Recombinant microorganism for improved production of alanine

Abstract

The present invention relates to a recombinant nucleic acid molecule, a recombinant microorganism, to a method for producing alanine and to the use of the recombinant nucleic acid molecule or the recombinant microorganism for the fermentative production of alanine.

1 . A recombinant microorganism comprising a reduced, repressed or deleted activity and/or expression of an asd gene or a gdhA gene. 
     
     
         2 . The recombinant microorganism of  claim 1  further comprising at least one of the features selected from the group of
 (a) an introduced, increased or enhanced activity and/or expression of an alaD gene encoding an alanine dehydrogenase, 
 (b) a reduced, repressed or deleted activity and/or expression of a pflB gene encoding a pyruvate formate lyase I, 
 (c) a reduced, repressed or deleted activity and/or expression of an adhE gene encoding a bifunctional acetaldehyde-CoA dehydrogenase/iron-dependent alcohol dehydrogenase/pyruvate-formate lyase deactivase, 
 (d) a reduced, repressed or deleted activity and/or expression of a ldhA gene encoding a NAO-dependent fermentative D-lactate dehydrogenase, 
 (e) a reduced, repressed or deleted activity and/or expression of a pta gene encoding a phosphate acetyltransferase and/or a reduced, repressed or deleted activity and/or expression of an ackA gene encoding an acetate kinase A and propionate kinase 2, 
 (f) a reduced, repressed or deleted activity and/or expression of a frdA gene encoding a fumarate reductase, and/or 
 (g) a reduced, repressed or deleted activity and/or expression of a dadX gene encoding an alanine racemase,
 wherein the reduction, repression, deletion, introduction, increase or enhancement of the activity and/or expression of a gene is determined compared to a respective reference microorganism. 
 
 
     
     
         3 - 8 . (canceled) 
     
     
         9 . The recombinant microorganism of  claim 1 , further comprising at least one feature selected from the group of:
 (a) a reduced, repressed or deleted activity and/or expression of a brnQ gene encoding a brnQ protein having a branched chain amino acid transporter activity,   (b) a reduced, repressed or deleted activity and/or expression of a gcvB gene encoding a non-protein encoding RNA,   (c) an increased and/or enhanced activity and/or expression of a zipA gene encoding cell division protein involved in Z ring assembly,   (d) an increased and/or enhanced activity and/or expression of a lpd gene encoding a lipoamide dehydrogenase,   a changed activity of a lpxD gene encoding encoding an UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase protein,   an increased and/or enhanced activity and/or expression of a gcvA gene encoding a DNA-binding protein, and/or   an increased and/or enhanced activity and/or expression of a ygaW gene encoding an alanine transporter.   
     
     
         10 - 15 . (canceled) 
     
     
         16 . The recombinant microorganism of  claim 1 , wherein the asd gene or the gdhA gene is selected from the group of
 (i) a nucleic acid molecule comprising a sequence of SEQ ID NO: 25 or SEQ ID NO: 37, or   (ii) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 25 or SEQ ID NO: 37, or   (iii) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 25 or SEQ ID NO: 37 under stringent conditions, or   (iv) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 26 or SEQ ID NO: 38, or   (v) a nucleic acid molecule encoding a polypeptide having at least 60% homology to the polypeptide of SEQ ID NO: 26 or SEQ ID NO: 38.   
     
     
         17 . The recombinant microorganism of  claim 2 , wherein the alaD gene is selected from the group consisting of
 (AA) a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, or   (BB) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 1, or   (CC) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 1 under stringent conditions, or   (DD) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 2, or   (EE) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 2.   
     
     
         18 . The recombinant microorganism of  claim 2 , wherein the pflB gene is selected from the group consisting of
 (A) a nucleic acid molecule comprising a sequence of SEQ ID NO: 5, or   (B) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 5, or   (C) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 5 under stringent conditions, or   (D) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 6, or   (E) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 6.   
     
     
         19 . The recombinant microorganism of  claim 2 , wherein the adhE gene is selected from the group consisting of
 (F) a nucleic acid molecule comprising a sequence of SEQ ID NO: 7, or   (G) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 7, or   (H) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 7 under stringent conditions, or   (I) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 8, or   (J) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 8.   
     
     
         20 . The recombinant microorganism of  claim 2 , wherein the ldhA gene is selected from the group consisting of
 (K) a nucleic acid molecule comprising a sequence of SEQ ID NO: 9, or   (L) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 9, or   (M) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 9 under stringent conditions, or   (N) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 10, or   (O) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 10.   
     
     
         21 . The recombinant microorganism of  claim 2 , wherein the pta gene is selected from the group consisting of
 (P1) a nucleic acid molecule comprising a sequence of SEQ ID NO: 11, or   (Q1) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 11, or   (R1) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 11 under stringent conditions, or   (S1) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 12, or   (T1) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 12 and wherein the ackA gene is selected from the group consisting of   (P2) a nucleic acid molecule comprising a sequence of SEQ ID NO: 32, or   (Q2) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 32, or   (R2) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 32 under stringent conditions, or   (S2) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 33, or   (T2) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 33.   
     
     
         22 . The recombinant microorganism of  claim 2 , wherein the frdA gene is selected from the group consisting of
 (U) a nucleic acid molecule comprising a sequence of SEQ ID NO: 13, or   (V) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 13, or   (W) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 13 under stringent conditions, or   (X) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 14, or   (Y) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 14.   
     
     
         23 . The recombinant microorganism of  claim 2 , wherein the dadX gene is selected from the group consisting of
 (Z) a nucleic acid molecule comprising a sequence of SEQ ID NO: 15, or   (FF) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 15, or   (GG) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 15 under stringent conditions, or   (HH) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 16, or   (II) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 16.   
     
     
         24 . The recombinant microorganism of  claim 9 , wherein the ygaW gene is selected from the group consisting of
 (JJ) a nucleic acid molecule comprising a sequence of SEQ ID NO: 17, or   (KK) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 17, or   (LL) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 17 under stringent conditions, or   (MM) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 18, or   (NN) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 18.   
     
     
         25 . The recombinant microorganism of  claim 9 , wherein the zipA gene is selected from the group consisting of
 (OO) a nucleic acid molecule comprising a sequence of SEQ ID NO: 19, or   (PP) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 19, or   (QQ) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 19 under stringent conditions, or   (RR) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 20, or   (SS) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 20.   
     
     
         26 . The recombinant microorganism of  claim 9 , wherein the lpd gene is selected from the group consisting of
 (TT) a nucleic acid molecule comprising a sequence of SEQ ID NO: 21, or   (UU) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 21, or   (VV) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 21 under stringent conditions, or   (WW) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 22, or   (XX) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 22.   
     
     
         27 . The recombinant microorganism of  claim 9 , wherein the brnQ gene is selected from the group consisting of
 (YY) a nucleic acid molecule comprising a sequence of SEQ ID NO: 23, or   (ZZ) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 23, or   (AAA) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 23 under stringent conditions, or   (BBB) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 24, or   (CCC) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 24.   
     
     
         28 . The recombinant microorganism of  claim 9 , wherein the lpxD gene is selected from the group consisting of
 (DDD) a nucleic acid molecule comprising a sequence of SEQ ID NO: 27, or   (EEE) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 28.   
     
     
         29 . The recombinant microorganism of  claim 9 , wherein the gcvA gene is selected from the group consisting of
 (FFF) a nucleic acid molecule comprising a sequence of SEQ ID NO: 29, or   (GGG) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 29, or   (HHH) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 29 under stringent conditions, or   (III) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 30, or   (JJJ) a nucleic acid molecule encoding a polypeptide having at least 60% homology to a polypeptide of SEQ ID NO: 30.   
     
     
         30 . The recombinant microorganism of  claim 9 , wherein the gcvB gene is selected from the group consisting of
 (KKK) a nucleic acid molecule comprising a sequence of SEQ ID NO: 31, or   (LLL) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 31, or   (MMM) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 31 under stringent conditions.   
     
     
         31 . The recombinant microorganism of  claim 1 , wherein the microorganism is selected from a genus of the group consisting of  Corynebacterium, Bacillus, Erwinia, Escherichia, Pantoea, Streptomyces, Zymomonas, Rhodococcus, Saccharomyces, Candida  or  Pichia.    
     
     
         32 - 36 . (canceled) 
     
     
         37 . A method of producing alanine comprising culturing one or more recombinant microorganism according to  claim 1  under conditions that allow for the production of alanine 
     
     
         38 - 43 . (canceled) 
     
     
         44 . The method of  claim 37 , comprising
 I) growing the one or more microorganism in a fermenter to obtain a fermentation broth, and   II) recovering alanine from the fermentation broth.