Methods and compositions for the production of xylitol from xylose utilizing dynamic metabolic control
The present disclosure is related to genetically engineered microbial strains and related bioprocesses for the production of xylitol. Specifically, the use of dynamically controlled synthetic metabolic valves to reduce the activity of certain enzymes, leads to increased xylitol production in a two-stage process.
1 . A genetically modified E. coli microorganism for producing xylitol from xylose comprising:
a first gene expression-silencing synthetic metabolic valve or an enzymatic degradation synthetic metabolic valve for overexpression of xylose reductase in a productive stationary phase;
a second gene modification comprising chromosomal modification of the xylA gene to provide for reducing the amount of xylose isomerase enzyme in a productive stationary phase; and
further comprising a gene-silencing synthetic metabolic valve or the enzyme degradation synthetic metabolic valve are directed to control of the gene encoding enoyl-ACP reductase, or the enoyl-ACP reductase enzyme in a productive stationary phase,
wherein the genetically modified E. coli microorganism will produce xylitol in a biofermentation process comprising growing the genetically modified E. coli microorganism in a medium in a growth phase, transitioning to a productive stationary phase, the transition comprising: slowing or stopping microorganism growth, inducing the first gene modification, to effect overexpression of xylose reductase and the second gene expression-silencing synthetic metabolic valve or an enzymatic degradation synthetic metabolic valve to decrease the total amount of xylose isomerase enzyme activity, and producing xylitol in the productive stationary phase,
wherein the biofermentation process occurs in a media in which xylose was substituted for glucose, 1 gram xylose for 1 gram glucose.
2 . The genetically modified E. coli microorganism of claim 1 , wherein the xylose reductase is an NADPH dependent xylose reductase.
3 . The genetically modified E. coli microorganism of claim 1 , wherein the xylose reductase is the xyrA gene of A. niger.
4 . The genetically modified E. coli microorganism of claim 1 , wherein the genetically modified microorganism produces xylitol from a xylose feedstock.
5 . The genetically modified E. coli microorganism of claim 1 , further comprising a gene-silencing synthetic metabolic valve or the enzyme degradation synthetic metabolic valve are directed to control of the gene encoding glucose-6-phosphate dehydrogenase (zwf) or the glucose-6-phosphate dehydrogenase (zwf) enzyme.
6 . The genetically modified E. coli microorganism of claim 1 , further comprising a gene-silencing synthetic metabolic valve or the enzyme degradation synthetic metabolic valve consist of:
silencing of a gene encoding glucose-6-phosphate dehydrogenase (zwf); and
enzyme degradation of glucose-6-phosphate dehydrogenase (zwf) and enoyl-ACP reductase (fabI) enzymes.
7 . The genetically modified E. coli microorganism of claim 1 , wherein the first or second synthetic metabolic valves are induced by nutrient depletion.
8 . The genetically modified E. coli microorganism of claim 1 , wherein the first or second synthetic metabolic valves are induced by phosphate depletion.
9 . The genetically modified E. coli microorganism of claim 1 , the microorganism further comprises a chromosomal deletion.
10 . The genetically modified E. coli microorganism of claim 1 , wherein the first or second synthetic metabolic valves effect gene silencing by CRISPR interference, the and the first, second or third synthetic metabolic valves further comprising a CASCADE guide array, the array comprising two or more genes encoding small guide RNAs each specific for targeting a different gene for simultaneous silencing of multiple genes.
11 . The genetically modified E. coli microorganism of claim 1 , wherein the microorganism produces a xylitol product titer of at least 20 g/L at twenty-four hours in a biofermentation process.
12 . The genetically modified E. coli microorganism of claim 1 , further comprises modification to the E. coli microorganism so that:
activity of a membrane bound transhydrogenase activity is induced;
activity of a pyruvate ferredoxin oxidoreductase is induced; and
activity of a NADPH dependent ferredoxin reductase is induced.
13 . A multi-stage fermentation bioprocess for producing xylitol from a genetically modified E. coli microorganism of claim 1 , comprising:
(a) providing the genetically modified E. coli microorganism;
(b) growing the genetically modified E. coli microorganism in a media with a xylose feedstock;
(c) transitioning from a growth phase to a xylitol producing stage by slowing or stopping the growth of the E. coli microorganism; and
inducing the first or second synthetic metabolic valves to effect overexpression of xylose reductase and reducing expression of xylose isomerase, thereby
(d) producing xylitol.
14 . A genetically modified E. coli microorganism for producing xylitol from xylose comprising:
a first gene expression-silencing synthetic metabolic valve or an enzymatic degradation synthetic metabolic valve for overexpression of xylose reductase in a productive stationary phase;
a second gene modification comprising chromosomal modification of the xylA gene to provide for reducing the amount of xylose isomerase enzyme in a productive stationary phase and
wherein the genetically modified E. coli microorganism will produce xylitol in a biofermentation process comprising growing the genetically modified E. coli microorganism in a medium in a growth phase, transitioning to a productive stationary phase, the transition comprising: slowing or stopping microorganism growth, inducing the first gene modification, to effect overexpression of xylose reductase and the second gene expression silencing synthetic metabolic valve or an enzymatic degradation synthetic metabolic valve to decrease the total amount of xylose isomerase enzyme activity, and producing xylitol in the productive stationary phase,
wherein the biofermentation process occurs in a media in which xylose was substituted for glucose, 1 gram xylose for 1 gram glucose.
15 . The genetically modified E. coli microorganism of claim 14 , wherein the xylose reductase is an NADPH dependent xylose reductase or the xyrA gene of A. niger.
16 . The genetically modified E. coli microorganism of claim 14 , wherein the E. coli microorganism further comprises a gene expression-silencing synthetic metabolic valve or an enzymatic degradation synthetic metabolic valve to regulate expression of a third gene or third enzyme that is glucose-6-phosphate dehydrogenase (zwf), enoyl-ACP reductase (fabI), soluble transhydrogenase (udhA) or citrate synthase (gltA).
17 . The genetically modified E. coli microorganism of claim 14 , wherein the induction occurs via nutrient depletion or phosphate depletion.
18 . The genetically modified E. coli microorganism of claim 14 , further comprising a chromosomal deletion.
19 . The genetically modified E. coli microorganism of claim 14 , wherein the silencing of gene expression comprises CRISPR interference, and the genetically modified microorganism further comprises a CASCADE guide array, the array comprising two or more genes encoding small guide RNAs each specific for targeting a different gene for simultaneous silencing of multiple genes.
20 . The genetically modified E. coli microorganism of claim 14 , wherein the microorganism produces a xylitol product titer of at least 20 g/L at twenty-four hours in a biofermentation process.
21 . The genetically modified E. coli microorganism of claim 14 , further comprising:
activity of a membrane bound transhydrogenase activity is induced;
activity of a pyruvate ferredoxin oxidoreductase is induced;
activity of a NADPH dependent ferredoxin reductase is induced and
wherein the microorganism produces at least one chemical product whose biosynthesis requires NADPH.
22 . A multi-stage fermentation bioprocess for producing xylitol from a genetically modified E. coli microorganism of claim 14 , comprising:
(a) providing the genetically modified E. coli microorganism,
(b) growing the genetically modified E. coli microorganism in a media with a xylose feedstock;
(c) transitioning from a growth phase to a xylitol producing stage by slowing or stopping the growth of the E. coli microorganism; and
inducing the first or second synthetic metabolic valves to effect overexpression of xylose reductase or reducing expression of xylose isomerase, thereby
(d) producing xylitol.