Method for Producing L-Amino Acids Using an Alkaliphilic Bacteria

A recombinant microorganism, useful in the production of an amino acid, wherein said microorganism has been engineered to overexpress one or more enzymes selected from the group consisting of:

• a) an alanine dehydrogenase comprising an amino acid sequence at least 85% identical to the sequence of SEQ ID NO:72;
• b) an enzyme of the hut cluster, selected from the group consisting of:
• i) a urocanate hydratase (hutU) enzyme, comprising a sequence at least 90% identical to the sequence of SEQ ID NO:192;
• ii) an imidazolonepropionase (hutI) enzyme comprising a sequence at least 90%, identical to the sequence of SEQ ID NO:194;
• iii) a histidine ammonia-lyase (hutH) enzyme comprising a sequence at least 90%, identical to the sequence of SEQ ID NO:196;
• iv) a formimidoylglutamase, characterized in that said enzyme has a sequence identity of at least 90% identical to the sequence of SEQ ID NO:198.

A recombinant microorganism engineered to overexpress an alanine dehydrogenase comprising an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:72, and further comprising one or more enzymes selected from the group consisting of:

• a) a threonine dehydratase (IlvA, EC 4.3.1.19) having a a sequence at least 95% identical to the sequence of SEQ ID NO:106;
• b) a subunit of an acetolactate synthase (IIvB) having a sequence at least 95% identical to SEQ ID NO:98;
• c) an isomer reductase (IlvC, EC 1.1.1.86) having a sequence at least 95% identical to the sequence of SEQ ID NO:100;
• d) a dihydroxyacid dehydratase (IlvD, EC 4.2.1.9) having a sequence at least 95%, identical to the sequence of SEQ ID NO:102;
• e) a transaminase (IlvE, EC 2.6.1.42) having a sequence at least 95%, identical to the sequence of SEQ ID NO:104;
• f) an acetolactate synthase (IlvH, EC 2.2.1.6) having a sequence at least 95% identical to the sequence of SEQ ID NO:122;
• g) a threonine synthase (ThrC, EC 4.2.3.1) having a sequence at least 95% identical to the sequence of SEQ ID NO:108;
• h) an optionally feedback-resistant isopropylmalate synthase (LeuA, EC 2.3.3.13) having a sequence at least 95% identical to the sequence of SEQ ID NO:110;
• i) an isopropylmalate dehydrogenase (LeuB, EC 1.1.1.85) having a sequence at least 95% identical to the sequence of SEQ ID NO:112;
• j) the subunits of an isopropylmalate isomerase (LeuCD, EC 4.2.1.33) having sequences at least 95% identical to the sequences of SEQ ID NO:114 or SEQ ID NO:116;
• k) a 3-methyl-2-oxobutanoate hydroxymethyltransferase (PanB, EC 2.1.2.11) having a sequence at least 95% identical to the sequence of SEQ ID NO:118;
• l) a pantothenate synthase (PanC, EC 6.3.2.1) having a sequence at least 95% identical to the sequence of SEQ ID NO:120;
• m) a glutamate dehydrogenase (Gdh) having a sequence at least 95%, identical to the sequence of SEQ ID NO:124;
• n) a glutamine synthetase (glutamine synthetase 1) having a sequence at least 95% identical to the sequence of SEQ ID NO:126;
• o) a glutamine synthetase (glutamine synthetase 2) having a sequence at least 95% identical to the sequence of SEQ ID NO:128;
• p) a glutamate synthase having a sequence at least 95% identical to the sequence of SEQ ID NO:130;
• q) an isocitrate dehydrogenase having a sequence at least 95% identical to the sequence of:
• SEQ ID NO:132;
• r) an aconitate hydrase having a sequence at least 95% identical to the sequence of SEQ ID NO:134;
• s) a citrate synthase having a sequence at least 95% identical to the sequence of SEQ ID NO:136,
• t) an aminopeptidase C (PepC) having a sequence at least 95% identical to the sequence of SEQ ID NO:138;
• u) a pyruvate dehydrogenase having a sequence at least 95% identical to the sequence of SEQ ID NO:140;
• v) a pyruvate kinase (pyruvate kinase 1) having a sequence at least 95% identical to the sequence of SEQ ID NO:142;
• w) a pyruvate kinase (pyruvate kinase 2) having a sequence at least 95%, identical to the sequence of SEQ ID NO:144;
• x) an enolase having a sequence at least 95% identical to the sequence of SEQ ID NO:146;
• y) a 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (GpmA) having a sequence at least 95% identical to the sequence of SEQ ID NO:148;
• z) a phosphoglycerate kinase (Pgk) having a sequence at least 95%, identical to the sequence of SEQ ID NO:150;
• aa) a glyceraldehyde-3-phosphate dehydrogenase (glycerol-3-phosphate dehydrogenase 1) having a sequence at least 95% identical to the sequence of SEQ ID NO:152;
• bb) a glyceraldehyde-3-phosphate dehydrogenase (glycerol-3-phosphate dehydrogenase 2) having a sequence at least 95% identical to the sequence of SEQ ID NO:154;
• cc) a triosephosphate isomerase (TpiA) having a sequence at least 95% identical to the sequence of SEQ ID NO:156;
• dd) a fructose bisphosphate aldolase having a sequence at least 95%, identical to the sequence of SEQ ID NO:158;
• ee) a 1-phosphofructokinase having a sequence at least 95% identical to the sequence of SEQ ID NO:160;
• ff) a 6-phosphofructokinase having a sequence at least 95% identical to the sequence of SEQ ID NO:162;
• gg) subunits of a succinyl-CoA ligase (SucCD, EC 6.2.1.5) coded for by attenuated polynucleotides (sucCD);
• hh) a DNA binding domain of type HTH tetR (McbR) having a sequence at least 95% identical to the sequence of SEQ ID NO:2;
• ii) a homoserine kinase (ThrB, EC 2.7.1.39) having a sequence at least 95%, identical to the sequence of SEQ ID NO:4;
• jj) a glucose-6-phosphate isomerase (Pgi, EC 5.3.1.9) having a sequence at last 95% identical to the sequence of SEQ ID NO:6;
• kk) a phosphoenolpyruvate carboxykinase (Pck, EC 4.1.1.32) having a sequence at least 95% identical to the sequence of SEQ ID NO:8;
• ll) a D-methionine-binding lipoprotein (MetQ) having a sequence at least 95% identical to the sequence of SEQ ID NO:10;
• mm) a methionine transporter (MetP) having a sequence at least 95%, identical to the sequence of SEQ ID NO:12;
• nn) an ATP-dependent methionine transporter (MetN) having a sequence at least 95%, identical to the sequence of SEQ ID NO:14;
• oo) an S-adenosylmethionine synthase (MetK) having a sequence at least 95% identical to the sequence of SEQ ID NO:16;
• pp) a methionine import system permease (MetI) having a sequence at least 95% identical to the sequence of SEQ ID NO:18;
• qq) a 4-hydroxy-tetrahydrodipicolinate synthase (DapA, EC 4.3.3.7) having a sequence at least 95% identical to the sequence of SEQ ID NO:20;
• rr) a cysteine synthase (CBS, CysK) having a sequence at least 95% identical to the sequence of SEQ ID NO:22;
• ss) a carboxylate-amine ligase having a sequence at least 95% identical to the sequence of SEQ ID NO:24;
• tt) a cystathionine beta-lyase (AecD) having a sequence at least 95%, identical to the sequence of SEQ ID NO:26;
• uu) an aspartate semialdehyde dehydrogenase (Asd, EC 1.2.1.11) having a sequence at least 95% identical to the sequence of SEQ ID NO:28;
• vv) a 5-methyltetrahydrofolate homocysteine methyltransferase (MetH, EC 2.1.1.13) coded for by an overexpressed polynucleotide (metH);
• ww) the smaller subunit of a transporter for branched-chain amino acids (BrnE) having a sequence at least 95% identical to the sequence of SEQ ID NO:30;
• xx) the larger subunit of a transporter for branched-chain amino acids (BrnF) having a sequence at least 95% identical to the sequence of SEQ ID NO:32;
• yy) a serine acetyltransferase (CysE) having a sequence at least 95% identical to the sequence of SEQ ID NO:34;
• zz) a cysteine synthase (CysK) having a sequence at least 95% identical to the sequence of SEQ ID NO:36;
• aaa) the H protein of a glycine cleavage system (GcvH) having a sequence at least 95% identical to the sequence of SEQ ID NO:38;
• bbb) the P protein of a glycine cleavage system (GcvP) having a sequence at least 95% identical to the sequence of SEQ ID NO:40;
• ccc) the T protein of a glycine cleavage system (GcvT) having a sequence at least 95% identical to the sequence of SEQ ID NO:4;
• ddd) a serine hydroxymethyltransferase (GlyA) having a sequence at least 95% identical to the sequence of SEQ ID NO:44;
• eee) an optionally feedback-resistant homoserine dehydrogenase (Hom, EC 1.2.1.11) having a sequence at least 95% identical to the sequence of SEQ ID NO:46;
• fff) a lipoyl synthase (LipA) having a sequence at least 95%, identical to the sequence of SEQ ID NO:48;
• ggg) a lipoyl transferase (LipB) having a sequence at least 95%, identical to the sequence of SEQ ID NO:50;
• hhh) a dihydrolipoyl dehyrogenase (Lpd) having a sequence at least 95%, identical to the sequence of SEQ ID NO:52;
• iii) a lipoate-protein ligase (LplA) having a sequence at least 95% identical to the sequence of SEQ ID NO:94;
• jjj) a dihydrolipoyl dehyrogenase (GcvL) having a sequence at least 95% identical to the sequence of SEQ ID NO:96;
• kkk) a feedback-resistant aspartate kinase (LysC, EC 2.7.2.4) having a sequence at least 95% identical to the sequence of SEQ ID NO:54;
• lll) a cystathionine gamma-synthase (MetB) having a sequence at least 95%, identical to the sequence of SEQ ID NO:56;
• mmm) a 5,10-methylenetetrahydrofolate reductase (MetF) having a sequence at least 95% identical to the sequence of SEQ ID NO:58;
• nnn) a homoserine O-acetyltransferase (MetX) having a sequence at least 95%, identical to the sequence of SEQ ID NO:60;
• ooo) an O-acetylhomoserine lyase (MetY) having a sequence at least 95% identical to the sequence of SEQ ID NO:62;
• ppp) a feedback-resistant pyruvate carboxylase (Pyc, EC 6.4.1.1) having a sequence at least 95% identical to the sequence of SEQ ID NO:64;
• qqq) an optionally feedback-resistant D-3-phosphoglycerate dehydrogenase (SerA) having a sequence at least 95%, identical to the sequence of SEQ ID NO:66;
• rrr) a phosphoserine phosphatase (SerB) having a sequence at least 95% identical to the sequence of SEQ ID NO:68;
• sss) a phosphoserine aminotransferase (SerC) having a sequence at least 95% identical to the sequence of SEQ ID NO:70;
• ttt) the subunit of a sulphate adenylyltransferase (CysD) having a sequence at least 95% identical to the sequence of SEQ ID NO:74;
• uuu) an adenosine phosphosulphate reductase (CysH), having a sequence at least 95% identical to the sequence of SEQ ID NO:76;
• vvv) a sulphite reductase (CysI) having a sequence at least 95%, identical to the sequence of SEQ ID NO:78;
• www) an NADPH-dependent glutamate synthase beta chain (CysJ) having a sequence at least 95%, identical to the sequence of SEQ ID NO:80;
• xxx) the subunit of a sulphate adenylyltransferase (CysN) having a sequence at least 95% identical to the sequence of SEQ ID NO:82;
• yyy) a cystathionine beta-synthase (CysY) having a sequence at least 95%, to the sequence of SEQ ID NO:84;
• zzz) a sulphate transporter (CysZ) having a sequence at least 95% identical to the sequence of SEQ ID NO:86;
• aaaa) a 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (MetE) having a sequence at least 95% identical to the sequence of SEQ ID NO:88;
• bbbb) a peptidyl-tRNA hydrolase 1 (PtH1) having a sequence at least 95%, identical to the sequence of SEQ ID NO:90;
• cccc) a peptidyl-tRNA hydrolase 2 (PtH2) having a sequence at least 95%, identical to the sequence of SEQ ID NO:92;
• dddd) a diaminopimelate dehrogenase (Ddh, EC 1.4.1.16) encoded by an overexpressed polynucleotide (ddh);
• eeee) a diaminopimelate decarboxylase (LysA, EC 4.1.1.20) having a sequence at least 95% identical to the sequence of SEQ ID NO:164;
• ffff) an aspartate aminotransferase (AaT, EC 2.6.1.1) having a sequence at least 95% identical to the sequence of SEQ ID NO:166;
• gggg) an L-lysine exporter (LysE, lysine efflux permease) having a sequence at least 95% identical to the sequence of SEQ ID NO:168;
• hhhh) a diaminopimelate epimerase (DapF, EC 5.1.1.7) encoded by an overexpressed polynucleotide (dapF);
• iiii) a dihydropicolinate reductase (DapB, EC 1.3.1.26) having a sequence at least 95% identical to the sequence of SEQ ID NO:170;
• jjjj) a glucose-6-phosphate dehydrogenase (EC 1.1.1.49) having a sequence at least 95% identical to the sequence of SEQ ID NO:172;
• kkkk) the Zwf subunit of a glucose-6-phosphate dehydrogenase (Zwf, EC 1.1.1.49) having a sequence at least 95% identical to the sequence of SEQ ID NO:186;
• llll) the OpcA subunit of a glucose-6-phosphate dehydrogenase (OpcA, EC 1.1.1.49) having a sequence at least 95%, identical to the sequence of SEQ ID NO:188;
• mmmm) a phosphogluconic acid dehydrogenase (Gnd, EC 1.1.1.44) having a sequence at least 95% identical to the sequence of SEQ ID NO:174;
• nnnn) a malate:
• quinone oxidoreductase (Mqo, EC 1.1.99.16) having a sequence at least 95% identical to the sequence of SEQ ID NO:176;
• oooo) the E1p subunit of a pyruvate dehydrogenase complex (AceE, EC 1.2.4.1) having a sequence at least 95% identical to the sequence of SEQ ID NO:178;
• pppp) a citrate synthase (GltA, EC 4.1.3.7) having a sequence at least 95% identical to the sequence of SEQ ID NO:
• 180;
• qqqq) a malate dehydrogenase (Mdh, EC 1.1.1.37) having a sequence at least 95% identical to the sequence of SEQ ID NO:182;
• and
• rrrr) a UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase (MurE, EC 6.3.2.13) having a sequence at least 95% identical to the sequence of SEQ ID NO:184.