BRENDA - Enzyme Database
show all sequences of 1.1.1.4

Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365

Schilling, C.; Ciccone, R.; Sieber, V.; Schmid, J.; Metab. Eng. 61, 381-388 (2020) View publication on PubMed

Data extracted from this reference:

Engineering
Protein Variants
Commentary
Organism
additional information
Paenibacillus polymyxa is used for the production of R-,R-2,3-butanediol in exceptionally high enantiomeric purity. Rational metabolic engineering efforts to increase productivity and product titers have been restricted due to limited genetic accessibility of the organism. By use of CRISPR-Cas9 mediated genome editing, six metabolic mutant variants are generated and compared in batch fermentations. Downstream processing is facilitated by completely eliminating exopolysaccharide formation through the combined knockout of the sacB gene and the clu1 region, encoding for the underlying enzymatic machinery of levan and paenan synthesis. Knockout constructs are generated to eliminate undesirable side-products of 2,3-BDL fermentations. Spore formation is inhibited by deletion of spoIIE, thereby disrupting the sporulation cascade of Paenibacillus polymyxa. Optimization of the carbon flux towards 2,3-butanediol is achieved by deletion of the lactate dehydrogenase ldh1 and decoupling of the butanediol dehydrogenase from its natural regulation via constitutive episomal expression. The improved strain shows 45% increased productivity, reaching a final concentration of 43.8 g/l butanediol. A yield of 0.43 g/g glucose is achieved, accounting for 86% of the theoretical maximum. Paenibacillus polymyxa is transformed by conjugation using Escherichia coli strain S17-1 harboring the various plasmids. Method optimization and evaluation, overview
Paenibacillus polymyxa
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
(R)-acetoin + NADH + H+
Paenibacillus polymyxa
high level production of (R,R)-butane-2,3-diol
(R,R)-butane-2,3-diol + NAD+
-
-
r
(R)-acetoin + NADH + H+
Paenibacillus polymyxa DSM 365
high level production of (R,R)-butane-2,3-diol
(R,R)-butane-2,3-diol + NAD+
-
-
r
Organism
Organism
UniProt
Commentary
Textmining
Paenibacillus polymyxa
-
-
-
Paenibacillus polymyxa DSM 365
-
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
(R)-acetoin + NADH + H+
-
761810
Paenibacillus polymyxa
(R,R)-butane-2,3-diol + NAD+
-
-
-
r
(R)-acetoin + NADH + H+
high level production of (R,R)-butane-2,3-diol
761810
Paenibacillus polymyxa
(R,R)-butane-2,3-diol + NAD+
-
-
-
r
(R)-acetoin + NADH + H+
-
761810
Paenibacillus polymyxa DSM 365
(R,R)-butane-2,3-diol + NAD+
-
-
-
r
(R)-acetoin + NADH + H+
high level production of (R,R)-butane-2,3-diol
761810
Paenibacillus polymyxa DSM 365
(R,R)-butane-2,3-diol + NAD+
-
-
-
r
Synonyms
Synonyms
Commentary
Organism
BDH
-
Paenibacillus polymyxa
butanediol dehydrogenase
-
Paenibacillus polymyxa
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
30
-
in vivo assay at
Paenibacillus polymyxa
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
6
-
in vivo assay at
Paenibacillus polymyxa
Cofactor
Cofactor
Commentary
Organism
Structure
NAD+
-
Paenibacillus polymyxa
NADH
-
Paenibacillus polymyxa
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NAD+
-
Paenibacillus polymyxa
NADH
-
Paenibacillus polymyxa
Engineering (protein specific)
Protein Variants
Commentary
Organism
additional information
Paenibacillus polymyxa is used for the production of R-,R-2,3-butanediol in exceptionally high enantiomeric purity. Rational metabolic engineering efforts to increase productivity and product titers have been restricted due to limited genetic accessibility of the organism. By use of CRISPR-Cas9 mediated genome editing, six metabolic mutant variants are generated and compared in batch fermentations. Downstream processing is facilitated by completely eliminating exopolysaccharide formation through the combined knockout of the sacB gene and the clu1 region, encoding for the underlying enzymatic machinery of levan and paenan synthesis. Knockout constructs are generated to eliminate undesirable side-products of 2,3-BDL fermentations. Spore formation is inhibited by deletion of spoIIE, thereby disrupting the sporulation cascade of Paenibacillus polymyxa. Optimization of the carbon flux towards 2,3-butanediol is achieved by deletion of the lactate dehydrogenase ldh1 and decoupling of the butanediol dehydrogenase from its natural regulation via constitutive episomal expression. The improved strain shows 45% increased productivity, reaching a final concentration of 43.8 g/l butanediol. A yield of 0.43 g/g glucose is achieved, accounting for 86% of the theoretical maximum. Paenibacillus polymyxa is transformed by conjugation using Escherichia coli strain S17-1 harboring the various plasmids. Method optimization and evaluation, overview
Paenibacillus polymyxa
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
(R)-acetoin + NADH + H+
Paenibacillus polymyxa
high level production of (R,R)-butane-2,3-diol
(R,R)-butane-2,3-diol + NAD+
-
-
r
(R)-acetoin + NADH + H+
Paenibacillus polymyxa DSM 365
high level production of (R,R)-butane-2,3-diol
(R,R)-butane-2,3-diol + NAD+
-
-
r
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
(R)-acetoin + NADH + H+
-
761810
Paenibacillus polymyxa
(R,R)-butane-2,3-diol + NAD+
-
-
-
r
(R)-acetoin + NADH + H+
high level production of (R,R)-butane-2,3-diol
761810
Paenibacillus polymyxa
(R,R)-butane-2,3-diol + NAD+
-
-
-
r
(R)-acetoin + NADH + H+
-
761810
Paenibacillus polymyxa DSM 365
(R,R)-butane-2,3-diol + NAD+
-
-
-
r
(R)-acetoin + NADH + H+
high level production of (R,R)-butane-2,3-diol
761810
Paenibacillus polymyxa DSM 365
(R,R)-butane-2,3-diol + NAD+
-
-
-
r
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
30
-
in vivo assay at
Paenibacillus polymyxa
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
6
-
in vivo assay at
Paenibacillus polymyxa
General Information
General Information
Commentary
Organism
metabolism
overview of 2,3-BDL biosynthesis pathway and byproducts in microaerobic conditions of Paenibacillus polymyxa strain DSM 365 starting from sucrose as a substrate
Paenibacillus polymyxa
General Information (protein specific)
General Information
Commentary
Organism
metabolism
overview of 2,3-BDL biosynthesis pathway and byproducts in microaerobic conditions of Paenibacillus polymyxa strain DSM 365 starting from sucrose as a substrate
Paenibacillus polymyxa
Other publictions for EC 1.1.1.4
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Synonyms
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
760423
Kuang
New insights into two yeast B ...
Saccharomyces cerevisiae, Saccharomyces cerevisiae YBA_08
Appl. Microbiol. Biotechnol.
104
6679-6692
2020
-
-
2
-
-
-
-
8
-
2
-
4
-
6
-
-
2
-
-
-
17
-
31
-
8
2
-
-
8
2
-
2
6
-
-
-
-
-
2
6
-
-
-
-
-
-
8
-
2
-
4
-
-
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2
-
-
17
-
31
-
2
-
-
8
2
-
2
-
-
2
2
-
8
8
760761
Subramanian
Phylogenetics-based identific ...
no activity in Serratia marcescens
Biotechnol. Biofuels
13
186
2020
-
-
-
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
-
-
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-
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-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
761810
Schilling
Engineering of the 2,3-butane ...
Paenibacillus polymyxa, Paenibacillus polymyxa DSM 365
Metab. Eng.
61
381-388
2020
-
-
-
-
1
-
-
-
-
-
-
2
-
4
-
-
-
-
-
-
-
-
4
-
2
1
-
-
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1
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-
2
-
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-
-
2
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
4
-
1
-
-
-
1
-
-
-
-
1
1
-
-
-
762382
Muschallik
-
Synthesis of alpha-hydroxy ke ...
Alkalihalobacillus clausii, Alkalihalobacillus clausii DSM 8716T
RSC Adv.
10
12206-12216
2020
-
-
1
-
1
-
-
-
-
1
-
2
-
2
-
-
1
-
-
-
-
-
8
-
5
1
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
1
-
2
-
-
-
1
-
-
-
-
8
-
1
-
-
-
1
-
-
-
-
1
1
-
-
-
760463
Gong
-
Cloning, expression and chara ...
Bacillus thuringiensis serovar kurstaki, Bacillus thuringiensis serovar kurstaki ACCC 10066
Biocatal. Agricult. Biotechnol.
22
10137
2019
1
-
1
-
-
-
2
2
-
1
-
4
-
2
-
-
1
-
-
-
-
1
8
1
3
2
-
1
-
2
-
1
2
-
1
-
1
-
1
2
-
-
-
-
2
-
2
-
1
-
4
-
-
-
1
-
-
-
1
8
1
2
-
1
-
2
-
1
1
-
1
1
-
-
-
761834
Zhang
Synthetic engineering of Cory ...
Bacillus subtilis subsp. subtilis, Bacillus subtilis subsp. subtilis 168
Microb. Cell Fact.
18
128
2019
-
-
1
-
1
-
-
-
-
-
-
2
-
5
-
-
-
-
-
-
-
-
2
-
2
1
-
-
-
1
-
-
2
-
-
-
-
-
1
2
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
756608
Liu
2,3-Butanediol catabolism in ...
Pseudomonas aeruginosa, Pseudomonas aeruginosa ATCC 15692, Pseudomonas aeruginosa 1C, Pseudomonas aeruginosa PRS 101, Pseudomonas aeruginosa DSM 22644, Pseudomonas aeruginosa CIP 104116, Pseudomonas aeruginosa LMG 12228, Pseudomonas aeruginosa JCM 14847
Environ. Microbiol.
20
3927-3940
2018
-
-
1
-
-
-
-
1
-
1
-
16
-
11
-
-
-
-
-
-
8
-
48
-
5
1
-
-
-
1
-
-
2
-
-
-
-
-
1
2
-
-
-
-
-
-
1
-
1
-
16
-
-
-
-
-
-
8
-
48
-
1
-
-
-
1
-
-
-
-
2
2
-
-
-
760411
Dias
2,3-Butanediol production by ...
Paenibacillus brasilensis, Paenibacillus brasilensis PB24
Appl. Microbiol. Biotechnol.
102
8773-8782
2018
-
1
1
-
-
-
-
-
-
-
-
2
-
2
-
-
-
-
-
-
-
-
4
-
4
-
-
-
-
-
-
-
1
-
-
-
-
1
1
1
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
2
2
-
-
-
740952
Liang
Selection of an endogenous 2,3 ...
Escherichia coli, Escherichia coli BW25113
Metab. Eng.
39
181-191
2017
-
-
-
-
-
-
-
2
-
-
-
-
-
32
-
-
-
-
-
-
-
-
4
-
-
-
-
-
2
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
2
-
-
-
-
-
1
1
-
2
2
760716
Yamada
Efficient production of 2,3-b ...
Bacillus amyloliquefaciens, Bacillus amyloliquefaciens UCMB5033
Biores. Technol.
245
1558-1566
2017
-
-
1
-
1
-
-
-
-
-
-
2
-
3
-
-
-
-
-
1
-
-
2
-
4
-
-
-
-
-
-
-
2
-
-
-
-
-
1
2
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
761565
Cui
Highly efficient bioreduction ...
Bacillus subtilis subsp. subtilis, Bacillus subtilis subsp. subtilis 168
J. Biotechnol.
243
1-9
2017
-
1
1
-
1
-
-
2
-
-
-
2
1
3
-
-
1
-
-
-
-
-
6
-
2
1
-
1
-
1
-
1
2
-
-
-
-
1
1
2
-
1
-
-
-
-
2
-
-
-
2
1
-
-
1
-
-
-
-
6
-
1
-
1
-
1
-
1
-
-
-
-
-
-
-
761646
Li
Reducing diacetyl production ...
Saccharomyces uvarum, Saccharomyces uvarum WY1
J. Ind. Microbiol. Biotechnol.
44
1541-1550
2017
-
-
1
-
1
-
-
-
-
-
-
2
-
5
-
-
-
-
-
-
-
-
2
-
3
-
-
-
-
-
-
-
2
-
-
-
-
-
1
2
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
1
2
2
1
-
-
762259
Zhang
Effect of deletion of 2,3-but ...
Bacillus subtilis, Bacillus subtilis BS168D
Prep. Biochem. Biotechnol.
47
761-767
2017
1
-
1
-
1
-
-
-
-
-
-
2
-
5
-
-
-
-
-
1
-
-
2
-
3
-
-
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-
-
-
-
2
-
-
-
1
-
1
2
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
2
2
-
-
-
737512
Rados
Stereospecificity of Corynebac ...
Corynebacterium glutamicum, Corynebacterium glutamicum ATCC 13032
Appl. Microbiol. Biotechnol.
100
10573-10583
2016
-
-
1
-
-
-
-
4
-
-
-
4
-
37
-
-
1
-
-
-
-
-
10
-
3
-
-
-
-
-
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
4
-
-
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4
-
-
-
1
-
-
-
-
10
-
-
-
-
-
-
-
-
-
-
-
-
-
4
4
737519
de Oliveira
Synthetic operon for (R,R)-2,3 ...
Bacillus subtilis, Bacillus subtilis WN1038
Appl. Microbiol. Biotechnol.
100
719-728
2016
-
-
1
-
-
-
-
-
-
-
2
2
-
8
-
-
-
-
-
-
-
-
2
2
1
-
-
-
-
-
-
-
1
-
-
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-
-
1
1
-
-
-
-
-
-
-
-
-
2
2
-
-
-
-
-
-
-
-
2
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
738345
Ge
-
Contracted but effective: Prod ...
Bacillus licheniformis, Bacillus licheniformis MW3
Green Chem.
18
4693-4703
2016
-
-
1
-
-
-
-
-
-
-
-
4
-
3
-
-
1
-
-
-
14
-
20
-
3
-
-
-
-
-
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
4
-
-
-
1
-
-
14
-
20
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
739694
Bae
Efficient production of acetoi ...
Saccharomyces cerevisiae, Saccharomyces cerevisiae BY4741
Sci. Rep.
6
27667
2016
-
-
-
-
-
-
-
-
-
-
-
-
-
76
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
1
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
737847
Kang
Enhanced production of 2,3-but ...
Bacillus sp. (in: Bacteria), Bacillus sp. (in: Bacteria) BRC1
Bioprocess Biosyst. Eng.
38
299-305
2015
-
-
1
-
-
-
-
4
-
1
-
2
-
2
-
-
1
-
-
-
-
-
14
-
2
1
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10
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5
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5
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10
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246407
Höhn-Bentz
Bacterial 2,3-butanediol dehyd ...
Gluconobacter oxydans, Aeromonas hydrophila, Bacillus subtilis, Paenibacillus polymyxa
Arch. Microbiol.
116
197-203
1978
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