Genetically engineered bacterium using glucose as substrate for de novo synthesis of vanillin and application thereof
The present invention discloses a genetically engineered bacterium using glucose as a substrate for de novo synthesis of vanillin and an application thereof, which belongs to the technical field of gene recombination and metabolic engineering. The genetically engineered bacterium using the glucose as the substrate for de novo synthesis of vanillin disclosed by the present invention is recombinant Corynebacterium glutamicum modified by chassis microorganisms and including a vanillin synthesis module and a methyl cyclic regeneration module. The genetically engineered bacteria constructed by the present invention are safe and non-toxic, can use the glucose for de novo synthesis of natural vanillin, and is low in production cost, high in yield, and promising in application prospect.
1 . A genetically engineered bacterium that uses glucose as a substrate for de novo synthesis of vanillin, wherein the genetically engineered bacterium is a recombinant Corynebacterium glutamicum comprising a vanillin synthesis module and a methyl cyclic regeneration module;
wherein the recombinant Corynebacterium glutamicum is a variant of a Corynebacterium glutamicum ATCC13032 cell obtained by knocking out the pcaHG, van, vdh and fud genes in said Corynebacterium glutamicum ATCC13032 cell, wherein the fud gene is an alcohol dehydrogenase (NADP+) gene, the alcohol dehydrogenase (NADP+) gene being a Zn-dependent alcohol dehydrogenase gene;
wherein the vanillin synthesis module comprises a transketolase gene, a 3-deoxy-7-phosphoheptulonate synthase gene, an O-methyltransferase gene, a carboxylic acid reductase gene and a 4′-phosphopantetheinyl transferase gene;
wherein the methyl cyclic regeneration module comprises a 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase gene, an S-adenosylmethionine synthase gene, a homoserine O-acetyltransferase gene and an adenosylhomocysteinase gene;
wherein the recombinant Corynebacterium glutamicum expresses any one of the combinations of genes selected from:
(a) an Escherichia coli tktA gene, an Escherichia coli K12 3-deoxy-7-phosphoheptulonate synthase gene having the sequence of SEQ ID NO: 45, a Rattus norvegicus O-methyltransferase gene having the sequence of SEQ ID NO: 46, a Nocardia iowensis carboxylic acid reductase gene having the sequence of SEQ ID NO: 47, a Mycobacterium marinum 4′-phosphopantetheinyl transferase gene having the sequence of SEQ ID NO: 48, an endogenous Corynebacterium glutamicum 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase gene having the sequence of SEQ ID NO: 49, an endogenous Corynebacterium glutamicum S-adenosylmethionine synthase gene having the sequence of SEQ ID NO: 50, an endogenous Corynebacterium glutamicum homoserine O-acetyltransferase gene having sequence of SEQ ID NO: 51, and an endogenous Corynebacterium glutamicum adenosylhomocysteinase gene having the sequence of SEQ ID NO: 52;
(b) an endogenous Corynebacterium glutamicum transketolase gene having the sequence of SEQ ID NO: 83, an endogenous Corynebacterium glutamicum 3-deoxy-7-phosphoheptulonate synthase gene having the sequence of SEQ ID NO: 84, an Arabidopsis thaliana O-methyltransferase gene having the sequence of SEQ ID NO: 85, a Mycobacterium marinum carboxylic acid reductase gene having the sequence of SEQ ID NO: 86, a Nocardia iowensis 4′-phosphopantetheinyl transferase gene having the sequence of SEQ ID NO: 87, an Escherichia coli K12 metE gene, an Escherichia coli K12 metK gene, a Saccharomyces cerevisiae S288C met2 gene, and an endogenous Corynebacterium glutamicum adenosylhomocysteinase gene having the sequence of SEQ ID NO: 52;
(c) a Saccharomyces cerevisiae S288C tkl1 gene, a Saccharomyces cerevisiae S288C aro4 gene, s Homo sapiens O-methyltransferase gene having the sequence of SEQ ID NO: 105, a Nocardia iowensis carboxylic acid reductase gene having the sequence of SEQ ID NO: 47, a Bacillus subtilis 4′-phosphopantetheinyl transferase gene having the sequence of SEQ ID NO: 106, a Saccharomyces cerevisiae S288C met6 gene, a Saccharomyces cerevisiae S288C SAM2 gene, a Saccharomyces cerevisiae S288C met2 gene, and an endogenous Corynebacterium glutamicum adenosylhomocysteinase gene having the sequence of SEQ ID NO: 52; and
(d) an Escherichia coli tktA gene, an Escherichia coli K12 3-deoxy-7-phosphoheptulonate synthase gene having the sequence of SEQ ID NO: 45, a Mus musculus O-methyltransferase gene having the sequence of SEQ ID NO: 137, a Nocardia iowensis carboxylic acid reductase gene having the sequence of SEQ ID NO: 47, a Mycobacterium marinum 4′-phosphopantetheinyl transferase gene having the sequence of SEQ ID NO: 48, an endogenous Corynebacterium glutamicum 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase gene having the sequence of SEQ ID NO: 49, an endogenous Corynebacterium glutamicum S-adenosylmethionine synthase gene having the sequence of SEQ ID NO: 50, an endogenous Corynebacterium glutamicum homoserine O-acetyltransferase gene having sequence of SEQ ID NO: 51, and an endogenous Corynebacterium glutamicum adenosylhomocysteinase gene having the sequence of SEQ ID NO: 52.
2 . A method for constructing the genetically engineered bacterium that uses glucose as a substrate for de novo synthesis of vanillin according to claim 1 , wherein said method comprises:
(i) disrupting the endogenous pcaHG, van, vdh, and fud genes in a Corynebacterium glutamicum ATCC13032 cell, wherein the fud gene is an alcohol dehydrogenase (NADP + ) gene, the alcohol dehydrogenase (NADP + ) gene being a Zn-dependent alcohol dehydrogenase gene, and
(ii) transforming the Corynebacterium glutamicum of (i) with any one of the combinations of genes selected from (a), (b), (c) and (d).