Amino acid and metabolite biosynthesis
Bacterial strains that are engineered to increase the production of amino acids, including aspartate-derived amino acids (e.g., methionine, lysine, threonine, isoleucine, and S-adenosylmethionine (S-AM)) and cysteine, and related metabolites are described. The strains can be genetically engineered to harbor one or more nucleic acid molecules (e.g., recombinant nucleic acid molecules) encoding a polypeptide (e.g., a polypeptide that is heterologous or homologous to the host cell) and/or they may be engineered to increase or decrease expression and/or activity of polypeptides (e.g., by mutation of endogenous nucleic acid sequences).
1 . An Enterobacteriaceae or coryneform bacterium comprising at least one isolated nucleic acid molecule selected from the group consisting of:
(a) a nucleic acid molecule comprising a sequence encoding a bacterial sulfate ABC transporter ATP-binding polypeptide or a functional variant thereof;
(b) a nucleic acid molecule comprising a sequence encoding a bacterial sulfate transport system permease W polypeptide or a functional variant thereof;
(c) a nucleic acid molecule comprising a sequence encoding a bacterial sulfate, thiosulfate transport system permease T polypeptide or a functional variant thereof;
(d) a nucleic acid molecule comprising a sequence encoding a bacterial sulfate adenylyltransferase subunit 1 polypeptide or a functional variant thereof;
(e) a nucleic acid molecule comprising a sequence encoding a bacterial sulfate adenylyltransferase subunit 2 polypeptide or a functional variant thereof;
(f) a nucleic acid molecule comprising a sequence encoding a bacterial adenylylsulfate kinase polypeptide or a functional variant thereof;
(g) a nucleic acid molecule comprising a sequence encoding a bacterial phosphoadenosine phosphosulfate reductase polypeptide or a functional variant thereof;
(h) a nucleic acid molecule comprising a sequence encoding a bacterial sulfite reductase alpha subunit polypeptide or a functional variant thereof;
(i) a nucleic acid molecule comprising a sequence encoding a bacterial sulfite reductase hemopolypeptide beta-component polypeptide or a functional variant thereof;
(j) a nucleic acid molecule comprising a sequence encoding a bacterial sulfite reductase (NADPH), flavopolypeptide beta subunit polypeptide or a functional variant thereof;
(k) a nucleic acid molecule comprising a sequence encoding a bacterial adenylyl-sulphate reductase alpha subunit polypeptide or a functional variant thereof;
(l) a nucleic acid molecule comprising a sequence encoding a bacterial phosphoglycerate dehydrogenase polypeptide or a functional variant thereof;
(m) a nucleic acid molecule comprising a sequence encoding a bacterial phosphoserine transaminase polypeptide or a functional variant thereof;
(n) a nucleic acid molecule comprising a sequence encoding a bacterial phosphoserine phosphatase polypeptide or a functional variant thereof;
(o) a nucleic acid molecule comprising a sequence encoding a bacterial serine O-acetyltransferase polypeptide or a functional variant thereof;
(p) a nucleic acid molecule comprising a sequence encoding a bacterial cysteine synthase A polypeptide or a functional variant thereof;
(q) a nucleic acid molecule comprising a sequence encoding a bacterial cysteine synthase B polypeptide or a functional variant thereof;
(r) a nucleic acid molecule comprising a sequence encoding a bacterial ABC-type vitamin B12 transporter permease component polypeptide or a functional variant thereof;
(s) a nucleic acid molecule comprising a sequence encoding a bacterial ABC-type vitamin B12 transporter ATPase component polypeptide or a functional variant thereof;
(t) a nucleic acid molecule comprising a sequence encoding a bacterial ABC-type cobalamin/Fe 3+ -siderophore transport system polypeptide or a functional variant thereof;
(u) a nucleic acid molecule comprising a sequence encoding a bacterial adenosyltransferase polypeptide or a functional variant thereof;
(v) a nucleic acid molecule comprising a sequence encoding a bacterial GTP cyclohydrolase I polypeptide or a functional variant thereof;
(w) a nucleic acid molecule comprising a sequence encoding a bacterial phoA, psiA, or psiF gene product polypeptide or a functional variant thereof;
(x) a nucleic acid molecule comprising a sequence encoding a bacterial dihydroneopterin aldolase polypeptide or a functional variant thereof;
(y) a nucleic acid molecule comprising a sequence encoding a bacterial 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase polypeptide or a functional variant thereof;
(z) a nucleic acid molecule comprising a sequence encoding a bacterial dihydropteroate synthase polypeptide or a functional variant thereof;
(aa) a nucleic acid molecule comprising a sequence encoding a bacterial dihydrofolate synthetase polypeptide or a functional variant thereof;
(ab) a nucleic acid molecule comprising a sequence encoding a bacterial dihydrofolate reductase polypeptide or a functional variant thereof;
(ac) a nucleic acid molecule comprising a sequence encoding a bacterial folylpolyglutamate synthetase polypeptide or a functional variant thereof;
(ad) a nucleic acid molecule comprising a sequence encoding a putative bacterial methionine (APC transporter superfamily) permease (YjeH) polypeptide or a functional variant thereof;
(ae) a nucleic acid molecule comprising a sequence encoding a bacterial transcriptional activator of MetE/H polypeptide or a functional variant thereof;
(af) a nucleic acid molecule comprising a sequence encoding a bacterial 6-phosphogluconate dehydrogenase polypeptide or a functional variant thereof;
(ag) a nucleic acid molecule comprising a sequence encoding a bacterial S-methylmethionine homocysteine methyltransferase polypeptide or a functional variant thereof;
(ah) a nucleic acid molecule comprising a sequence encoding a bacterial S-adenosylhomocysteine hydrolase polypeptide or a functional variant thereof;
(ai) a nucleic acid molecule comprising a sequence encoding a bacterial site-specific DNA methylase polypeptide or a functional variant thereof;
(aj) a nucleic acid molecule comprising a sequence encoding a bacterial methionine export sytem protein 1 polypeptide or a functional variant thereof;
(ak) a nucleic acid molecule comprising a sequence encoding a bacterial methionine export sytem protein 2 polypeptide or a functional variant thereof;
(al) a nucleic acid molecule comprising a sequence encoding a bacterial ABC transport system ATP-binding protein (MetN) polypeptide or a functional variant thereof;
(am) a nucleic acid molecule comprising a sequence encoding a bacterial ABC transport system permease protein (MetP) polypeptide or a functional variant thereof;
(an) a nucleic acid molecule comprising a sequence encoding a bacterial ABC transport system substrate-binding protein (MetQ) polypeptide or a functional variant thereof;
(ao) a nucleic acid molecule comprising a sequence encoding a bacterial aspartokinase polypeptide or a functional variant thereof;
(ap) a nucleic acid molecule comprising a sequence encoding a bacterial aspartate semialdehyde dehydrogenase or a functional variant thereof;
(aq) a nucleic acid molecule comprising a sequence encoding a bacterial homoserine dehydrogenase polypeptide or a functional variant thereof;
(ar) a nucleic acid molecule comprising a sequence encoding a bacterial O-homoserine acetyl transferase polypeptide or a functional variant thereof;
(as) a nucleic acid molecule comprising a sequence encoding a bacterial O-acetylhomoserine sulfhydrylase polypeptide or a functional variant thereof;
(at) a nucleic acid molecule comprising a sequence encoding a bacterial cobalamin-dependent methionine synthase polypeptide or a functional variant thereof;
(au) a nucleic acid molecule comprising a sequence encoding a bacterial cobalamin-independent methionine synthase polypeptide or a functional variant thereof;
(av) a nucleic acid molecule comprising a sequence encoding a bacterial homoserine kinase polypeptide or a functional variant thereof;
(aw) a nucleic acid molecule comprising a sequence encoding a bacterial methionine adenosyltransferase polypeptide or a functional variant thereof;
(ax) a nucleic acid molecule comprising a sequence encoding a bacterial O-succinylhomoserine (thio)-lyase polypeptide or a functional variant thereof;
(ay) a nucleic acid molecule comprising a sequence encoding a bacterial cystathionine beta-lyase polypeptide or a functional variant thereof;
(az) a nucleic acid molecule comprising a sequence encoding a bacterial 5,10-methylenetetrahydrofolate reductase polypeptide or a functional variant thereof;
(ba) a nucleic acid molecule comprising a sequence encoding a bacterial dihydrodipicolinate synthase polypeptide or a functional variant thereof;
(bb) a nucleic acid molecule comprising a sequence encoding a bacterial pyruvate carboxylase polypeptide or a functional variant thereof;
(bc) a nucleic acid molecule comprising a sequence encoding a bacterial glutamate dehydrogenase polypeptide or a functional variant thereof;
(bd) a nucleic acid molecule comprising a sequence encoding a bacterial diaminopimelate dehydrogenase polypeptide or a functional variant thereof;
(be) a nucleic acid molecule comprising a sequence encoding a bacterial methionine and cysteine biosynthesis repressor (McbR) polypeptide or a functional variant thereof;
(bf) a nucleic acid molecule comprising a sequence encoding a bacterial lysine exporter protein polypeptide or a functional variant thereof;
(bg) a nucleic acid molecule comprising a sequence encoding a bacterial phosphoenolpyruvate carboxykinase polypeptide or a functional variant thereof;
(bh) a nucleic acid molecule comprising a sequence encoding a bacterial phosphoenolpyruvate carboxylase polypeptide or a functional variant thereof;
(bi) a nucleic acid molecule comprising a sequence encoding a bacterial glycine dehydrogenase (decarboxylating) polypeptide or a functional variant thereof;
(bj) a nucleic acid molecule comprising a sequence encoding a bacterial H polypeptide (involved in the glycine cleavage system) or a functional variant thereof;
(bk) a nucleic acid molecule comprising a sequence encoding a bacterial aminomethyl transferase polypeptide or a functional variant thereof;
(bl) a nucleic acid molecule comprising a sequence encoding a bacterial dihydrolipoamide dehydrogenase polypeptide or a functional variant thereof;
(bm) a nucleic acid molecule comprising a sequence encoding a bacterial lipoate-protein ligase A polypeptide or a functional variant thereof;
(bn) a nucleic acid molecule comprising a sequence encoding a bacterial lipoic acid synthase polypeptide or a functional variant thereof;
(bo) a nucleic acid molecule comprising a sequence encoding a bacterial lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase polypeptide or a functional variant thereof;
(bp) a nucleic acid molecule comprising a sequence encoding a bacterial fructose 1,6 bisphosphatase polypeptide or a functional variant thereof;
(bq) a nucleic acid molecule comprising a sequence encoding a bacterial glucose 6 phosphate dehydrogenase polypeptide or a functional variant thereof;
(br) a nucleic acid molecule comprising a sequence encoding a glucose-6-phosphate isomerase polypeptide or a functional variant thereof; and
(bs) a nucleic acid molecule comprising a sequence encoding a bacterial NCgl2640 polypeptide or a functional variant thereof
2 . The bacterium of claim 1 wherein the bacterium comprises at least two of nucleic acid molecules (a)-(bs).
3 . The bacterium of claim 1 wherein the bacterium comprises at least three of nucleic acid molecules (a)-(bs).
4 . The bacterium of claim 1 wherein the bacterium comprises at least four of nucleic acid molecules (a)-(bs).
5 . The bacterium of claim 1 wherein the bacterium comprises at least five of nucleic acid molecules (a)-(bs).
6 . The bacterium of claim 1 wherein at least one of the polypeptides is heterologous to the bacterium.
7 . The bacterium of claim 1 wherein at least two of the polypeptides are heterologous to the bacterium.
8 . The bacterium claim 1 wherein the bacterium is a Corynebacterium glutamicum bacterium.
9 . The bacterium of claim 1 , wherein the bacterium comprises (aj) and (ak).
10 . The bacterium of claim 1 , wherein the bacterium comprises (r), (s) and (t).
11 . The bacterium of claim 1 , wherein the bacterium comprises (a), (b) and (c).
12 . The bacterium of claim 1 , wherein the bacterium comprises (d) and (e).
13 . The bacterium of claim 1 , wherein the bacterium comprises (i) and (j).
14 . The bacterium of claim 1 , wherein the bacterium comprises (l) and (o).
15 . The bacterium of claim 1 , wherein the bacterium comprises (p) and (q).
16 . The bacterium of claim 1 , wherein the bacterium comprises (bi), (bj), and (bk).
17 . The bacterium of claim 1 , wherein the bacterium comprises (bi), (bj), (bk) and (bl).
18 . The bacterium of claim 1 , wherein the bacterium comprises (bi), (bj), (bk) and at least one of: (1) (bm) or (2) (bn) and (o).
19 . The bacterium of claim 1 , wherein the bacterium comprises (bi), (bj), (bk) (bl) and at least one of: (1) (bm) or (2) (bn) and (bo).
20 . A method of producing an amino acid or a related metabolite, the method comprising:
cultivating the bacterium claim 1 under conditions that allow the amino acid or the related metabolite to be produced, and collecting a composition that comprises the amino acid or related metabolite from the culture.
21 . The method of claim 1 wherein the amino acid is selected from: methionine, S-adenosylmethionine, lysine, theronine and cysteine.
22 . The bacterium of claim 1 comprising at least one isolated nucleic acid molecule selected from the group consisting of (a)-(an) and at least one isolated nucleic acid molecule selected from the group consisting of (ao)-(bs).
23 . The bacterium of claim 1 comprising at least one isolated nucleic acid molecule selected from the group consisting of (a)-(an) and at least two isolated nucleic acid molecules selected from the group consisting of (ao)-(bs).
24 . The bacterium of claim 1 comprising at least two isolated nucleic acid molecules selected from the group consisting of (a)-(an) and at least one isolated nucleic acid molecule selected from the group consisting of (ao)-(bs).
25 . The bacterium of claim 1 comprising at least two isolated nucleic acid molecules selected from the group consisting of (a)-(an) and at least two isolatd nucleic acid molecules selected from the group consisting of (ao)-(bs).
26 . The bacterium of claim 1 comprising: an isolated nucleic acid molecule encoding a variant aspartokinase with reduced feedback inhibition, a variant homoserine dehydrogenase with reduced feedback inhibition or a variant O-acetylhomoserine sulfhydrylase with reduced feedback inhibition.