. HMSC055A01, HMSC065D07,

and HMSC078C09

WP_070435059.1

Corynebacterium sp. HMSC28B08

WP_070423128.1

Corynebacterium sp. HMSC05C01

WP_070420257.1

Corynebacterium sp. HMSC06C06

WP_070362427.1

Corynebacterium sp. NML98-0116 and HMSC08D02

WP_062035107.1

C. simulans PES1; Corynebacterium sp. HMSC06G04

WP_061922333.1

C. simulans Wattiau and 1B08; Corynebacterium

sp. HMSC06D04, HMSC08C04, HMSC077D03, HMSC071F07;

Corynebacterium accolens AH4003

WP_049193712.1

C. jeikeium 401_CJE1; Corynebacterium

sp. HMSC055G02

WP_046649108.1

C. aurimucosum 944_CAUR; Corynebacterium

sp. HMSC068G04 and HMSC056F09; C. minutissimum 1941

WP_043015176.1

C. jeikeium Cj30184; Corynebacterium

sp. HMSC058E07

WP_005531605.1

C. striatum 216, ATCC 6940, 797_CAUR, 587_CAUR,

542_CAUR, 1329_CAUR, 1327_CAUR, 2230, 2245, 2023, and 2247;

Corynebacterium sp. HMSC05D08

OHF39428.1

Corynebacterium sp. HMSC074A01

WP_066493224.1

Corynebacterium sp. DNF00584, CMW7794,

HMSC072G08, HMSC077B05, HMSC077D10, and HMSC0665A05

WP_066839768.1

C. stationis

WP_066794092.1

C. stationis

WP_066525378.1

C. bouchesdurhonense

WP_049167646.1

C. propinquum

WP_049158908.1

C. striatum

WP_049148304.1

C. propinquum

WP_065421559.1

C. halotolerans

WP_064833329.1

Corynebacterium sp. EPI-003-04-2554_SCH2473622

WP_063936745.1

C. afermentans

WP_058832265.1

C. pseudotuberculosis

WP_006063324.1

C. durum

WP_058093366.1

C. pseudotuberculosis

WP_055178564.1

C. lowii

WP_055121854.1

C. oculi

AKK06005.1

C. mustelae

WP_053412142.1

C. lactis

WP_052204189.1

C. riegelii

WP_038628980.1

C. jeikeium 805_CJEI; Corynebacterium sp.

ATCC 6931, HMSC11H10, HMSC074C11, HMSC063A05, HMSC064E10,

and HMSC074C04

WP_049049964.1

C. jeikeium

WP_049378186.1

C. aurimucosum

WP_048758558.1

C. vitaeruminis

WP_048737982.1

C. falsenii

WP_016422736.1

C. jeikeium 239_CJEI and 212_CJEI; C. urealyticum

1055_CURE; Corynebacterium sp. HFH0082, HMSC14H10,

HMSC064H12, HMSC063G05, HMSC077C02, HMSC064E08, HMSC070B05,

HMSC061H03, HMSC075F02; C. amycolatum_ICIS

53 and ICIS 9

WP_048381636.1

C. renale

WP_047263492.1

C. mustelae

WP_047259618.1

C. uterequi

WP_047253131.1

C. testudinoris

WP_047240275.1

C. epidermidicanis

WP_005526431.1

C. matruchotii

WP_046650939.1

C. xerosis

WP_046645947.1

C. striatum

WP_046204153.1

C. argentoratense

WP_046202297.1

C. kroppenstedtii

WP_046095943.1

C. ulcerans

AHW64140.1

C. glyciniphilum AJ 3170

WP_042621478.1

C. marinum

WP_042530750.1

C. singulare

WP_042404782.1

C. jeddahense

WP_040354603.1

C. ammoniagenes

WP_040085900.1

C. humireducens (GenBank Accession No. AJE33249.1,

SEQ ID NO: 25)

WP_039675253.1

C. minutissimum

WP_038620584.1

C. ulcerans

WP_038611699.1

C. ureicelerivorans

WP_035113157.1

C. auriscanis

WP_035104698.1

C. camporealensis

WP_035012334.1

C. jeikeium

WP_035010427.1

C. jeikeium

WP_035008179.1

C. jeikeium

WP_035003885.1

C. jeikeium

WP_035001868.1

C. jeikeium

WP_034998920.1

C. ihumii

WP_034987985.1

C. jeikeium

WP_034981960.1

C. jeikeium

WP_034974411.1

C. jeikeium

WP_034967970.1

C. jeikeium

WP_034663424.1

C. tuscaniense

WP_027020497.1

C. sputi

WP_027012302.1

C. freiburgense

WP_025402713.1

C. falsenii (GenBank Accession No. AHI03141.1),

SEQ ID NO: 89

WP_025252883.1

C. vitaeruminis (GenBank Accession No. AHI22863.1);

SEQ ID NO: 87

WP_023022439.1

Corynebacterium sp. KPL1859, KPL1855, and KPL1814

WP_023021500.1

Corynebacterium sp. KPL1860, KPL1857, KPL1856,

KPL1821, KPL1817, and KPL1856

WP_023019439.1

Corynebacterium sp. KPL1995 and KPL1989

WP_023017462.1

Corynebacterium sp. KPL2004, KPL1998, KPL1996,

KPL1986, KPL1824, and KPL1996

WP_005279118.1

C. accolens ATCC 49725; Corynebacterium

sp. KPL1818

WP_022862033.1

C. massiliense

WP_021353956.1

C. pseudodiphtheriticum

WP_020976309.1

C. argentoratense

WP_005324852.1

C. tuberculostearicum SK141;

C. pseudogenitalium ATCC 33035

WP_020934820.1

C. maris

WP_020441165.1

C. terpenotabidum (GenBank Accession No. AGP30804.1),

SEQ ID NO: 93

WP_019194592.1

C. timonense

WP_018582009.1

C. pilosum

WP_018339807.1

C. caspium

WP_018295562.1

C. lubricantis

WP_018119593.1

C. propinquum

WP_018118857.1

C. mastitidis

WP_018023988.1

C. ulceribovis

WP_018021704.1

C. doosanense

WP_018019762.1

C. ciconiae

WP_018017013.1

C. capitovis

WP_016457552.1

C. pyruviciproducens (GenBank Accession No. EPD70204.1),

SEQ ID NO: 91

WP_010265064.1

C. bovis

WP_010186759.1

C. aurimucosum

EFG82419.1

C. ammoniagenes DSM 20306

WP_006840267.1

C. lipophiloflavum

WP_005521071.1

C. matruchotii (GenBank Accession No. EEG26974.1),

SEQ ID NO: 88

WP_005510779.1

C. amycolatum

WP_005389976.1

C. glucuronolyticum

WP_005294809.1

C. jeikeium

WP_005290056.1

C. genitalium

WP_005283075.1

C. accolens

WP_015400950.1

C. halotolerans (GenBank Accession No. AGF72531.1),

SEQ ID NO: 90

WP_014525815.1

C. ulcerans

WP_014367098.1

C. pseudotuberculosis

WP_014306922.1

C. diphtheriae

WP_014009994.1

C. variabile

WP_013911577.1

C. ulcerans

WP_013888447.1

C. resistens

WP_013241959.1

C. pseudotuberculosis

WP_012731602.1

C. kroppenstedtii

WP_012360244.1

C. urealyticum

WP_011273572.1

C. jeikeium

WP_003851588.1

C. diphtheriae

TABLE 4B

NCBI Reference Numbers for Corynebacterial OpcA Proteins

Reference Number
Species and/or Strain

WP_075348233.1

C. glutamicum XV

WP_003862265.1

C. glutamicum ATCC 14067, ATCC 21831, AR1,

USDA-ARS-USMARC-56828, YI, SYPS-062, SYPS-062-33a, and

ATCC 21493; B. flavum ATCC 15168

WP_040967467.1

C. glutamicum

WP_003856048.1

C. glutamicum SCgG1, SCgG2, S9114, Z118,

CS176, AS 1.542, TG-13; C. crenatum MT

WP_074508163.1

C. glutamicum

WP_011897297.1

C. glutamicum

WP_011014458.1

C. glutamicum

WP_087497587.1

C. glutamicum

WP_066565817.1

C. crudilactis

WP_096456023.1

C. glutamicum

WP_053544982.1

C. deserti

WP_015651366.1

C. callunae

WP_080794830.1

Corynebacterium sp. Marseille-P2417

WP_006767698.1

C. efficiens

k. Insertion of at Least One Additional Copy of lysA, ask, asd, ddh, and/or dapB

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise at least one additional copy of at least one gene selected from the group consisting of lysA, ask, asd, ddh, and dapB. 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.

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

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.

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.

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.

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.

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.

l. Replacement of the Start Codon of aceE

In some embodiments, the start codon of the native aceE gene in the genome of any of the engineered strains of Corynebacteria described above is replaced. 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.

m. Replacement of the Native pyc Gene Promoter

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise a pyc replacement promoter in place of the native pyc gene promoter. The pyc 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 pyc replacement promoter is promoter Pcg1860 (SEQ ID NO:11; see US 2017/0159045).

n. Insertion of a Promoter in Front of the zwf Open Reading Frame

Engineered strains of Corynebacteria comprising any of the structural alterations described above can also comprise 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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).

o. Insertion of a Codon-Optimized lysA Coding Sequence

Engineered strains of Corynebacteria comprising any of the structural alterations described above can comprise 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 Nešvera et al., 2012; Pátek et al., 2003(a); Pátek et al., 2003(b); Pátek 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 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).

Nucleic Acids and Vectors

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-11474, the Corynebacterium deposited as NRRL B-67439, 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.

Nucleic acids that can be used to engineer the alterations described above are well known and are described, for example, in U.S. Pat. Nos. 7,368,276, 6,927,046, US 2017/0159045, and WO 2017/00376 and in the Examples, below. Nucleic acids encoding the altered corynebacterial rnaJ, accDA, and cg1144 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. 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.

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; Knoppová et al., 2007; and Pátek & Nešvera, 2013.

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.

Engineered Strains of Corynebacteria

This disclosure provides strains of Corynebacteria engineered to contain at least one of the four alterations described above—i.e., (a) insertion of a replacement promoter in front of the cg1383 start codon; (b) replacement of the native phoU promoter; (c) replacement of the native cg3210 promoter; and (d) replacement of the native cg0800 promoter—as well as at least one of the following alterations:

- (e) alteration of the rnaJ coding sequence to encode a ribonuclease J protein comprising a G448S substitution;
- (f) alteration of the native accDA coding sequence to encode an acetyl-CoA carboxylase carboxyl transferase subunit alpha/beta protein comprising a G310E substitution;
- (g) alteration of the native cg1144 coding sequence to encode a cg1144 protein comprising a P66S substitution;
- (h) insertion of a promoter in front of the cg2766 start codon;
- (i) replacement of the native actA promoter;
- (j1) replacement of the native opcA coding sequence by coding sequence for an opcA protein of a different Corynebacterium species or strain;
- (j2) replacement of the native opcA coding sequence by coding sequence for an opcA protein of a different Corynebacterium species or strain under the control of an inserted opcA promoter;
- (k) insertion of at least one additional copy of at least one gene selected from the group consisting ask, asd, ddh, and dapB;
- (l) replacement of the start codon of the native aceE gene;
- (m) replacement of the native pyc gene promoter;
- (n) insertion of an additional promoter in front of the zwf open reading frame; and
- (o) insertion of a codon-optimized lysA coding sequence.

The following non-limiting embodiments of engineered Corynebacteria fall within the description above.

Embodiment 1. The Corynebacterium deposited with the Agriculture Research Culture Collection (NRRL) International Depository Authority, 1815 N. University Street, Peoria, IL 61604 on Dec. 22, 2017, under the provisions of the Budapest Treaty and assigned Accession No. NRRL B-67535.

Embodiment 2. A Corynebacterium having a bacterial genome consisting essentially of the genome of the bacterium deposited as NRRL B-67439 but for up to 4 structural alterations selected from the group consisting of:

- (a) insertion of a promoter in front of the cg1383 start codon;
- (b) replacement of the native phoU promoter;
- (c) replacement of the native cg3210 promoter; and
- (d) replacement of the native cg0800 promoter.

Embodiment 3. A Corynebacterium having a bacterial genome consisting essentially of the genome of the bacterium deposited as NRRL B-11474 but for

- (i) at least one structural alteration selected from the group consisting of:
  - (a) insertion of a promoter in front of the cg1383 start codon;
  - (b) replacement of the native phoU promoter;
  - (c) replacement of the native cg3210 promoter; and
  - (d) replacement of the native cg0800 promoter; and
- (ii) at least one additional structural alteration selected from the group consisting of:
  - (e) alteration of the rnaJ coding sequence to encode a corynebacterial rnaJ protein comprising a G448S substitution;
  - (f) alteration of the native accDA coding sequence to encode a corynebacterial accDA protein comprising a G310E substitution;
  - (g) alteration of the native cg1144 coding sequence to encode a corynebacterial cg1144 protein comprising a P66S substitution;
  - (h) insertion of a promoter in front of the cg2766 start codon;
  - (i) replacement of the native actA promoter;
  - (j1) replacement of the native opcA coding sequence by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium;
  - (j2) replacement of the native opcA coding sequence by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium under the control of an inserted opcA promoter;
  - (k) insertion of at least one additional copy of at least one gene selected from the group consisting of lysA, ask, asd, ddh, and dapB;
  - (l) replacement of the native start codon of aceE by GTG, CTG, or TTG;
  - (m) replacement of the native pyc gene promoter by promoter Pcg1860 (SEQ ID NO:11);
  - (n) insertion of promoter Pcg0007_39 (SEQ ID NO:20) in front of the zwf open reading frame; and
  - (o) insertion of a codon-optimized lysA coding sequence.

Embodiment 4. The Corynebacterium of Embodiment 2 or 3, wherein the promoter in front of the cg1383 start codon is promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 5. The Corynebacterium of any of Embodiments 2-4, wherein the native phoU promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 6. The Corynebacterium of any of Embodiments 2-5, wherein the native cg3210 promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 7. The Corynebacterium of any of Embodiments 2-6, wherein the native cg0800 promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 8. The Corynebacterium of any of Embodiments 3-7, in which (e) the native rnaJ coding sequence is altered to encode the corynebacterial rnaJ protein comprising a G448S substitution.

Embodiment 9. The Corynebacterium of any of Embodiments 3-8, in which (e) the native rnaJ coding sequence is altered to encode the amino acid sequence SEQ ID NO:3.

Embodiment 10. The Corynebacterium of any of Embodiments 3-9, in which (f) the native accDA coding sequence is altered to encode the corynebacterial accDA protein comprising the G310E substitution.

Embodiment 11. The Corynebacterium bacterium of any of Embodiments 3-10, in which (f) the native accDA coding sequence is altered to encode the amino acid sequence SEQ ID NO:6.

Embodiment 12. The Corynebacterium of any of Embodiments 3-11, in which (g) the native cg1144 coding sequence is altered to encode the corynebacterial cg1144 protein comprising the P66S substitution.

Embodiment 13. The Corynebacterium bacterium of any of Embodiments 3-12, in which (g) the native cg1144 coding sequence is altered to encode the amino acid sequence SEQ ID NO:9.

Embodiment 14. The Corynebacterium bacterium of any of Embodiments 3-13, in which (h) the promoter is inserted in front of the cg2766 start codon.

Embodiment 15. The Corynebacterium of any of Embodiments 3-14, in which (h) the promoter inserted in front of the cg2766 start codon is promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 16. The Corynebacterium bacterium of any of Embodiments 3-15, in which (i) the native actA promoter is replaced.

Embodiment 17. The Corynebacterium of any of Embodiments 3-16 in which (i) the native actA promoter is replaced by promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 18. The Corynebacterium bacterium of any of Embodiments 3-17, in which (j) the native opcA coding sequence is replaced by a replacement opcA coding sequence for an opcA protein of a different Corynebacterium.

Embodiment 19. The Corynebacterium of any of Embodiments 3-18, 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 20. The Corynebacterium of any of Embodiments 3-19, 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 21. The Corynebacterium of any of Embodiments 3-20, in which the replacement opcA coding sequence is under control of an inserted opcA promoter.

Embodiment 22. The Corynebacterium of Embodiment 21, in which the inserted opcA promoter is promoter Pcg0007_39 (SEQ ID NO:20).

Embodiment 23. The Corynebacterium bacterium of any of Embodiments 3-22, in which (k) 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 24. The Corynebacterium bacterium of Embodiment 23, in which the genome of the bacterium contains (a) at least one additional copy of lysA, ask, asd, ddh, and dapB.

Embodiment 24. The Corynebacterium bacterium of Embodiment 23, 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 25. The Corynebacterium bacterium of Embodiment 23, 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 26. The Corynebacterium bacterium of Embodiment 23, 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 27. The Corynebacterium bacterium of Embodiment 23, 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 28. The Corynebacterium bacterium of any of Embodiments 3-27, in which the start codon of aceE is replaced by TTG, GTG, or CTG.

Embodiment 29. The Corynebacterium bacterium of any of Embodiments 3-28, in which the start codon of aceE is replaced by TTG.

Embodiment 30. The Corynebacterium bacterium of any of Embodiments 3-29, in which the native pyc gene promoter is replaced.

Embodiment 31. The Corynebacterium bacterium of any of Embodiments 3-30, in which the native pyc gene promoter is replaced by promoter Pcg1860 (SEQ ID NO:11).

Embodiment 32. The Corynebacterium bacterium of any of Embodiments 3-31, in which a promoter is inserted in front of the zwf open reading frame.

Embodiment 33. The Corynebacterium bacterium of any of Embodiments 3-32, in which promoter Pcg0007_39 (SEQ ID NO:20) is inserted in front of the zwf open reading frame.

Embodiment 34. The Corynebacterium of any of Embodiments 3-33, which contains (o) a codon-optimized lysA coding sequence.

Embodiment 35. The Corynebacterium of Embodiment 34, in which the codon-optimized lysA coding sequence is under control of Pcg0007_39 (SEQ ID NO:20).

Embodiment 36. The Corynebacterium of Embodiment 34 or 35, which comprises a heterologous terminator for the codon-optimized lysA coding sequence.

Embodiment 37. The Corynebacterium bacterium of any of Embodiments 34-36, which comprises the nucleotide sequence SEQ ID NO:17.

Production of Lysine

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. Pat. Nos. 8,048,649, 7,635,579, 7,504,242, 7,300,777, 7,267,967, 7,160,711, 7,141,388, 7,122,369, 6,927,046, 6,830,903, US 2002/0086371, US 2004/0043458, US 2009/0325244, EP 1246921, and EP 1619252.

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

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-orj2′ cassette (4Go) was digested from pFC3-ask-asd-dapB-ddh plasmid (U.S. Pat. No. 7,368,276) with PmeI and XmaI and ligated into pDElia11 (U.S. Pat. No. 6,927,046) linearized with HincII and XmaI to construct pD11-KBDH.

To generate a 9.4 kb homology region containing a SpeI site, a DNA fragment was amplified from NRRL B-11474 genomic DNA using primers 18417 (SEQ ID NO:70) and 184F8 (SEQ ID NO:71) and cloned into the pBKMS vector NdeI/PstI sites by IN-FUSION® (Clontech). The resulting plasmid pBKMS 184 3p was digested with SpeI and a second PCR product similarly obtained from NRRL B-11474 genomic DNA using primers 161-184f5 and 162-184r6 was inserted by IN-FUSION® reaction to generate pBKMS184. The 5.4 kb 4Go cassette was amplified from pD11-KDBH with primers 244-4Go F2 Inf SpeI (SEQ ID NO:72) and 253-4Go R3 (SEQ ID NO:73), digested with SpeI and cloned by ligation into pBKMS 184 SpeI site.

Example 1B. Construction of a Vector Containing the Lysine Biosynthesis Genes ask-asd-dapB-ddh and a FarI Homology Region for Targeted Integration

The 3′ FarI homology region from a NRRL B-11474 derived strain was amplified by PCR using primers FarI 3P InfusF (SEQ ID NO:30) and FarI 3P InfusR AvrII (SEQ ID NO:31). The purified PCR fragment was cloned by IN-FUSION® reaction (Clontech) into pBKMS digested with NdeI and PstI to generate pBKMS FarI 3p. Similarly, the 5′ FarI homology region from a NRRL B-11474-derived strain was PCR amplified with primers FarI 5p InfusF (SEQ ID NO:32) and FarI 5p InfusR (SEQ ID NO:33) and cloned into the XbaI/AvrII sites of pBKMS FarI 3p to generate pBKMS FarI.

The 5.4 kb ask-asd-ddh-dapB-orj2′ cassette (4Go) was digested from pFC3-ask-asd-dapB-ddh plasmid (U.S. Pat. No. 7,368,276) with PmeI and XmaI and ligated into pDElia11 (U.S. Pat. No. 6,927,046) linearized with HincII and XmaI. The resulting vector pD11-KBDH was digested with NruI and SwaI to eliminate a ddh-dapB 2.9-kb fragment to generate pD11KD. pD11KD was then digested with SmaI and a 1.36 kb PCR fragment amplified from pD11KDBH using primers 685 (SEQ ID NO:74) and 686 (SEQ ID NO:75) was inserted using IN-FUSION® (Clontech) to generate pD11KDH. A 986-bp dapB fragment was then amplified from pD11KDBH with primers 687 (SEQ ID NO:76) and 693 (SEQ ID NO:77) and cloned into the pD11KDH SbfI site by IN-FUSION® (Clontech) reaction. The KBDH fragment was excised from the resulting pD11KBDH R plasmid by restriction with SpeI and cloned into the AvrII site of pBKMS FarI to generate pBKMSFarI4GRA.

Example 2. Allelic Replacement in C. glutamicum

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 μg/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 5) 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.

TABLE 5

Media used in Examples 2 and 3.

Medium
Medium
Medium
Medium
Medium
Medium

B
C
D
E
F
G
units

Volume
1000
1000
1000
1000
1000
1000
ml

glucose

30
75
35
50
g

Sucrose
50
50

g

L-alanine
0.5

mg

L-methionine
0.5

mg

l-threonine
0.25

mg

KH₂PO₄

1
g

K₂HPO₄
3
3

0.75
1

g

Urea
3
3

g

MgSO₄*7H₂O
0.5
0.5

0.3
0.4
0.4
g

yeast extract

35

g

Polypeptone
20

g

peptone (BBL)

Beef Extract
5

g

(BBL)

d-Biotin
0.756
0.756
3.024
0.225
0.3
0.3
mg

Thiamine*HCl
3
3
3

mg

Niacinamide
0.125
0.125
0.125

g

pH (KOH)
7.3
7.3

7.4
7.4
7.3

MnSO₄*H₂O

0.01
0.0075
0.01
0.01
g

FeSO4*7H₂O

0.1
0.01
g

(NH₄)₂SO₄

37.5
50
50
g

CSL Roquette

20
15
20
20
g

Raffinae¹

5

g

CaCO₃

50
50
50
50
g

Example 3. Genomic DNA Extraction

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×g (4° C.) for 10 minutes. The pellets were suspended in 10 ml of a solution containing 25 mM Tris-HCl pH 8.0, 10 mM EDTA, 50 mM 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 μl 10 mg/ml RNAse A, and 167 μl 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

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-11474 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 pK18mobsacB (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 10-Beta cells (New England Biolabs) with 50 μg/ml kanamycin selection before transformation into C. glutamicum as described in Example 2.

Example 4B. Construction of Transformation Vector to Introduce rnaJ_G448SAllele

Two homology arms were amplified using PCR from NRRL B-11474 genomic DNA obtained as described in Example 3.

The approximately 2.1 kb downstream homology arm (nucleotides 2026 to 4101 from SEQ ID NO:1) was amplified using primers SNP_084_P1 (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:1) was amplified using primers SNP_084_P3 (SEQ ID NO:40) and SNP_084_P4 (SEQ ID NO:41). 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-11474 and its derived strains as described in Example 2. Resulting strains carry the altered rnaJ coding sequence (SEQ ID NO:2), encoding the rnaJ amino acid sequence with the G448S substitution (SEQ ID NO:3).

Example 4C. Construction of Transformation Vector to Introduce accDA_G310EAllele

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_P1 (SEQ ID NO:46) and SNP_033_P2 (SEQ ID NO:47). Primer SNP_033_P1 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-11474 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 cg1144_P66SAllele

Two homology arms were amplified from NRRL B-11474 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:51). Primer SNP_316_P1 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_316_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_316_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-11474 and its derived strains as described in Example 2. Resulting strains carry the altered cg1144 coding sequence (SEQ ID NO:8), encoding the cg1144 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 Pcg1860

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 Pcg1860_pyc_P1 (SEQ ID NO:42) and Pcg1860_pyc_P2 (SEQ ID NO:43). Similarly, the 2050 bp downstream homology arm (nucleotides 2161 to 4210 from SEQ ID NO:10) was amplified using primers Pcg1860_pyc_P3 (SEQ ID NO:44) and Pcg1860_pyc_P4 (SEQ ID NO:45). The 93-bp Pcg1860 nucleotide sequence (SEQ ID NO:11) was obtained by overlap of the 5′ extensions of primers Pcg1860_pyc_P2 and Pcg1860-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 Pcg1860 (SEQ ID NO:11) instead of the pyc promoter (SEQ ID NO:12).

Example 4F. Construction of Transformation Vector to Introduce aceE_atg>ttgAllele

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_P1 (SEQ ID NO:54) and SNP_aceE_P2 (SEQ ID NO:55). Primer SNP_aceE_P1 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

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 −2 kb 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^COcontaining 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

Two homology arms were amplified from a NRRL B-11474 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_P1 (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-11474 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 zwf ORF (SEQ ID NO:21).

Example 4I. Replacement of Native opcA Coding Sequence by the opcA Coding Sequence of C. humireducens NBRC 106098

Two homology arms were amplified from NRRL B-11474-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_P1 (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:61). 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-11474 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 4J. Construction of Transformation Vector to Insert Promoter Pcg0007_39 in Front of the cg2766 Start Codon

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_P1 (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-11474 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

Two homology arms were amplified from NRRL B-11474 genomic DNA (Example 3) by PCR.

The 2021 bp downstream homology arm (nucleotides 2263 to 4283 from SEQ ID NO:28) was amplified using primers Pcg0007_39-actA_P1 (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-actA_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-11474 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 4L. Construction of Transformation Vector to Insert Promoter Pcg0007_39 in Front of the cg1383 Start Codon

Two homology arms were amplified from a NRRL B-11474 genomic DNA by PCR. The 2043 bp upstream homology arm (nucleotides 1 to 2043 of SEQ ID NO:98) was amplified using primers Pcg0007_39-cg1383_P1 (SEQ ID NO:99) and Pcg0007_39-cg1383_P2 (SEQ ID NO:100). Similarly, the 2050 bp downstream homology arm (nucleotides 2044 to 4093 of SEQ ID NO:98) was amplified using primers Pcg0007_39-cg1383_P3 (SEQ ID NO:101) and Pcg0007_39-cg1383_P4 (SEQ ID NO:102). The 93-bp Pcg0007_39 nucleotide sequence was obtained by overlap of the 5′ extensions of primers Pcg0007_39-cg1383_P2 and Pcg0007_39-cg1383_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 Pcg0007_39 promoter inserted in front of cg1383 start codon (SEQ ID NO:103).

Example 4M. Construction of Transformation Vector to Replace the Native phoU Promoter with Promoter Pcg0007_39

Two homology arms were amplified from a NRRL B-11474 genomic DNA by PCR. The 1935 bp upstream homology arm (nucleotides 1 to 1935 of SEQ ID NO:104) was amplified using primers Pcg0007_39-phoU_P1 (SEQ ID NO:105) and Pcg0007_39-phoU_P2 (SEQ ID NO:106). Similarly, the 2050 bp downstream homology arm (nucleotides 1995 to 4044 of SEQ ID NO:104) was amplified using primers Pcg0007_39-phoU_P3 (SEQ ID NO:107) and Pcg0007_39-phoU_P4 (SEQ ID NO:108). The 93-bp Pcg0007_39 nucleotide sequence is obtained by overlap of the 5′ extensions of primers Pcg0007_39-phoU_P2 and Pcg0007_39-phoU_P3. The two homology arms are 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 Pcg0007_39 promoter in front of phoU start codon (SEQ ID NO:109) and replacing nucleotides 1936-1994 of SEQ ID NO:104.

Example 4N. Construction of Transformation Vector to Replace the Native Cg3210 Promoter with Promoter Pcg0007_39

Two homology arms were amplified from a NRRL B-11474 genomic DNA by PCR. The 1935 bp upstream homology arm (nucleotides 1 to 2030 of SEQ ID NO:110) were amplified using primers Pcg0007_39-cg3210_P1 (SEQ ID NO:111) and Pcg0007_39-cg3210_P2 (SEQ ID NO:112). Similarly, the 2047 bp downstream homology arm (nucleotides 2090 to 4136 of SEQ ID NO:110) were amplified using primers Pcg0007_39-cg3210_P3 (SEQ ID NO:113) and Pcg0007_39-3210_P4 (SEQ ID NO:114). The 93-bp Pcg0007_39 nucleotide sequence was obtained by overlap of the 5′ extensions of primers Pcg0007_39-cg3210_P2 and Pcg0007_39-cg3210_P3. The two homology arms are 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 Pcg0007_39 replacing nucleotides 2031 to 2089 of SEQ ID NO:110, and inserted in front of cg3210 start codon (SEQ ID NO:115).

Example 4O. Construction of Transformation Vector to Replace the Native Cg0800 Promoter with Promoter Pcg0007_39

Two homology arms were amplified from a NRRL B-11474 genomic DNA by PCR. The 2043 bp upstream homology arm (nucleotides 1 to 2043 of SEQ ID NO:116) was amplified using primers Pcg0007_39-cg0800_P3 (SEQ ID NO:117) and Pcg0007_39-cg0800_P4 (SEQ ID NO:118). Similarly, the 2050 bp downstream homology arm (nucleotides 2184 to 4233 of SEQ ID NO:116) was amplified using primers Pcg0007_39-cg0800_P1 (SEQ ID NO:119) and Pcg0007_39-0800_P2 (SEQ ID NO:120). The 93-bp Pcg0007_39 nucleotide sequence was obtained by overlap of the 5′ extensions of primers Pcg0007_39-cg0800_P2 and Pcg0007_39-cg0800_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 Pcg0007_39 promoter inserted in front of cg0800 start codon (SEQ ID NO:121), and replacing nucleotides 2044-2183 of SEQ ID NO:116.

Example 5. Evaluation of Altered C. glutamicum Strains for Lysine Production in Microtiter Plates
Example 5A. Lysine Production by Altered C. glutamicum Strains

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 or 96 hours. Culture conditions were 32° C., 1000 rpm (Infors HT).

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.

The amount of lysine (mM) produced by each recombinant strain and its immediate parent strain is shown in the Table 6. Table 7A is a list of genomic alterations, and Table 7B identifies which alterations are present in the tested strains with respect to the native genome of the bacterium deposited as NRRL B-11474.

TABLE 6

Parent
Modified
Change from Parent to
Lysine production (mM) - 24 hrs
Lysine production (mM) - 96 hrs

Strain ID
Strain ID
Modified Strain
Parent Strain
Modified Strain
Parent Strain
Modified Strain

34507
88274
0007_39-cg0800
5.51 +/− 3.24
4.84 +/− 1.85
47.62 +/− 6.82
54.13 +/− 1.02

121645
139804
0007_39-cg0800
20.22 +/− 4.62
14.59 +/− 1.54
49.84 +/− 4.51
53.08 +/− 1.41

151834
174547
0007_39-cg0800
5.33 +/− 9.59
12.71 +/− 10.45
57.98 +/− 4.13
56.78 +/− 4.55

174205
182533
0007_39-cg0800
12.82 +/− 7.93
17.86 +/− 2.46
55.51 +/− 5.11
61.54 +/− 2.09

174275
182550
0007_39-cg0800
18.06 +/− 5.8
20.24 +/− 5.46
58.89 +/− 4.31
57.48 +/− 3.8

174275
182557
0007_39-cg0800
18.06 +/− 5.8
19.3 +/− 2.73
58.89 +/− 4.31
63.14 +/− 2.55

175088
182566
0007_39-cg0800
19.65 +/− 5.47
19.83 +/− 5.4
59.7 +/− 3.89
57.61 +/− 3.73

175093
182574
0007_39-cg0800
26.29 +/− 8.54
21.54 +/− 6.77
55.33 +/− 6.84
60.49 +/− 2.8

175109
182591
0007_39-cg0800
24.07 +/− 6.91
16.86 +/− 6.38
53.94 +/− 3.57
56.96 +/− 2.37

175109
182598
0007_39-cg0800
24.07 +/− 6.91
33.71 +/− 5.63
53.94 +/− 3.57
58.56 +/− 2.41

175784
182601
0007_39-cg0800
26.06 +/− 11.12
17.64 +/− 6.7
57.43 +/− 2.77
59.8 +/− 2.57

175784
182608
0007_39-cg0800
26.06 +/− 11.12
25.56 +/− 2.25
57.43 +/− 2.77
61.07 +/− 2.22

176538
182609
0007_39-cg0800
25.6 +/− 6.2
28.81 +/− 2.81
57.82 +/− 5.05
62.76 +/− 1.23

176538
182616
0007_39-cg0800
25.6 +/− 6.2
24.35 +/− 2.7
57.82 +/− 5.05
64.08 +/− 1.6

176509
182617
0007_39-cg0800
23.21 +/− 11.77
9.03 +/− 3.72
62.92 +/− 4.61
66.83 +/− 3.8

176509
182624
0007_39-cg0800
23.21 +/− 11.77
15.26 +/− 2.45
62.92 +/− 4.61
66.74 +/− 1.18

174547
183652
0007_39-cg0800
12.71 +/− 10.45
20.12 +/− 6.82
56.78 +/− 4.55
61.19 +/− 1.97

175155
183691
0007_39-cg0800
21.4 +/− 5.14
21.27 +/− 2.26
56.03 +/− 3.66
52.58 +/− 2.62

175174
183700
0007_39-cg0800
23.97 +/− 5.96
24.65 +/− 5.13
57.11 +/− 2.8
55.95 +/− 4.59

175776
183708
0007_39-cg0800
30.83 +/− 10.18
20.58 +/− 4.21
58.82 +/− 4.55
55.64 +/− 4.54

175785
183715
0007_39-cg0800
26.69 +/− 6.46
26.39 +/− 2.62
57.18 +/− 3.68
53.79 +/− 2.97

175785
183716
0007_39-cg0800
26.69 +/− 6.46
23.11 +/− 5.68
57.18 +/− 3.68
51.52 +/− 2.25

178745
187964
0007_39-cg0800
18.28 +/− 9.23
8.24 +/− 1.95
60.96 +/− 4.7
63.09 +/− 1.94

180398
187972
0007_39-cg0800
23.43 +/− 12.62
10.26 +/− 1.19
57.63 +/− 3.9
59.75 +/− 1.86

180402
187984
0007_39-cg0800
22.94 +/− 15.61
13.82 +/− 2.75
52.86 +/− 5.07
57.71 +/− 2.64

178726
188102
0007_39-cg0800
15.7 +/− 12.23
5.54 +/− 3.79
62.47 +/− 3.79
60.2 +/− 1.47

178726
188104
0007_39-cg0800
15.7 +/− 12.23
4.62 +/− 4.5
62.47 +/− 3.79
61.32 +/− 1.82

180390
188154
0007_39-cg0800
26.07 +/− 11.03
19.31 +/− 8.45
57.26 +/− 2.63
58.87 +/− 2.1

176538
188162
0007_39-cg0800
25.6 +/− 6.2
22.58 +/− 2.65
57.82 +/− 5.05
61.01 +/− 1.67

176538
188169
0007_39-cg0800
25.6 +/− 6.2
14.06 +/− 4.59
57.82 +/− 5.05
61.24 +/− 1.71

175229
188170
0007_39-cg0800
14.97 +/− 7.27
15.19 +/− 3.38
42.82 +/− 18.51
64.79 +/− 1.95

181567
188178
0007_39-cg0800
16.82 +/− 11.51
4.96 +/− 3.88
58.84 +/− 5.97
61.7 +/− 3.84

180384
188186
0007_39-cg0800
27.09 +/− 13.8
23.88 +/− 12.17
56.9 +/− 3.43
60.93 +/− 2.15

182562
190037
0007_39-cg0800
15.13 +/− 6.02
18.61 +/− 1.81
61.26 +/− 3.72
60.35 +/− 1.59

182563
190045
0007_39-cg0800
23.62 +/− 7.18
19.63 +/− 1.79
62.13 +/− 3.81
60.13 +/− 1.86

182612
190053
0007_39-cg0800
21.67 +/− 4.04
17.96 +/− 6.16
62.94 +/− 4.44
58.86 +/− 2.58

182620
190061
0007_39-cg0800
19.34 +/− 6.03
18.17 +/− 5.41
61.92 +/− 4.76
63.53 +/− 3.46

182623
190069
0007_39-cg0800
23.86 +/− 4.55
25.51 +/− 7.13
58.63 +/− 5.35
59.76 +/− 3.09

179967
191527
0007_39-cg0800
9.16 +/− 5.93
9.28 +/− 4.58
61.7 +/− 5.41
58.22 +/− 4.37

182566
192419
0007_39-cg0800
19.83 +/− 5.4
22.34 +/− 3.99
57.61 +/− 3.73
53.9 +/− 2.05

176538
193279
0007_39-cg0800
25.6 +/− 6.2
26.4 +/− 2.41
57.82 +/− 5.05
56.47 +/− 4.58

182612
193295
0007_39-cg0800
21.67 +/− 4.04
24.95 +/− 5.13
62.94 +/− 4.44
56.12 +/− 5.52

191541
197907
0007_39-cg0800
13.96 +/− 7.55
19.08 +/− 3.27
63.49 +/− 3.11
62.35 +/− 2.05

191520
206822
0007_39-cg0800
14.26 +/− 5.09
14.33 +/− 2.29
64.17 +/− 3.8
61.85 +/− 1.8

193318
213002
0007_39-cg0800
24 +/− 6.35
21.79 +/− 3.62
61.14 +/− 3.26
57.62 +/− 1.17

193318
213012
0007_39-cg0800
24 +/− 6.35
24.84 +/− 3.03
61.14 +/− 3.26
61.88 +/− 4.03

00000
219284
0007_39-cg0800
5.06 +/− 5.44
3.82 +/− 3.9
24.04 +/− 7.01
36.76 +/− 4.58

121645
219308
0007_39-cg0800
20.22 +/− 4.62
20.06 +/− 8.89
49.84 +/− 4.51
52.49 +/− 4.39

34507
87642
0007_39-cg1383
5.51 +/− 3.24
10.65 +/− 2.82
47.62 +/− 6.82
48.74 +/− 4.66

81651
121624
0007_39-cg 1383
9.15 +/− 2.55
9.27 +/− 2.27
43.53 +/− 2.69
47.46 +/− 2.64

99349
121638
0007_39-cg1383
16.22 +/− 3.6
17.45 +/− 1.02
53.98 +/− 3.54
52.36 +/− 1

99370
121643
0007_39-cg 1383
13.58 +/− 3.8
11.72 +/− 2.78
51.8 +/− 4.46
49.6 +/− 1.97

71075
132568
0007_39-cg 1383
23.93 +/− 5.69
27.33 +/− 3.66
51.37 +/− 5.54
56.21 +/− 2.84

112373
134579
0007_39-cg 1383
19.39 +/− 4.03
17.46 +/− 1.34
52.21 +/− 4.81
51.73 +/− 1.92

121645
134603
0007_39-cg1383
20.22 +/− 4.62
18.52 +/− 3.41
49.84 +/− 4.51
52.89 +/− 2.6

108568
144042
0007_39-cg1383
26.79 +/− 7.43
14.54 +/− 8.65
52.58 +/− 5.38
60.47 +/− 5.14

108569
144051
0007_39-cg 1383
13.76 +/− 8.08
20.82 +/− 0.97
58.16 +/− 6.61
64.88 +/− 2.74

132598
148418
0007_39-cg1383
25.69 +/− 4.05
23.55 +/− 1.52
50.53 +/− 3.56
53.64 +/− 1.32

132581
148431
0007_39-cg1383
10.81 +/− 3.05
9.17 +/− 1.73
48.17 +/− 9.27
54.36 +/− 1.53

134595
148999
0007_39-cg1383
10.48 +/− 3.33
13.14 +/− 1.15
50.76 +/− 2.94
53 +/− 0.93

132599
149034
0007_39-cg1383
19.33 +/− 4.23
23.52 +/− 1.53
52.51 +/− 5.41
49.83 +/− 0.84

134610
149042
0007_39-cg1383
18.73 +/− 4.67
19.1 +/− 3.06
48.27 +/− 2.83
48.46 +/− 1.81

102381
149058
0007_39-cg1383
26.82 +/− 8.13
33.01 +/− 3.67
49.78 +/− 3.59
49.01 +/− 1.14

148401
151595
0007_39-cg1383
19.21 +/− 5.8
9.29 +/− 7.05
52.95 +/− 4.06
54.43 +/− 2.93

148401
151611
0007_39-cg 1383
19.21 +/− 5.8
−11.43 +/− 1.47
52.95 +/− 4.06
0 +/− 0

148936
151753
0007_39-cg1383
20.42 +/− 10.42
29.69 +/− 4.69
44.38 +/− 7.76
52.61 +/− 7.57

148409
151764
0007_39-cg1383
11.55 +/− 4.46
10.69 +/− 2.77
56.02 +/− 2.26
56.33 +/− 1.12

148416
151804
0007_39-cg 1383
19.71 +/− 5.15
10.16 +/− 1.63
52.61 +/− 2
51.87 +/− 1.61

148989
151840
0007_39-cg 1383
22.37 +/− 3.54
23.75 +/− 3.91
41.08 +/− 3.55
40.95 +/− 3.54

149046
151904
0007_39-cg1383
18.43 +/− 5.34
19.02 +/− 3.73
52.81 +/− 5.31
50.01 +/− 4.06

149046
151914
0007_39-cg1383
18.43 +/− 5.34
17.83 +/− 3.84
52.81 +/− 5.31
46.8 +/− 0.96

148930
151920
0007_39-cg 1383
23.07 +/− 3.68
15.93 +/− 5.26
50.11 +/− 3.78
55.57 +/− 5.07

138706
151965
0007_39-cg1383
26.08 +/− 4.76
18.24 +/− 6.28
50.84 +/− 4.61
54.03 +/− 5.93

108568
152452
0007_39-cg 1383
26.79 +/− 7.43
25.7 +/− 1.89
52.58 +/− 5.38
54.45 +/− 0.96

102381
152468
0007_39-cg 1383
26.82 +/− 8.13
29.39 +/− 4.95
49.78 +/− 3.59
49.8 +/− 3.74

132590
152500
0007_39-cg 1383
26.43 +/− 7.04
24.96 +/− 2.09
43.76 +/− 5.2
44.2 +/− 0.9

151863
154589
0007_39-cg1383
17.14 +/− 4.77
22.8 +/− 1.45
55.32 +/− 4.32
52.28 +/− 2.72

151867
154600
0007_39-cg1383
19.54 +/− 4.74
18.29 +/− 5.44
48.04 +/− 5.55
49.91 +/− 5.85

151834
155462
0007_39-cg 1383
5.33 +/− 9.59
16.23 +/− 0.93
57.98 +/− 4.13
49.11 +/− 1.17

151834
155466
0007_39-cg 1383
5.33 +/− 9.59
15.89 +/− 1.64
57.98 +/− 4.13
49.56 +/− 0.95

151834
155469
0007_39-cg1383
5.33 +/− 9.59
17.14 +/− 1.1
57.98 +/− 4.13
49.3 +/− 1.13

151906
162735
0007_39-cg 1383
19.77 +/− 3.91
20.74 +/− 1.45
54.38 +/− 4.77
49.16 +/− 0.76

152616
163111
0007_39-cg 1383
12.28 +/− 4.92
10.65 +/− 2.71
55.25 +/− 2.57
53.29 +/− 1.51

152672
163167
0007_39-cg1383
22.62 +/− 6.35
16.67 +/− 2.9
53.52 +/− 4.63
48.45 +/− 1.79

154572
172144
0007_39-cg 1383
21.71 +/− 6.13
15.03 +/− 2.39
39.85 +/− 4.03
40.41 +/− 2.88

154591
172152
0007_39-cg 1383
17.14 +/− 5.9
12.96 +/− 1.6
50.61 +/− 3.77
52.72 +/− 2.16

154599
172168
0007_39-cg1383
20.27 +/− 4.57
15.68 +/− 3.72
50.86 +/− 3
51.08 +/− 1.63

155481
174249
0007_39-cg1383
16.87 +/− 6.35
14.66 +/− 13.4
55.17 +/− 4.66
56.8 +/− 4.07

162769
175204
0007_39-cg1383
9.47 +/− 2.63
9.35 +/− 0.75
51.09 +/− 4.23
57.99 +/− 1.77

155479
175787
0007_39-cg1383
25.76 +/− 13.89
14.66 +/− 7.05
54.62 +/− 4.15
55.9 +/− 2.03

158120
175821
0007_39-cg1383
24.09 +/− 11.01
14.42 +/− 6.62
55.15 +/− 5.86
53.72 +/− 1.55

155470
176476
0007_39-cg1383
19.35 +/− 8.82
25.29 +/− 6.91
54.58 +/− 4.66
59.02 +/− 3.23

155475
176484
0007_39-cg 1383
8.82 +/− 10.28
15.14 +/− 7.35
57.55 +/− 4.53
55.73 +/− 1.1

155475
176485
0007_39-cg 1383
8.82 +/− 10.28
13.75 +/− 8.3
57.55 +/− 4.53
55.32 +/− 1.25

163159
176545
0007_39-cg 1383
11.22 +/− 9.89
8.07 +/− 6.14
56.13 +/− 2.65
57.41 +/− 1.5

163159
176546
0007_39-cg1383
11.22 +/− 9.89
7.11 +/− 4.97
56.13 +/− 2.65
57.38 +/− 1.61

174232
180360
0007_39-cg1383
15.11 +/− 8.67
0.76 +/− 6.4
55.05 +/− 5.29
57.54 +/− 3.5

174547
183641
0007_39-cg1383
12.71 +/− 10.45
27.95 +/− 4.06
56.78 +/− 4.55
58.42 +/− 1.32

182550
193331
0007_39-cg 1383
20.24 +/− 5.46
24.15 +/− 2.8
57.48 +/− 3.8
58.09 +/− 1.39

193318
210851
0007_39-cg 1383
24 +/− 6.35
20.06 +/− 2.12
61.14 +/− 3.26
62.29 +/− 3.93

71075
101738
0007_39-cg3210
23.93 +/− 5.69
18.26 +/− 0.81
51.37 +/− 5.54
55.17 +/− 2.13

121645
139570
0007_39-cg3210
20.22 +/− 4.62
21.73 +/− 2.3
49.84 +/− 4.51
47.31 +/− 1.93

151834
174360
0007_39-cg3210
5.33 +/− 9.59
15.7 +/− 5.87
57.98 +/− 4.13
59.32 +/− 5.04

212960
279235
0007_39-cg3210
19.01 +/− 7.66
8.94 +/− 4.87
65.29 +/− 2.97
66.82 +/− 2.72

242504
279255
0007_39-cg3210
8.19 +/− 8.46
18.38 +/− 6.79
46.34 +/− 21.04
68.67 +/− 2.51

224392
279327
0007_39-cg3210
17.04 +/− 7.51
15.54 +/− 9.31
62.15 +/− 4.39
63.25 +/− 2.23

212953
279352
0007_39-cg3210
22.18 +/− 7.38
16.91 +/− 9.64
66.66 +/− 4.4
66.6 +/− 1.73

176509
182623
0007_39-cg3210
23.21 +/− 11.77
23.86 +/− 4.55
62.92 +/− 4.61
58.63 +/− 5.35

148936
151762
0007_39-phoU
20.42 +/− 10.42
26.53 +/− 2.19
44.38 +/− 7.76
56.79 +/− 3.2

148409
151773
0007_39-phoU
11.55 +/− 4.46
10.75 +/− 1.81
56.02 +/− 2.26
59.85 +/− 1.48

148401
151832
0007_39-phoU
19.21 +/− 5.8
14.31 +/− 10.23
52.95 +/− 4.06
56.79 +/− 1.82

149046
151913
0007_39-phoU
18.43 +/− 5.34
18.56 +/− 4.04
52.81 +/− 5.31
48.29 +/− 3.71

148930
151929
0007_39-phoU
23.07 +/− 3.68
16.8 +/− 3.51
50.11 +/− 3.78
47.53 +/− 2.28

138706
151969
0007_39-phoU
26.08 +/− 4.76
23.62 +/− 3.26
50.84 +/− 4.61
48.12 +/− 1.63

149004
151978
0007_39-phoU
14.69 +/− 3.98
12.23 +/− 2.18
50.89 +/− 2.53
47.9 +/− 0.94

132590
152448
0007_39-phoU
26.43 +/− 7.04
25.78 +/− 5.19
43.76 +/− 5.2
34.64 +/− 11.84

108568
152464
0007_39-phoU
26.79 +/− 7.43
21.13 +/− 4.3
52.58 +/− 5.38
49.87 +/− 3.36

102381
152480
0007_39-phoU
26.82 +/− 8.13
21.83 +/− 3
49.78 +/− 3.59
46.3 +/− 2.46

134595
152496
0007_39-phoU
10.48 +/− 3.33
8.37 +/− 6.33
50.76 +/− 2.94
53.53 +/− 2.61

151790
152592
0007_39-phoU
6.73 +/− 3.54
6.04 +/− 2.46
38.81 +/− 6.33
39.33 +/− 5.41

151772
152616
0007_39-phoU
15.86 +/− 5.08
12.28 +/− 4.92
54.33 +/− 2.55
55.25 +/− 2.57

151778
152628
0007_39-phoU
17.31 +/− 1.59
16.09 +/− 1.57
47.45 +/− 2.14
49.53 +/− 2.67

151605
152636
0007_39-phoU
18.94 +/− 5.76
19.7 +/− 4.58
54.45 +/− 3
55.89 +/− 2.5

151834
152648
0007_39-phoU
5.33 +/− 9.59
23 +/− 3.03
57.98 +/− 4.13
52.13 +/− 1.52

151739
152672
0007_39-phoU
26.26 +/− 4.46
22.62 +/− 6.35
45.11 +/− 2.72
53.52 +/− 4.63

151920
155490
0007_39-phoU
15.93 +/− 5.26
17.69 +/− 4.34
55.57 +/− 5.07
48.22 +/− 2.57

151920
155510
0007_39-phoU
15.93 +/− 5.26
17.55 +/− 2.09
55.57 +/− 5.07
47.51 +/− 2.64

151904
155525
0007_39-phoU
19.02 +/− 3.73
18.57 +/− 1.27
50.01 +/− 4.06
50.17 +/− 2.74

151965
155545
0007_39-phoU
18.24 +/− 6.28
21.53 +/− 5.21
54.03 +/− 5.93
54.69 +/− 3.12

151863
162721
0007_39-phoU
17.14 +/− 4.77
22.19 +/− 3.3
55.32 +/− 4.32
0 +/− 0

151906
162743
0007_39-phoU
19.77 +/− 3.91
21.93 +/− 1.14
54.38 +/− 4.77
49.46 +/− 1.14

152645
162789
0007_39-phoU
25.2 +/− 7.97
16.31 +/− 1.1
51.26 +/− 2.62
46.29 +/− 2.06

152645
163154
0007_39-phoU
25.2 +/− 7.97
0 +/− 0
51.26 +/− 2.62
48.62 +/− 1.16

154572
172143
0007_39-phoU
21.71 +/− 6.13
17.81 +/− 2.22
39.85 +/− 4.03
42.4 +/− 2.67

154591
172151
0007_39-phoU
17.14 +/− 5.9
14.07 +/− 1.89
50.61 +/− 3.77
50.45 +/− 2.24

154650
172199
0007_39-phoU
26.32 +/− 3.56
0 +/− 0
50.5 +/− 2.27
50.32 +/− 1.7

154660
172228
0007_39-phoU
22.3 +/− 7.62
23.55 +/− 1.24
60.29 +/− 4.52
53.48 +/− 3.04

155464
174199
0007_39-phoU
16.27 +/− 5.92
0 +/− 0
50.22 +/− 2.48
52.48 +/− 1.7

155475
174232
0007_39-phoU
8.82 +/− 10.28
15.11 +/− 8.67
57.55 +/− 4.53
55.05 +/− 5.29

155479
174240
0007_39-phoU
25.76 +/− 13.89
0 +/− 0
54.62 +/− 4.15
53.38 +/− 2.96

155481
174248
0007_39-phoU
16.87 +/− 6.35
0 +/− 0
55.17 +/− 4.66
53.97 +/− 1.38

155554
174264
0007_39-phoU
23.09 +/− 7.12
17.48 +/− 3.64
54.58 +/− 4.71
52.23 +/− 1.66

159431
174281
0007_39-phoU
22.48 +/− 6.07
19.72 +/− 1.98
51.6 +/− 3.61
47.51 +/− 2.1

155470
175029
0007_39-phoU
19.35 +/− 8.82
12.01 +/− 6.57
54.58 +/− 4.66
50.94 +/− 3.23

155475
175046
0007_39-phoU
8.82 +/− 10.28

0 +/−0
57.55 +/− 4.53
51.7 +/− 2.26

155479
175062
0007_39-phoU
25.76 +/− 13.89

0 +/−0
54.62 +/− 4.15
53.76 +/− 1.99

155554
175086
0007_39-phoU
23.09 +/− 7.12
15.63 +/− 2.23
54.58 +/− 4.71
52.66 +/− 1.56

159431
175102
0007_39-phoU
22.48 +/− 6.07
17.03 +/− 2.67
51.6 +/− 3.61
50.82 +/− 1.13

161403
175165
0007_39-phoU
19.67 +/− 3.35
21.39 +/− 3.92
52.14 +/− 2.96
57.3 +/− 2.23

162718
175181
0007_39-phoU
20.15 +/− 3.26
17.88 +/− 0.5
55.8 +/− 3.85
58.37 +/− 1.64

162753
175183
0007_39-phoU
14.14 +/− 3.34
16.72 +/− 3.58
52.11 +/− 5.03
58.46 +/− 1.78

162753
175184
0007_39-phoU
14.14 +/− 3.34
17.1 +/− 3.23
52.11 +/− 5.03
55.09 +/− 1.33

162769
175200
0007_39-phoU
9.47 +/− 2.63
12.16 +/− 1.03
51.09 +/− 4.23
57.07 +/− 1.7

158120
175816
0007_39-phoU
24.09 +/− 11.01
13.14 +/− 7.43
55.15 +/− 5.86
52.89 +/− 1.62

155554
176509
0007_39-phoU
23.09 +/− 7.12
23.21 +/− 11.77
54.58 +/− 4.71
62.92 +/− 4.61

163159
176542
0007_39-phoU
11.22 +/− 9.89
10.93 +/− 9.9
56.13 +/− 2.65
59.53 +/− 1.93

174275
183629
0007_39-phoU
18.06 +/− 5.8
18.36 +/− 1.97
58.89 +/− 4.31
59.27 +/− 5.68

174547
186920
0007_39-phoU
12.71 +/− 10.45
18.02 +/− 6.25
56.78 +/− 4.55
54.57 +/− 2.8

182566
187910
0007_39-phoU
19.83 +/− 5.4
10.81 +/− 2.17
57.61 +/− 3.73
64.1 +/− 2.74

182574
187918
0007_39-phoU
21.54 +/− 6.77
15.3 +/− 3.93
60.49 +/− 2.8
60.85 +/− 2.78

182591
187926
0007_39-phoU
16.86 +/− 6.38
14.86 +/− 4.26
56.96 +/− 2.37
58.1 +/− 2.44

182601
187934
0007_39-phoU
17.64 +/− 6.7
6.77 +/− 4.44
59.8 +/− 2.57
61.9 +/− 1.68

182617
187942
0007_39-phoU
9.03 +/− 3.72
7.02 +/− 1.84
66.83 +/− 3.8
65.52 +/− 2.46

182550
187990
0007_39-phoU
20.24 +/− 5.46
10.05 +/− 1.68
57.48 +/− 3.8
65.34 +/− 1.65

175776
188149
0007_39-phoU
30.83 +/− 10.18
20.56 +/− 2.06
58.82 +/− 4.55
65.39 +/− 1.41

180390
188159
0007_39-phoU
26.07 +/− 11.03
19.93 +/− 2.96
57.26 +/− 2.63
57.94 +/− 1.42

183661
190101
0007_39-phoU
25 +/− 6.72
17.15 +/− 5.25
55.74 +/− 3.95
58.42 +/− 3.41

175776
193273
0007_39-phoU
30.83 +/− 10.18
19.93 +/− 3.32
58.82 +/− 4.55
61.28 +/− 4.94

182566
193289
0007_39-phoU
19.83 +/− 5.4
25.28 +/− 3.47
57.61 +/− 3.73
56.86 +/− 3.79

182566
193293
0007_39-phoU
19.83 +/− 5.4
22.06 +/− 3.24
57.61 +/− 3.73
59.08 +/− 4.96

183175
196697
0007_39-phoU
25.54 +/− 6.32
25.38 +/− 2.97
59.81 +/− 3.17
58.08 +/− 1.95

187911
197877
0007_39-phoU
23.32 +/− 7.65
23.58 +/− 2.87
59.21 +/− 2.88
60.42 +/− 1.91

187929
197885
0007_39-phoU
6.36 +/− 5.48
4.56 +/− 4.95
60.03 +/− 3.8
63.01 +/− 3.09

191496
197954
0007_39-phoU
23.88 +/− 6.7
24.32 +/− 2.9
56.21 +/− 3.28
55.7 +/− 2.66

188141
201612
0007_39-phoU
20.98 +/− 6.13
27.79 +/− 8.07
57.51 +/− 4.3
56.12 +/− 1.65

191500
201628
0007_39-phoU
27.42 +/− 5.91
19.4 +/− 4.61
52.16 +/− 3.99
51.01 +/− 2.82

191504
201644
0007_39-phoU
17.11 +/− 5.12
7.49 +/− 1.61
58.22 +/− 4.58
61.16 +/− 2.28

191520
201660
0007_39-phoU
14.26 +/− 5.09
14.07 +/− 4.87
64.17 +/− 3.8
62.95 +/− 2.53

190123
201676
0007_39-phoU
13.81 +/− 3.49
9.42 +/− 7.47
63.37 +/− 3
62.34 +/− 1.92

191520
206820
0007_39-phoU
14.26 +/− 5.09
13.92 +/− 1.51
64.17 +/− 3.8
62.34 +/− 3.9

191500
206860
0007_39-phoU
27.42 +/− 5.91
28.84 +/− 2.6
52.16 +/− 3.99
53.4 +/− 1.89

199384
212930
0007_39-phoU
15.95 +/− 5.44
12.93 +/− 0.97
64.3 +/− 3.56
63.47 +/− 1.84

190061
212954
0007_39-phoU
18.17 +/− 5.41
17.23 +/− 2.08
63.53 +/− 3.46
61.59 +/− 2.24

TABLE 7A

Abbreviation
Description

4GoB
insertion of lysine biosynthesis genes ask, asd, dapB, and ddh in the bioD

region (Example 1)

4GR
insertion of lysine biosynthesis genes ask, asd, dapB, and ddh in a FarI

homology region (Example 1)

0007_39-zwf
insertion of promoter Pcg0007_39 in front of the zwf open reading frame

DSS_084
replacement of native maJ coding sequence with maJG448S allele

1860-pyc
replacement of native pyc promoter with promoter Pcg1860

DSS_033
replacement of native accDA coding sequence with accDAG310E allele

DSS_316
replacement of native cg1144 coding sequence with cg1144P66S allele

aceE_ATG > TTG
replacement of aceE start codon with TTG

lisa68
insertion of codon-optimized lysA coding sequence under the control of

promoter Pcg0007_39 and including sod terminator (SEQ ID NO: 17)

0007_39_opca_P4
replacement of native opcA coding sequence with opcA coding sequence

for C. humireducens under the control of promoter Pcg0007_39

0007_39-actA
replacement of native actA promoter with promoter Pcg0007_39

0007_39-cg1383
insertion of promoter Pcg0007_39 in front of cg1383 start codon

0007_39-cg2766
insertion of promoter Pcg0007_39 in front of cg2766 start codon

0007_39-phoU
replacement of native phoU promoter with promoter Pcg0007_39

0007_39-cg3210
replacement of native cg3210 promoter with promoter Pcg0007_39

0007_39-cg0800
replacement of native cg0800 promoter with promoter Pcg0007_39

Vhb04
nitric oxide dioxygenase [Fischerella thermalis]

WP_009454354.1 under the control of Pcg0007_39

fumC_atg > gtg
replacement of fumC start codon with GTG

0007_39-lysA
insertion of codon-optimized lysA sequence under the control of promoter

Pcg0007_39 and having a sod terminator

0007_39-CAA
insertion of codon-optimized glcp sequence (accession CAA34119.1) under

the control of promoter Pcg0007_39 and codon optimized ppgK sequence

(accession cg2091) under the control of Pcg1860

1860-xerD
replacement of native xerD promoter with promoter Pcg1860

0007_39-lpd
replacement of native lpd promoter with promoter Pcg0007_39

1860-aceF
replacement of native aceF promoter by promoter Pcg1860

0007_39-cg0725
replacement of native cg0725 promoter with promoter Pcg0007_39

DSS_121
replacement of native cg3092 coding sequence with cg3092D213N allele

DSS_422
replacement of native cg3180 coding sequence with cg3180W297* allele

0007_39-SCO
insertion of codon-optimized SCO5578 sequence (accession NP_629713.1)

under the control of promoter Pcg0007_39 and codon optimized ppgK

sequence (accession cg2091) under the control of Pcg1860

1860-cg1768
replacement of native cg1768 promoter by promoter Pcg1860

0007_39-rpoB
replacement of native rpoB promoter with promoter Pcg0007_39

0007_39-rpoB
same as 0007_39-rpoB

gene-383

0007_39BL1631
insertion of codon-optimized BL1631 sequence (accession NP_696783.1)

under the control of promoter Pcg0007_39 and codon optimized ppgK

sequence (accession cg2091) under the control of Pcg1860

0007_39-glf
replacement of native glf promoter with promoter Pcg0007_39

glk
insertion of codon-optimized glf sequence (accession AAA27691.1) under

the control of promoter Pcg0007_39 and codon optimized glk sequence

(accession AHJ72364.1) under the control of Pcg1860 (at Ni487)

glk_2966
same construct as above integrated at different insertion site (Ni2966)

0007_39-NCgl1262
replacement of native NCgl1262 promoter with promoter Pcg0007_39

Pcg0007-amtB
replacement of native amtB promoter with Pcg0007

Pcg0007_39-cg800
replacement of native cg0800 promoter with Pcg0007_39

3121-mutM2
replacement of native mutM promoter with promoter Pcg3121

1860-cg1144
replacement of native cg1144 promoter with promoter Pcg1860

3121-gltD
replacement of native gltD promoter with promoter Pcg3121

SCO_Ni2966
same construct as 0007_39-SCO, integrated at Ni2966

1860-tkt
replacement of native tkt promoter with promoter Pcg1860

3381-cg1410
replacement of native cg1410 promoter by promoter Pcg3381 (WO

2017/100376)

Pcg3121-prpB2
replacement of the native promoter of cg0760 with Pcg0760

0007_39-nusG
replacement of native nusG promoter with promoter Pcg0007_39

0007_39-aroP
replacement of native aroP promoter with promoter Pcg0007_39

0007_39-rho
replacement of native rho promoter with promoter Pcg0007_39

0007_39-cg1503
replacement of native cg1503 promoter with promoter Pcg0007_39

0007-39_ureR
replacement of native ureR promoter with promoter Pcg0007_39

0007_39-kgd
replacement of native kgd promoter with promoter Pcg0007_39

0007_39-cg2466
replacement of native cg2466 promoter with promoter Pcg0007_39

0007_39-cg2899
replacement of native cg2899 promoter with promoter Pcg0007_39

3121-cg1081
replacement of native cg1081 promoter with promoter Pcg3121

0007_39-cg1486
replacement of native cg1486 promoter with promoter Pcg0007_39

0007_39-Ncgl0304
replacement of native Ncg10304 promoter with promoter Pcg0007_39

0007_39-Ncgl0304
replacement of native Ncgl0304 promoter with promoter Pcg0007_39

0007_39-ncgl1511
replacement of native Ncgl1511 promoter with promoter Pcg0007_39

0007_39-Ncgl0767
replacement of native Ncgl0767 promoter with promoter Pcg0007_39

0755-dapA
replacement of native dapA promoter with promoter Pcg0755

0007_39-glpX
replacement of native glpX promoter with promoter Pcg0007_39

TABLE 7B

Genotypes of Modified Strains

Modified

Strain
Changes with respect to genome of NRRL B-11474

88274
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; fumC_atg > gtg; 0007_39-opca_P4; 0007_39-

cg0800

139804
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-opcA_P4;

0007_39-cg0800

174547
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg0800

182533
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg0725; 0007-_39-rho; 0007_39-cg0800

182550
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800

182557
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800

182566
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-cg0800

182574
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-kgd; 0007_39-cg0800

182591
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; Pcg0007-amtB; 0007_39-kgd; 0007_39-cg0800

182598
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; Pcg0007-amtB; 0007_39-kgd; 0007_39-cg0800

182601
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 3121-cg1081; 0007_39-cg0800

182608
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 3121-cg1081; 0007_39-cg0800

182609
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg0800

182616
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg0800

182617
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU;0007_39-cg0800

182624
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-cg0800

183652
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg800; Pcg0007_39-cg800

183691
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-phoU; 0007_39-kgd; 0007_39-cg0800

183700
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 1860-cg1768; glk; 0007_39-cg0725; 0007_39-cg2766; 0007_39-kgd; 0007_39-

cg0800

183708
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-glf; 0007_39-cg1383; 0007_39-aroP; 0007_39-kgd; 0007_39-cg0800

183715
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 0007_39-cg2466; 0007_39-cg0800

183716
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 0007_39-cg2466; 0007_39-cg0800

187964
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-nusG; 0007_39-

cg0800

187972
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; Pcg0007-amtB; 0007_39-kgd; 0007_39-cg1503; 0007_39-

cg0800

187984
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; Pcg0007-amtB; 0007_39-kgd; 1860-xerD; 0007_39-cg0800

188102
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-mutM2; 0007_39-aroP; 0007_39-ureR; 0007_39-

cg0800

188104
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-mutM2; 0007_39-aroP; 0007_39-ureR; 0007_39-

cg0800

188154
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-kgd; 0007_39-

cg0800

188162
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg0800

188169
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg0800

188170
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; lisa_68;

0007_39-rpoB; 0007_39-cg2766; 0007_39-aroP; 0007_39-cg0800

188178
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg2899;

0007_39-cg0800

188186
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-cg2899; 0007_39-kgd; 0007_39-

cg0800

190037
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-cg2899;

0007_39-cg0800

190045
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-ureR; 0007_39-

cg0800

190053
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg1486; 0007_39-cg0800

190061
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU; 0007_39-cg1486;

0007_39-cg0800

190069
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU; 0007_39-cg3210;

0007_39-cg0800

191527
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2960007_39-NCgl1262; 0007_39-cg0800

192419
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg1383; 0007_39-rpob; 0007_39-cg0800

193279
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg0800

193295
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg1486; 0007_39-cg0800

197907
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU; 0007_39-cg1486;

0007_39-Ncgl0304; 0007_39-cg0800

206822
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800;

0007_39-NCgl1262; 0007_39-cg0800

213002
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg1486; 1860-xerD; 0007_39-cg0800

213012
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg1486; 1860-xerD; 0007_39-cg0800

219284
0007_39-cg0800

219308
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; 0007_39-

cg0800

87642
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; fumC_atg > gtg; 0007_39-opca_P4; 0007_39-

cg1383

121624
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; fumC_ATG > TTG; opcA_P_4; Vhb04; 0007_39-

cg1383

121638
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

0007_39-cg1383

121643
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; fumC_ATG > GTG; opcA_P_4; 1860-aceF;

0007_39-lysA; 0007-39_BL1631; 0007_39-cg1383

132568
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg1383

134579
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg1383

134603
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; 0007_39-

cg1383

144042
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa_68; DSS_121;

0007_39-cg1383

144051
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa_68; DSS_422;

0007_39-cg1383

148418
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; fumC_ATG > GTG; opcA_P4; 1860-aceF;

0007_39-CAA; 0007_39-lysA; 0007_39-cg1383

148431
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; fumC_ATG > GTG; opcA_P4; 1860-aceF;

0007_39-lysA; 0007_39-SCO; 0007_39-cg1383

148999
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-rpoB; 0007_39-cg1383

149034
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

0007_39-CAA; 0007_39-cg1383

149042
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; lisa_68;

0007_39-cg1383

149058
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 1860-xerD; 0007_39-

cg1383

151595
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-cg1383

151611
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-cg1383

151753
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-glf; 0007_39-cg1383

151764
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-cg1383

151804
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; 0007_39-

cg2766; 0007_39-cg1383

151840
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; 0007_39-lysA;

0007_39-SCO; opcA_P_4; 0007_39-cg1383

151904
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; lisa_68;

0007_39-rpoB-gene-383; 0007_39-cg1383

151914
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; lisa_68;

0007_39-rpoB-gene-383; 0007_39-cg1383

151920
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg2766;

opcA_P4; 0007_39-cg1383

151965
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383

152452
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; DSS_121;

0007_39-cg1383

152468
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 1860-xerD; 0007_39-

cg1383

152500
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; 0007_39-lysA;

0007_39-SCO; 0007_39-cg1383

154589
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 1860-cg1768; glk; 0007_39-cg0725; 0007_39-cg1383

154600
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 1860-cg1768; glk; 1860-cg1144; 0007_39-cg1383

155462
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg1383

155466
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg1383

155469
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg1383

162735
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; lisa_68;

0007_39-rpoB-gene-383; 0007_39-cg2766; 0007_39-cg1383

163111
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-cg1383

163167
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; 0007_39-lysA;

0007_39-SCO; 0007_39-cg2766; opcA_P4; 0007_39-phoU; 0007_39-cg1383

172144
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; 0007_39-lysA;

0007_39-SCO; opcA_P_4; 0007_39-cg0725; 0007_39-aroP; 0007_39-cg1383

172152
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 1860-cg1768; glk; 0007_39-cg0725; 0007_39-cg2899; 0007_39-cg1383

172168
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 1860-cg1768; glk; 1860-cg1144; 0007_39-cg2766; 0007_39-cg1383

174249
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-mutM2; 0007_39-cg1383

175204
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg0725;

opcA_P4; lisa68; 0007_39-nusG; 1860-xerD; 0007_39-cg1383

175787
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 1860-tkt; 0007_39-cg1383

175821
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3381-cg1410; 0007_39-cg1383

176476
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007-_39-rho; 0007_39-cg1383

176484
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 0007_39-cg1383

176485
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 0007_39-cg1383

176545
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-gltD; 0007_39-cg1503; 0007_39-cg1383

176546
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-gltD; 0007_39-cg1503; 0007_39-cg1383

180360
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 0007_39-phoU; 0007_39-cg1383

183641
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg800; 0007_39-cg1383

193331
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800;

0007_39-cg1383

210851
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU; 0007_39-rpoB; 0007_39-kgd;

0007_39-cg1486; 1860-xerD; 0007_39-cg1383

101738
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg3210

139570
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-opca_P4;

9: 0007_39-cg3210

174360
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA@ni487; 0007_39-

opca_P4; 0007_39-acta; 0007_39-cg2766; 0007_39-cg3210

279235
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU; 0007_39-cg3210;

0007_39-cg0800; 1860-cg1768; 0007_39-cg3210

279255
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-0800; 0007_39-

cg1486; 1860-NCgl1262; 0007_39-cg3210

279327
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-0800; 0007_39-

cg1486; 0007_39-cg3210

279352
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU; 0007_39-0800; 0007_39-

cg1486; 0007_39-cg3210

182623
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU; 0007_39-cg3210

151762
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-glf; 0007_39-phoU

151773
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-phoU

151832
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-phoU

151913
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; lisa_68;

0007_39-rpoB-gene-383; 0007_39-phoU

151929
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg2766;

opcA_P4; 0007_39-phoU

151969
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-phoU

151978
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-rpoB; lisa_68; 0007_39-phoU

152448
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; 0007_39-lysA;

0007_39-SCO; 0007_39-phoU

152464
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; DSS_121;

0007_39-phoU

152480
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 1860-xerD; 0007_39-

phoU

152496
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-rpoB; 0007_39-phoU

152592
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg0725;

opcA_P4; lisa68; 0007_39-phoU

152616
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; 0007_39-actA; 0007_39-phoU

152628
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-cg2766; glk; 0007_39-phoU

152636
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; glk_2966; 0007_39-phoU

152648
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-phoU

152672
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; 0007_39-lysA;

0007_39-SCO; 0007_39-cg2766; opcA_P4; 0007_39-phoU

155490
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg2766;

opcA_P4; 0007_39-cg1383; 0007_39-phoU

155510
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg2766;

opcA_P4; 0007_39-cg1383; 0007_39-phoU

155525
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; lisa_68;

0007_39-rpoB-gene-383; 0007_39-cg1383; 0007_39-phoU

155545
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-phoU

162721
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 1860-cg1768; glk; 0007_39-cg0725; 0007_39-phoU

162743
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; opcA_P4; lisa_68;

0007_39-rpoB-gene-383; 0007_39-cg2766; 0007_39-phoU

162789
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-gltD; 0007_39-phoU

163154
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-gltD; 0007_39-phoU

172143
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; 0007_39-lysA;

0007_39-SCO; opcA_P_4; 0007_39-cg0725; 0007_39-aroP; 0007_39-phoU

172151
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 1860-cg1768; glk; 0007_39-cg0725; 0007_39-cg2899; 0007_39-phoU

172199
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; SCO_Ni2966; 0007_39-phoU

172228
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-glf; 0007_39-cg1383; 0007_39-aroP; 0007_39-phoU

174199
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg0725; 0007_39-phoU

174232
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 0007_39-phoU

174240
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 1860-tkt; 0007_39-phoU

174248
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-mutM2; 0007_39-phoU

174264
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU

174281
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; Pcg0007-amtB; 0007_39-phoU

175029
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007-_39-rho; 0007_39-phoU

175046
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 0007_39-phoU

175062
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 1860-tkt; 0007_39-phoU

175086
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU

175102
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; Pcg0007-amtB; 0007_39-phoU

175165
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 3121-prpB28; 0007_39-phoU

175181
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 1860-cg1768; glk; 0007_39-cg0725; 0007_39-cg2766; 0007_39-phoU

175183
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg0725;

opcA_P4; lisa68; 0007_39-actA; 0007_39-nusG; 0007_39-phoU

175184
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg0725;

opcA_P4; lisa68; 0007_39-actA; 0007_39-nusG; 0007_39-phoU

175200
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-cg0725;

opcA_P4; lisa68; 0007_39-nusG; 1860-xerD; 0007_39-phoU

175816
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3381-cg1410; 0007_39-phoU

176509
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU

176542
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 3121-gltD; 0007_39-cg1503; 0007_39-phoU

183629
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-phoU

186920
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg0800; 0007_39-phoU

187910
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-cg0800;

0007_39-phoU

187918
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-kgd; 0007_39-cg0800; 0007_39-

phoU

187926
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; Pcg0007-amtB; 0007_39-kgd; 0007_39-cg0800; 0007_39-

phoU

187934
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 3121-cg1081; 0007_39-cg0800; 0007_39-phoU

187942
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU;0007_39-cg0800;

0007_39-phoU

187990
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800;

0007_39-phoU

188149
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-glf; 0007_39-cg1383; 0007_39-aroP; 0007_39-kgd; 0007_39-phoU

188159
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-kgd; 0007_39-

phoU

190101
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; 0007_39-cg800; 0007_39-cg2899; 0007_39-phoU

193273
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; 0007_39-lysA; 0007_39-lpd;

opcA_P4; 0007_39-glf; 0007_39-cg1383; 0007_39-aroP; 0007_39-kgd; 0007_39-phoU

193289
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-cg0800;

0007_39-phoU

193293
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-cg0800;

0007_39-phoU

196697
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-phoU

197877
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-cg0800;

0007_39-cg1486; 0007_39-phoU

197885
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-cg2766; 0007_39-actA; glk_2966; 3121-cg1081; 0007_39-cg0800; 0007_39-

ncgl1511; 0007_39-phoU

197954
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800;

0007_39-Ncgl0304; 0007_39-phoU

201612
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800;

0007_39-cg1486; 0007_39-phoU

201628
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800; 0755-

dapA; 0007_39-phoU

201644
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800;

0007_39-Ncgl0767; 0007_39-phoU

201660
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800;

0007_39-NCgl1262; 0007_39-phoU

201676
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-cg0800;

0007_39-NCgl1262; 0007_39-phoU

206820
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800;

0007_39-NCgl1262; 0007_39-phoU

206860
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-ureR; 0007_39-cg0800; 0755-

dapA; 0007_39-phoU

212930
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-rpoB; 0007_39-cg0800;

0007_39-cg1486; 0007_39-glpX; 0007_39-phoU

212954
0007_39-zwf; DSS_084; 1860-pyc; DSS_033; DSS_316; aceE_ATG > TTG; lisa68; opcA_P4;

0007_39-actA; 0007_39-cg1383; 0007_39-cg2766; 0007_39-phoU; 0007_39-cg1486;

0007_39-cg0800; 0007_39-phoU

TABLE 8

Sequence Listing

SEQ ID NO:
sequence

1

C. glutamicum NRRL B-11474, RnaJ homology region

2
coding sequence for altered RnaJ protein

3
altered RnaJ protein

4

C. glutamicum NRRL B-11474, accDA homology region

5
coding sequence for altered accDA protein

6
altered accDA protein

7

C. glutamicum NRRL B-11474, cg1144 homology region

8
coding sequence for altered cg1144 protein

9
altered cg1144 protein

10

C. glutamicum NRRL B-11474, pyc homology region

11
promoter 1860

12
promoter 1860 and pyc coding sequence

13

C. glutamicum NRRL B-11474, aceE homology region

14
altered aceE coding sequence

15

C. glutamicum NRRL B-11474, lysA insertion homology region

16
codon-optimized lysA coding sequence

17
promoter cg0007_39-codon-optimized lysA coding sequence-sod

terminator

18

C. glutamicum NRRL B-11474, lysA

19

C. glutamicum NRRL B-11474 zwf homology region

20
Promoter cg0007_39

21
Promoter cg0007_39 insertion and zwf coding region

22

C. glutamicum NRRL B-11474, opcA homology region

23

C. humireducens opcA coding sequence

24
promoter cg0007_39-C. humireducense opcA coding sequence

25

C. humireducens opcA protein

26

C. glutamicum NRRL B-11474, cg2766 homology region

27
Promoter cg0007_39-cg2766

28

C. glutamicum NRRL B-11474, actA homology region

29
promoter cg0007_39-actA

30
primer FarI 3p InfusF

31
primer FarI 3p InfusR AvrII

32
primer FarI 5p InfusF

33
primer FarI 5p Infus R

34
primer Pcg0007_39-zwf_P1

35
primer Pcg0007_39-zwf_P2

36
primer Pcg0007_39-zwf_P3

37
primer Pcg0007_39-zwf_P4

38
primer SNP_084_P1

39
primer SNP_084_P2

40
primer SNP_084_P3

41
primer SNP_084_P4

42
primer Pcg1860_pyc_P1

43
primer Pcg1860_pyc_P2

44
primer Pcg1860_pyc_P3

45
primer Pcg1860_pyc_P4

46
primer SNP_033_P1

47
primer SNP_033_P2

48
primer SNP_033_P3

49
primer SNP_033_P4

50
primer SNP_316_P1

51
primer SNP_316_P2

52
primer SNP_316_P3

53
primer SNP_316_P4

54
primer SCS_aceE_P1

55
primer SCS_aceE_P2

56
primer SCS_aceE_P3

57
primer SCS_aceE_P4

58
primer opcA_P1

59
primer opcA_P2

60
primer opcA_P3

61
primer opcA_P4

62
primer pcg0007_39-cg2766_p1

63
primer pcg0007_39-cg2766_P2

64
primer pcg0007_39-cg2766_P3

65
primer pcg0007_39-cg2766_P4

66
primer pcg0007_39-actA_P1

67
primer pcg0007_39-actA_P2

68
primer pcg0007_39-actA_P3

69
primer pcg0007_39-actA_P4

70
primer 184f7

71
primer 184F8

72
primer 244-4Go F2 Inf SpeI

73
primer 253-4Go R3

74
primer 685

75
primer 686

76
primer 687

77
primer 693

78

C. glutamicum RnaJ protein

79

C. glutamicum RnaJ protein

80

C. ulcerans RnaJ protein

81

C. glutamicum accDA protein

82

C. glutamicum MB001 accDA protein

83

C. crudilactis accDA protein

84

C. glutamicum cg1144 protein

85

C. crudilactis cg1144 protein

86

C. efficiens YS-314cg1144 protein

87

C. vitaeruminis DSM 20294 opcA protein

88

C. matruchotii ATCC 33806 opcA protein

89

C. falsenii DSM 44353 opcA protein

90

C. halotolerans YIM 70093 = DSM 44683 opcA protein

91

C. pyruviciproducens ATCC BAA-1742 opcA protein

92

C. casei UCMA 3821 opcA protein

93

C. terpenotabidum Y-11 opcA protein

94

C. glutamicum NRRL B-11474 opcA protein

95
promoter 3121

96
promoter 0755

97
promoter 3381

98

C. glutamicum cg1383 homology region

99
primer Pcg0007_39-cg1383_P1

100
primer Pcg0007_39-cg1383_P2

101
primer Pcg0007_39-cg1383_P3

102
primer Pcg0007_39-cg1383_P4

103
Pcg0007_39 promoter inserted in front of cg1383 start codon

104

C. glutamicum phoU homology region

105
primer Pcg0007_39-phoU_P1

106
primer Pcg0007_39-phoU_P2

107
primer Pcg0007_39-phoU_P3

108
primer Pcg0007_39-phoU_P4

109
Pcg0007_39 promoter in front of phoU start codon

110

C. glutamicum cg3210 homology region

111
Pcg0007_39-cg3210-p1

112
Pcg0007_39-cg3210-p2

113
Pcg0007_39-cg3210-p3

114
Pcg0007_39-cg3210-p4

115
Pcg0007_39 in front of cg3210

116

C. glutamicum cg0800 homology region

117
Pcg0007_39-cg0800-p1

118
Pcg0007_39-cg0800-p2

119
Pcg0007_39-cg0800-p3

120
Pcg0007_39-cg0800-p4

121
Pcg0007_39 in front of cg0800

REFERENCES

Becker et al., “Systems level engineering of Corynebacterium glutamicum—Reprogramming translational efficiency for superior production,” Engineering in Life Sciences 10, 430-38, 2010

Buchholz et al., “Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of 1-Lysine, 1-Valine, and 2-Ketoisovalerate,” Applied Env. Microbiol. 79, 5566-75, 2013

Knoppová et al., Curr. Microbiol. 55, 234-39, 2007

Lee, J Microbiol Biotechnol. 2014 January; 24(1):70-9

Nešvera et al., Subcell. Biochem. 2012; 64:203-21

Pátek & Nešvera, “Promoters and Plasmid Vectors of Corynebacterium glutamicum,” in Corynebacterium glutamicum, Yukawa & Inui, eds., Springer-Verlag, Berlin 2013, pp. 51-88

Pátek et al., J. Biotechnol. 104, 311-23, 2003(a)

Pátek et al., J. Biotechnol. 104, 325-34, 2003(b)

Pátek et al., Microb. Biotechnol. 2013 March; 6(2):103-17

Pfeifer-Sancar et al., BMC Genomics 14, 888, 2013

Rytter et al., Appl. Microbiol Biotechnol. 2014 March; 98(6):2617-23

Shang et al., Biotechnol. Lett. 2017 Nov. 21. doi: 10.1007/s10529-017-2479-y

Yim et al., Biotechnol. Bioeng. 2013 November; 110(11):2959-69

ENGINEERED STRAINS OF CORYNEBACTERIA

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

PCT Information

Provisional Applications (1)