# Patent

## Title
Biosynthetic pathways and methods

## Abstract

This disclosure describes a recombinant microbial cells and methods of making and using such recombinant microbial cells. Generally, the recombinant cells may be modified to exhibit increased biosynthesis of a TCA derivative compared to a wild-type control. In some embodiments, the TCA derivative can include 1,4-butanediol. In various embodiments, the microbial cell is a fungal cell or a bacterial cell. In some embodiments, the increased biosynthesis of the TCA derivative can include an increase in xylose dehydrogenase activity, xylonolactonase activity, xylonate dehydratase activity, or 2-keto-3-deoxyaldonic acid dehydratase activity.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is the § 371 U.S. National Stage of International Application No. PCT/US2013/076118, filed 18 Dec. 2013, which claims priority to U.S. Provisional Patent Application Ser. No. 61/738,752, filed Dec. 18, 2012 and U.S. Provisional Patent Application Ser. No. 61/821,490, filed May 9, 2013, each of which is incorporated herein by reference.


SUMMARY
This disclosure describes, in one aspect, a recombinant microbial cell modified to exhibit increased biosynthesis of a TCA derivative compared to a wild-type control. In some embodiments, the TCA derivative can include 1,4-butanediol. In various embodiments, the microbial cell is a fungal cell or a bacterial cell. In some embodiments, the increased biosynthesis of the TCA derivative can include an increase in xylose dehydrogenase activity, xylonolactonase activity, xylonate dehydratase activity, or 2-keto-3-deoxyaldonic acid dehydratase activity.
In another aspect, this disclosure describes a method that generally includes incubating any embodiments of the recombinant cell summarized above in medium that includes a carbon source under conditions effective for the recombinant cell to produce a TCA derivative. In some embodiments, the TCA derivative can include 1,4-butanediol. In some embodiments, the carbon source can include xylose, arabinose, glucaric acid, galactaric acid, or hydroxyproline. In some embodiments, the increased biosynthesis of the TCA derivative can include an increase in pentose dehydrogenase activity, pentonolactonase activity, aldonic acid dehydratase activity, or 2-keto-3-deoxyaldonic acid dehydratase activity. In other embodiments, the increased biosynthesis of the TCA derivative can include an increase in hexic acid dehydratase activity or 5-dehydro-4-deoxyglucarate dehydratase activity.
In another aspect, this disclosure describes a method that generally includes introducing into a host cell a heterologous polynucleotide encoding at least one polypeptide that catalyzes conversion of a carbon source to a TCA derivative, wherein the at least one polypeptide is operably linked to a promoter so that the modified host cell catalyzes conversion of the carbon source to TCA derivative. In some embodiments, the TCA derivative can include 1,4-butanediol. In some embodiments, the carbon source can include xylose.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. The 1,4-butanediol synthetic pathway in E. coli. (A) The synthetic pathway for 1,4-butanediol from glucose and xylose. Abbreviations: G-6P, glucose-6-phosphate; F-6P, fructose-6-phosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate. (B) Synthetic operon for protein overexpression to drive the xylose towards 2,5-dioxopentanoic acid (left), and then drive 2,5-dioxopentanoic acid towards to 1,4-butanediol (right).
FIG. 2. A, scheme of the organization of conserved genetic clusters involved in the pentose, hexaric acid, and hydroxyproline degradation. Analogous functions are indicated in the same degree of shading. Coding region sizes and distances are not to scale. Protein family numbers are displayed below each coding region according to Clusters of Orthologous Groups of proteins classification system. The coding regions indicated in white or gray encode the following proteins: araA, transcriptional regulator; araF-araH, 1-Ara ABC transporter (periplasmic 1-Ara binding protein, ATP-binding protein, permease); rrnAC3038, heat shock protein X; ycbE, glucarate/galactarate permease; ycbG, transcriptional regulator; PP1249, hydroxyproline permease. B, schematic representation of the convergence of catabolic pathways for pentoses, hexaric acids, and hydroxyproline at the level of 2,5-dioxopentanoate. Enzymatic activities are indicated by their EC number. Dashed lines indicate proposed spontaneous reactions.
FIG. 3. An engineered 1,4-butanediol synthetic pathway in E. coli. 
FIG. 4. An exemplary engineered metabolic pathway from 2,5-dioxopentanoic acid to 1,4-butanediol.
FIG. 5. An exemplary engineered metabolic pathway from 2,5-dioxopentanoic acid to 1,4-butanediol.
FIG. 6. An exemplary engineered metabolic pathway from D-arabinose to 2,5-dioxopentanoic acid.
FIG. 7. An exemplary engineered metabolic pathway from D-xylose to 2,5-dioxopentanoic acid.
FIG. 8. An exemplary engineered metabolic pathway from L-arabinose to 2,5-dioxopentanoic acid.
FIG. 9. An exemplary engineered metabolic pathway from D-glucaric acid to 2,5-dioxopentanoic acid.
FIG. 10. An exemplary engineered metabolic pathway from D-galactaric acid to 2,5-dioxopentanoic acid.
FIG. 11. An exemplary engineered metabolic pathway from 4(R)-hydroxy-L-proline to 2,5-dioxopentanoic acid.
FIG. 12. A plasmid map of YEplac195-xylBCDX (13945 bp).
FIG. 13. A plasmid map of YEplac11-KivDyqhD (9687 bp).
FIG. 14. Gas chromatography data showing production of 1,4-butanediol by genetically engineered S. cerevisiae. 



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
This disclosure describes a novel full biosynthetic pathway to biosynthesize high-volume TCA derivatives such as succinate, amino acids, and 1,4-butanediol from xylose by an engineered microbe. The TCA cycle can lead to many commercially important biobased chemicals such as, for example, amino acids (e.g., glutamate, threonine and lysine) and organic acids (e.g., succinate, maleate and fumarate). Here we report the engineering of a shortcut metabolic pathway to TCA cycle. The process from xylose to TCA only involves five steps as compared to conventional published pathways that include more than 20 steps. Because our pathway includes fewer steps from xylose to the TCA cycle, our pathway can produce TCA derivatives with the production of less by-product and, therefore, achieve higher yields than conventional biosynthetic pathways.
We have selected the TCA derivative 1,4-butanediol as a model product to demonstrate the generality of our novel biosynthetic pathway. 1,4-butanediol is a major commodity chemical; 2.5 million tons of 1,4-butanediol are used per year to make, for example, plastics, polyesters, and spandex fibers. 1,4-butanediol also can react, for example, with dicarboxylic acids to yield polyesters, with diisocyanates to yield polyurethanes, and with phosgene to yield chloroformates. Because our pathway permits the biosynthesis of 1,4-butanediol from, for example, xylose in only six steps from xylose to 1,4-butanediol, 1,4-butanediol may be biosynthesized with less by-product being formed and, therefore, a higher yield. For example, our pathway can produce 1.0 g/L 1,4-butanediol from 20 g/L xylose.
1,4-butanediol currently is manufactured from petroleum-based feedstocks such as acetylene, butane, propylene, and butadiene. Given the industrial importance of 1,4-butanediol as a chemical intermediate and the issues associated with petroleum feedstocks, alternative low-cost renewable biosynthetic routes from sugars have been sought. However, the highly reduced nature of 1,4-butanediol relative to carbohydrates has thwarted attempts thus far to develop effective pathways and organisms for direct production.
1,4-butanediol has been reported to be synthesized from glucose and xylose by engineered E. coli in which the succinyl-CoA intermediate was converted into succinate semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, 4-hydroxybutyraldehyde, and 1,4-butanediol by multiple enzymes from various organisms. This process involves around 20 chemical steps that include the pentose phosphate pathway, glycolysis, the TCA cycle, and designed artificial downstream metabolic steps. In contrast, this disclosure describes a shortcut pathway that requires only six steps (FIG. 1A).
D-xylose is converted by Caulobacter crescentus sequentially to D-xylonolactone, D-xylonate (D-xylonoic acid), 2-keto-3-deoxy-xylonate (2-oxo-4(S),5-dihydroxy-pentanoic acid), then α-ketoglutaric semialdehyde (2,5-dioxopentanoic acid) by, respectively, xylose dehydrogenase (xylB), xylonolactonase (xylC), xylonate dehydrogenase (xylD), Kda dehydratase (xylX). We cloned the coding regions of these enzymes into a single plasmid (pBDO-1), which was then transformed into an E. coli host cell. The host cell was then further modified to include a second plasmid that included a decarboxylase and an alcohol dehydrogenase. The decarboxylase converts the α-ketoglutaric semialdehyde to succinaldehyde; the alcohol dehydrogenase reduces the succinaldehyde to 1,4-butanediol (FIG. 1A). In some embodiments, the second plasmid was identified as pBDO-3 and included the coding regions of benzoylformate decarboxylase BFD (Pseudomonas putida) and an alcohol dehydrogenase of yqhD (E. coli). In other embodiments, the second plasmid was identified as pBDO-4 and included the decarboxylase of KIVD (Lactococcus lactis) and alcohol dehydrogenase of yqhD (E. coli).
The E. coli host cell possesses an endogenous xylose metabolism pathway that includes xylA, yjhH and yagE. To improve the product yield from xylose to 1,4-butanediol, expression of these three coding regions were inhibited. The host cell strain SBDO-1 is based on E. coli BW25113 in which xylA, yjhH, and yagE are knocked out so that SBDO-1 cannot metabolize xylose. Strain SBDO-2, carrying plasmid pBDO-1, also cannot metabolize xylose.
The strain SBDO-3, which is based on SBDO-2 but carries plasmid pBDO-2 that expresses α-ketoglutaric semialdehyde dehydrogenase xylA, can consume xylose quickly. These results indicate that the endogenous xylose utilization pathway in E. coli was blocked fully by the ΔxylA, ΔyjhH, and ΔyagE deletions in SBDO-1. Moreover, these results demonstrate that the C. crescentus enzymes function in E. coli. Consequently, xylose metabolism observed in SBDO-4 and SBDO-5 is attributable to the xylose pathway from C. crescentus that we engineered into the host cell. To produce 1,4-butanediol, plasmids pBDO-3 or pBDO-4, each of which expresses the same alcohol dehydrogenase but a different decarboxylase, were introduced into strain SBDO-2 strain. After two days of fermentation, strain SBDO-4 (carrying pBDO-3) produced 0.25 g/L 1,4-butanediol with 0.1 g/L 1,2,4-butanetriol (a by-product); strain SBDO-5 (carrying pBDP-4) produced 1.0 g/L 1,4-butanediol with 4.0 g/L 1,2,4-butanetriol. Thus, the kivD encoded on pBDO-4 and carried by strain SBDO-5 provides better yield of 1,4-butanediol than BFD. Other than 1,2,4-butanetriol, no other byproducts were detected in significant amounts in the fermentation broth, suggesting that our new 1,4-butanediol producing pathway has higher 1,4-butanediol yield as compared with the published pathway (Yim et al., 2011. Nat. Chem. Biol. 7:445-452).
Thus, in one aspect, the invention provides recombinant microbial cell modified to exhibit increased biosynthesis of a TCA derivative compared to a wild-type control.
While described above in the context of an exemplary embodiment in which the TCA derivative is a 1,4-butanediol, the recombinant cells and methods described herein can provide TCA derivatives other than 1,4-butanediol. Exemplary alternative TCA derivatives include, for example, succinate, fumarate, malate, glutamate, lysine, threonine, 4-hydroxybutyrate, and products synthesizable from a product of the TCA cycle in one, two, three, four, or five enzymatic steps. In some of these embodiments, one or more enzymes involved in the synthesis of the TCA derivative may be heterologous to the host cell and, therefore, provided recombinantly. Exemplary TCA derivative products and exemplary enzymes involved in the synthesis of the exemplary TCA derivative products are listed in Table 1. For any embodiment in which the identified enzyme is not endogenous to a host cell, the enzyme may be introduced into the host cell to produce a recombinant cell as described herein.







TABLE 1







Exemplary enzymes, enzyme sources, native substrates, and TCA derivative products















Encoding
Accession No.;

TCA derivative
SEQ


Common Name
Organism
gene
GI No.
Native Substrate
product
ID NO













D-arabinose dehydrogenase
















alcohol
Sulfolobus
SSO1300
NP_342747.1;
D-arabinose
D-arabinonic
1


dehydrogenase
solfataricus

GI:15898142

acid from D-



(AraDH)




arabinose











D-arabinonate dehydratase
















arabinonate
Sulfolobus
SSO3124
NP_344435.1;
D-arabinonic
2-oxo-4(S),5-
6


dehydratase (AraD)
solfataricus

GI:15899830
acid
dihydroxy-








pentanoic acid











2-Keto-3-deoxy-D-arabinonate Dehydratase
















2-keto-4-
Sulfolobus
SSO3118
NP_344431.1;
2-oxo-4(S),5-
2,5-
11


pentenoate
solfataricus

GI:15899826
dihydroxy-
dioxopentanoic



hydratase (KdaD)



pentanoic acid
acid











2,5-dioxopentanoate dehydrogenase
















aldehyde
Sulfolobus
SSO3117
NP_344430.1;
2,5-
2-oxoglutaric
16


dehydrogenase
solfataricus

GI:15899825
dioxopentanoic
acid



(DopDH)



acid












2,5-dioxovalerate dehydrogenase
















2,5-dioxovalerate
Bacillus
YcbD
NP_388129.1;
2,5-
2-oxoglutaric
21


dehydrogenase
subtilis

GI:16077316
dioxopentanoic
acid



(YcbD)



acid












D-xylose dehydrogenase
















D-Xylose
Caulobacter
CC0821
YP_002516237.1;
D-xylose
D-xylonolactone
26


dehydrogenase
crescentus

GI:221233801





(XylB)
















D-xylonolactonase
















D-xylonolactonase
Caulobacter
CC0820
YP_002516236.1;
D-xylonolactone
D-xylonic acid
31


(XylC)
crescentus

GI:221233800













D-xylonate dehydratase
















D-xylonate
Caulobacter
CC0819
NP_419636.1
D-xylonic acid
2-oxo-4(S),5-
36


dehydratase (XylD)
crescentus

GI:16125072

dihydroxy-








pentanoic acid











2-Keto-3-deoxy-D-arabinonate dehydratase
















2-keto-4-
Caulobacter
CC0823
NP_419640.1;
2-oxo-4(S),5-
2,5-
41


pentenoate
crescentus

GI:16125076
dihydroxy-
dioxopentanoic



hydratase (XylX)



pentanoic acid
acid











L-arabinose dehydrogenase
















dehydrogenase
Burkholderia
BTH_II1629
YP_439823.1;
L-arabinose
L-
46


(AraE)
thailandensis

GI:83716868

arabinonolactone




E264



from L-arabinose











L-arabinonolactonase
















L-
Burkholderia
BTH_II1625
YP_439819.1;
L-
L-arabinonic acid
51


arabinonolactonase
thailandensis

GI:83717359
arabinonolactone




(AraI)
E264















L-arabinonate dehydratase
















L-
Burkholderia
BTH_II1632
YP_439826.1;
L-arabinonic acid
2-oxo-4(R),5-
56


arabinonatedehydratase
thailandensis

GI:83718062

dihydroxy-



(AraB)
E264



pentanoic acid











2-Keto-3-deoxy-L-arabinonate Dehydratase
















dihydrodipicolinate
Burkholderia
BTH_II1630
YP_439824.1;
2-oxo-4(R),5-
2,5-
61


synthase (AraD)
thailandensis

GI:83717217
dihydroxy-
dioxopentanoic




E264


pentanoic acid
acid











D-glucarate dehydratase
















D-glucarate
Bacillus
YcbF
NP_388131.2;
D-glucaric acid
4-deoxy-5-keto-
66


dehydratase
subtilis

GI:255767063

D-glucaric acid



(YcbF)
















D-galactarate dehydratase
















D-galactarate
Bacillus
YcbH
NP_388133.2;
D-galactaric acid
4-deoxy-5-keto-
71


dehydratase
subtilis

GI:255767065

D-glucaric acid



(YcbH)
















5-dehydro-4-deoxyglucarate dehydratase
















5-dehydro-4-
Bacillus
YcbC
NP_388128.2;
4-deoxy-5-keto-
2,5-
76


deoxyglucarate
subtilis

GI:255767061
D-glucaric acid
dioxopentanoic



dehydratase




acid



(YcbC)
















Amino acid transporter LysE
















Amino acid
Pseudomonas
PP_1248
NP_743408.1;
4(R)-hydroxy-L-
4(R)-hydroxy-D-
81


transporter LysE
putida

GI:26987983
proline
proline



(HypE)
















PP_1245
















Hypothetical
Pseudomonas
PP_1245
NP_743405.1;
4(R)-hydroxy-D-
2-carboxy-4(R)-
86


protein of PP_1245
putida

GI:26987980
proline
hydroxy-pyrroline











PP_1247
















Hypothetical
Pseudomonas
PP_1247
NP_743407.1;
2-carboxy-4(R)-
2,5-
91


protein of PP_1247
putida

GI:26987982
hydroxy-pyrroline
dioxopentanoic








acid











PP_1246
















Hypothetical
Pseudomonas
PP_1246
NP_743406.1;
2,5-
2-oxoglutaric
93


protein of PP_1246
putida

GI:26987981
dioxopentanoic
acid







acid












Alpha-ketoisovalerate decarboxylase
















alpha-
Lactococcus
KivD
YP_003353820.1;
2,5-
Succinaldehyde
98


ketoisovalerate
lactis

GI:281491840
dioxopentanoic




decarboxylase



acid












Alcohol dehydrogenase (YqhD)
















alcohol
E. coli
yqhD
YP_001459806.1;
Succinaldehyde
1,4-butanediol
103


dehydrogenase


GI:157162488









In addition to the enzymes listed in Table 1, homologs of the listed enzymes may be used. Thus, as an alternative to AraDH (SEQ ID NO:1), one may use, for example, any of the polypeptides depicted in SEQ ID NO:2-5; as an alternative to AraD (SEQ ID NO:6), one may use, for example, any of the polypeptides depicted in SEQ ID NO: 7-10; as an alternative to Kda (SEQ ID NO:11), one may use, for example, any of the polypeptides depicted in SEQ ID NO: 12-15; as an alternative to DopDH (SEQ ID NO:16), one may use, for example, any of the polypeptides depicted in SEQ ID NO:17-20; as an alternative to YcbD (SEQ ID NO:21), one may use, for example, any of the polypeptides depicted in SEQ ID NO:22-25; as an alternative to XylB (SEQ ID NO:26), one may use, for example, any of the polypeptides depicted in SEQ ID NO:27-30; as an alternative to XylC (SEQ ID NO:31), one may use, for example, any of the polypeptides depicted in SEQ ID NO:32-35; as an alternative to XylD (SEQ ID NO:36), one may use, for example, any of the polypeptides depicted in SEQ ID NO:37-40; as an alternative to XylX (SEQ ID NO:41), one may use, for example, any of the polypeptides depicted in SEQ ID NO:42-45; as an alternative to AraE (SEQ ID NO:46), one may use, for example, any of the polypeptides depicted in SEQ ID NO:47-50; as an alternative to AraI (SEQ ID NO:51), one may use, for example, any of the polypeptides depicted in SEQ ID NO:52-55; as an alternative to AraB (SEQ ID NO:56), one may use, for example, any of the polypeptides depicted in SEQ ID NO:57-60; as an alternative to AraD (SEQ ID NO:61), one may use, for example, any of the polypeptides depicted in SEQ ID NO:62-65; as an alternative to YcbF (SEQ ID NO:66), one may use, for example, any of the polypeptides depicted in SEQ ID NO:67-70; as an alternative to YcbH (SEQ ID NO:71), one may use, for example, any of the polypeptides depicted in SEQ ID NO:72-75; as an alternative to YcbC (SEQ ID NO:76), one may use, for example, any of the polypeptides depicted in SEQ ID NO:77-80; as an alternative to HypE (SEQ ID NO:81), one may use, for example, any of the polypeptides depicted in SEQ ID NO:82-85; as an alternative to PP_1245 (SEQ ID NO:86), one may use, for example, any of the polypeptides depicted in SEQ ID NO:87-90; as an alternative to PP_1247 (SEQ ID NO:91), one may use, for example, the polypeptide depicted in SEQ ID NO:92; as an alternative to PP_1246 (SEQ ID NO:93), one may use, for example, any of the polypeptides depicted in SEQ ID NO:94-97; as an alternative to alpha-ketoisovalerate decarboxylase (SEQ ID NO:98), one may use, for example, any of the polypeptides depicted in SEQ ID NO:99-102; as an alternative to YqhD (SEQ ID NO:103), one may use, for example, any of the polypeptides depicted in SEQ ID NO:104-107.
In some cases, the wild-type control may be unable to produce the TCA derivative and, therefore, an increase in the biosynthesis of a particular product may reflect any measurable biosynthesis of that product. In certain embodiments, an increase in the biosynthesis of a TCA derivative can include biosynthesis sufficient for a culture of the microbial cell to accumulate the TCA derivative to a predetermine concentration.
The predetermined concentration may be any predetermined concentration of the product suitable for a given application. Thus, a predetermined concentration may be, for example, a concentration of at least 0.1 g/L such as, for example, at least 0.25 g/L, at least 0.5 g/L, at least 1.0 g/L, at least 2.0 g/L, at least 3.0 g/L, at least 4.0 g/L, at least 5.0 g/L, at least 6.0 g/L, at least 7.0 g/L, at least 8.0 g/L, at least 9.0 g/L, at least 10 g/L, at least 20 g/L, at least 50 g/L, at least 100 g/L, or at least 200 g/L.
While described above in the context of an exemplary embodiment in which the host cell is E. coli, the recombinant cells described herein can be constructed, and the methods of making and using the recombinant cells can be performed, using any suitable host cell.
Thus, the recombinant cell can be, or be derived from, any suitable microbe including, for example, a prokaryotic microbe or a eukaryotic microbe. As used herein, the term “or derived from” in connection with a microbe simply allows for the “host cell” to possess one or more genetic modifications before being modified to exhibit the indicated increased biosynthetic activity. Thus, the term “recombinant cell” encompasses a “host cell” that may contain nucleic acid material from more than one species before being modified to exhibit the indicated biosynthetic activity.
In some embodiments, the host cell may be selected to possess one or more natural physiological activities. For example, the host cell may be photosynthetic (e.g., cyanobacteria) or may be cellulolytic (e.g., Clostridium cellulolyticum).
In some embodiments, the recombinant cell may be, or be derived from, a eukaryotic microbe such as, for example, a fungal cell. In some of these embodiments, the fungal cell may be, or be derived from, a member of the Saccharomycetaceae family such as, for example, Saccharomyces cerevisiae, Candida rugosa, or Candida albicans. 
In other embodiments, the recombinant cell may be, or be derived from, a prokaryotic microbe such as, for example, a bacterium. In some of these embodiments, the bacterium may be a member of the phylum Protobacteria. Exemplary members of the phylum Protobacteria include, for example, members of the Enterobacteriaceae family (e.g., Escherichia coli) and, for example, members of the Pseudomonaceae family (e.g., Pseudomonas putida). In other cases, the bacterium may be a member of the phylum Firmicutes. Exemplary members of the phylum Firmicutes include, for example, members of the Bacillaceae family (e.g., Bacillus subtilis), members of the Clostridiaceae family (e.g., Clostridium cellulolyticum) and, for example, members of the Streptococcaceae family (e.g., Lactococcus lactis). In other cases, the bacterium may be a member of the phylum Cyanobacteria.
In some embodiments, the increased biosynthesis of the TCA derivative compared to a wild-type control can include an increase in activity of one or more enzymes involved in the metabolism of the carbon source (e.g., xylose or arabinose). Such enzymes may be found in the proteome of microbes such as, for example, Sulfolobus solfataricus, Caulobacter crescentus, Burkholderia thailandensis, Haloarcula marismortui, Bacillus subtilis, and Pseudomonas putida. Exemplary enzymes, shown in the context of their native metabolic pathways, are shown in FIG. 2. So, for example, increased biosynthesis of the TCA derivative can include an increase in activity of one or more enzymes involved in the metabolism of D-xylose in Caulobacter crescentus such as, for example, xylose dehydrogenase (FIG. 2, CC0821) activity, xylonolactonase (FIG. 2, CC0819) activity, xylonate dehydrogenase (FIG. 2, CC0822) activity, and 2-keto-3-deoxyaldonic acid dehydratase (FIG. 2, CC0823) activity compared to the wild-type control.
In some embodiments, the increased biosynthesis of the TCA derivative compared to a wild-type control can further include an increase in benzoylformate decarboxylase activity and an increase in alcohol dehydrogenase activity. In some of these embodiments, the benzoylformate decarboxylase can include BFD of Pseudomonas putida. In some of these embodiments, the alcohol dehydrogenase can include yqhD of E. coli. 
In some embodiments, the increased biosynthesis of the TCA derivative compared to a wild-type control can further include an increase in decarboxylase activity and an increase in alcohol dehydrogenase activity. In some of these embodiments, the decarboxylase can include KIVD of Lactococcus lactis. In some of these embodiments, the alcohol dehydrogenase can include yqhD of E. coli. See, e.g., Example 2 and FIGS. 12-14.
In some embodiments, the recombinant cell can include an engineered metabolic pathway designed to permit the recombinant cell to increase its consumption of a particular carbon source compared to a wild-type control. Exemplary metabolic pathways are illustrated in, for example, FIG. 6 through FIG. 11. Accordingly, exemplary carbon sources include, for example, arabinose, xylose, arabinose, glucaric acid, galactaric acid, or hydroxyproline. In other embodiments, the recombinant cell may be designed to consume a uronic acid such as, for example, galacturonic acid and/or glucuronic acid as a carbon source. In such embodiments, a heterologous polynucleotide that encodes a uronate dehydrogenase enzyme may be introduced into the recombinant cell to confer to the recombinant cell the ability to convert uronic acid to aldonic acid. In still other embodiments, the recombinant cell can utilize a carbon source that includes, for example, glucose, cellulose, galacturonic acid, glucuronic acid, CO2, or glycerol. In some of these embodiments, the recombinant cell may be further modified to convert the carbon source (e.g., glucose) to one or more of the carbon sources (e.g., xylose and/or a hexaric acid such as, e.g., glucaric acid) that is an entry point to one or more of the engineered pathways described herein.
FIG. 6 shows an exemplary metabolic pathway that permits a recombinant cell to use D-arabinose as a carbon source for the production of 2,5-dioxopentanoic acid. In this example, the recombinant cell can include an enzyme that can convert D-arabinose into D-arabinolactone such as, for example, a pentose dehydrogenase. One example of a suitable pentose dehydrogenase includes AraDH from Sulfolobus solfataricus. The pentose dehydrogenase can provide catalytic conversion of D-arabinose into D-arabinolactone that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 6 also includes an enzyme that can convert D-arabinonic acid to 2-oxo-4(S),5-dihydroxy-pentanoic acid such as, for example, an aldonic acid dehydratase. One example of a suitable aldonic acid dehydratase includes AraD from Sulfolobus solfataricus. The aldonic acid dehydratase can provide catalytic conversion of D-arabinonic acid into 2-oxo-4(S),5-dihydroxy-pentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 6 also includes an enzyme that can convert 2-oxo-4(S),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid such as, for example, a 2-keto-3-deoxyaldonic acid dehydratase. On example of a suitable 2-keto-3-deoxyaldonic acid dehydratase includes KdaD from Sulfolobus solfataricus. The 2-keto-3-deoxyaldonic acid dehydratase can provide catalytic conversion of 2-oxo-4(S),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
FIG. 7 shows an exemplary metabolic pathway that permits a recombinant cell to use D-xylose as a carbon source for the production of 2,5-dioxopentanoic acid. In this example, the recombinant cell can include an enzyme that can convert D-xylose to D-xylonolactone such as, for example, a pentose dehydrogenase. Exemplary suitable pentose dehydrogenases include XylB from Caulobacter crescentus or rrnAC3034 from Haloarcula marismortui. The pentose dehydrogenase can provide catalytic conversion of D-xylose to D-xylonolactone that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 7 also includes an enzyme that can convert D-xylonolactone to D-xylonic acid such as, for example, a pentonolactonase. Exemplary suitable pentonolactonases include XylC from Caulobacter crescentus or rrnAC3033 from Haloarcula marismortui. The pentonolactonase can provide catalytic conversion of D-xylonolactone to D-xylonic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 7 also includes an enzyme that can convert D-xylonic acid to 2-oxo-4(S),5-dihydroxy-pentanoic acid such as, for example, an aldonic acid dehydratase. Exemplary suitable aldonic acid dehydratases include XylD front Caulobacter crescentus or rrnAC3032 from Haloarcula marismortui. The aldonic acid dehydratase can provide catalytic conversion of D-xylonic acid into 2-oxo-4(S),5-dihydroxy-pentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 7 also includes an enzyme that can convert 2-oxo-4(S),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid such as, for example, a 2-keto-3-deoxyaldonic acid dehydratase. Exemplary suitable 2-keto-3-deoxyaldonic acid dehydratases include XylX from Caulobacter crescentus or rrnAC3039 from Haloarcula marismortui. The 2-keto-3-deoxyaldonic acid dehydratase can provide catalytic conversion of 2-oxo-4(S),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
FIG. 8 shows an exemplary metabolic pathway that permits a recombinant cell to use L-arabinose as a carbon source for the production of 2,5-dioxopentanoic acid. In this example, the recombinant cell can include an enzyme that can convert L-arabinose to L-arabinolactone such as, for example, a pentose dehydrogenase. One example of a suitable pentose dehydrogenase includes AraE from Burkholderia thailandensis. The pentose dehydrogenase can provide catalytic conversion of L-arabinose to L-arabinolactone that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 8 also includes an enzyme that can convert L-arabinolactone to L-arabinonic acid such as, for example, a pentonolactonase. One example of a suitable pentonolactonase includes AraI from Burkholderia thailandensis. The pentonolactonase can provide catalytic conversion of L-arabinolactone to L-arabinonic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 8 also includes an enzyme that can convert L-arabinonic acid to 2-oxo-4(R),5-dihydroxy-pentanoic acid such as, for example, an aldonic acid dehydratase. One example of a suitable aldonic acid dehydratase includes AraB from Burkholderia thailandensis. The aldonic acid dehydratase can provide catalytic conversion of L-arabinonic acid to 2-oxo-4(R),5-dihydroxy-pentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 8 also includes an enzyme that can convert 2-oxo-4(R),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid such as, for example, a 2-keto-3-deoxyaldonic acid dehydratase. One example of a suitable 2-keto-3-deoxyaldonic acid dehydratase includes AraD from Burkholderia thailandensis. The 2-keto-3-deoxyaldonic acid dehydratase can provide catalytic conversion of 2-oxo-4(R),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
FIG. 9 shows an exemplary metabolic pathway that permits a recombinant cell to use D-glucaric acid as a carbon source for the production of 2,5-dioxopentanoic acid. In this example, the recombinant cell can include an enzyme that can convert D-glucaric acid to 4-deoxy-5-keto-D-glucaric acid such as, for example, an aldonic acid dehydratase. Suitable exemplary aldonic acid dehydratases include YcbF from Bacillus subtilis. The aldonic acid dehydratase can provide catalytic conversion of D-glucaric acid to 4-deoxy-5-keto-D-glucaric acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 9 also includes an enzyme that can convert 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid such as, for example, a 2-keto-3-deoxyaldonic acid dehydratase. One example of a suitable 2-keto-3-deoxyaldonic acid dehydratase includes YcbC from Bacillus subtilis. The 2-keto-3-deoxyaldonic acid dehydratase can provide catalytic conversion of 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
FIG. 10 shows an exemplary metabolic pathway that permits a recombinant cell to use D-galactaric acid as a carbon source for the production of 2,5-dioxopentanoic acid. In this example, the recombinant cell can include an enzyme that can convert D-galactaric acid to 4-deoxy-5-keto-D-glucaric acid such as, for example, an aldonic acid dehydratase. Suitable exemplary aldonic acid dehydratases include YcbH from Bacillus subtilis. The aldonic acid dehydratase can provide catalytic conversion of D-galactaric acid to 4-deoxy-5-keto-D-glucaric acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 9 also includes an enzyme that can convert 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid such as, for example, a 2-keto-3-deoxyaldonic acid dehydratase. One example of a suitable 2-keto-3-deoxyaldonic acid dehydratase includes YcbC from Bacillus subtilis. The 2-keto-3-deoxyaldonic acid dehydratase can provide catalytic conversion of 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
FIG. 11 shows an exemplary metabolic pathway that permits a recombinant cell to use 4(R)-hydroxy-D-proline as a carbon source for the production of 2,5-dioxopentanoic acid. In this example, the recombinant cell can include an enzyme that can convert 4(R)-hydroxy-D-proline to 4(R)-hydroxy-D-proline. One suitable exemplary enzyme for this embodiment includes, amino acid transporter LysE (HypE) from Pseudomonas. The enzyme can provide catalytic conversion of 4(R)-hydroxy-D-proline to 4(R)-hydroxy-D-proline that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 11 also includes an enzyme that can convert 4(R)-hydroxy-D-proline to 2-carboxy-4(R)-hydroxy-δ-pyrroline. One suitable exemplary enzyme for this embodiment includes, for example, HypOX from Pseudomonas. The enzyme can provide catalytic conversion of 4(R)-hydroxy-D-proline to 2-carboxy-4(R)-hydroxy-δ-pyrroline that is, for example, at least 110% greater than that exhibited by a wild-type control.
The exemplary metabolic pathway illustrated in FIG. 11 also includes an enzyme that can convert 2-oxo-4(R)-5-aminopentanoic acid to 2,5-dioxopentanoic acid such as, for example, a 2-keto-3-deoxyaldonic acid dehydratase. One exemplary 2-keto-3-deoxyaldonic acid dehydratase includes PP1247 from Pseudomonas. The 2-keto-3-deoxyaldonic acid dehydratase can provide catalytic conversion of 2-oxo-4(R)-5-aminopentanoic acid to 2,5-dioxopentanoic acid that is, for example, at least 110% greater than that exhibited by a wild-type control.
The recombinant cell can be engineered to convert the 2,5-dioxopentanoic acid to any desirable TCA derivative. In some embodiments, the recombinant cell can include an α-ketoglutaric semialdehyde dehydrogenase to shunt the 2,5-dioxopentanoic acid into the TCA cycle. In this manner, TCA cycle derivatives such as, for example, succinate, fumarate, malate, glutamate, lysine, threonine, 4-hydroxybutyrate may be produced.
In some embodiments, however, the recombinant cell may be further modified to possess a metabolic pathway for the conversion of 2,5-dioxopentanoic acid to 1,4-butanediol. Exemplary metabolic pathways are illustrated, for example, in FIG. 4 and FIG. 5. The exemplary pathway illustrated in FIG. 4 includes an enzyme that can convert 2,5-dioxopentonoic acid to succinaldehyde such as, for example, a 2-ketoacid decarboxylase or a 2-oxoglutarate decarboxylase. Suitable exemplary enzymes include, for example, Kivd, BFD, and IPDC. The exemplary pathway illustrated in FIG. 4 also includes an enzyme that can convert succinaldehyde to 1,4-butanediol such as, for example, an alcohol dehydrogenase. Suitable exemplary alcohol dehydrogenases include YqhD, ADH6, YjgB, and YahK.
The exemplary pathway illustrated in FIG. 5 includes an enzyme that can convert 2,5-dioxopentonoic acid to 2-keto-5-hydroxy-pentonate such as, for example, an alcohol dehydrogenase. Here again, suitable exemplary alcohol dehydrogenases include, for example, YqhD, ADH6, YjgB, and YahK. The exemplary pathway illustrated in FIG. 5 includes an enzyme that can convert 2-keto-5-hydroxy-pentonate to 4-hydroxy-1-butyraldehyde such as, for example, a 2-ketoacid decarboxylase or a 2-oxoglutarate decarboxylase. Suitable exemplary enzymes include, for example, Kivd, BFD, and IPDC. The exemplary pathway illustrated in FIG. 5 includes conversion of 4-hydroxy-1-butyraldehyde into 1,4-butanediol. This conversion may be catalyzed by an alcohol dehydrogenase such as, for example, YqhD, ADH6, YjgB, and YahK. For the metabolic pathway illustrated in FIG. 5, therefore, the recombinant cell can include one or more alcohol dehydrogenases.
In some embodiments, the host cell can include one or more genetic modifications to reduce endogenous metabolism of the carbon source so that metabolism of the carbon source is directed toward the production of the TCA derivative. For example, in embodiments in which the carbon source is xylose and the host cell is E. coli, the host cell can include one or more modifications to decrease endogenous metabolism of xylose. In the case of E. coli, such modifications can include for example, a decrease in α-ketoglutaric semialdehyde dehydrogenase activity, aldolase activity, and/or 2-keto-3-deoxy gluconate aldolase activity. Such modifications can include modifications to coding regions of, or regulatory regions that control expression of, xylA, yjhH, and/or yagE. Such modifications can include, for example, a deletion of a sufficient amount of one or more coding regions that the enzymatic activity is reduced.
As used herein, the terms “activity” with regard to particular enzyme refers to the ability of a polypeptide, regardless of its common name or native function, to catalyze the conversion of the enzyme's substrate to a product, regardless of whether the “activity” as less than, equal to, or greater than the native activity of the identified enzyme. Methods for measuring the biosynthetic activities of cells are routine and well known to those of ordinary skill in the art.
As used herein, an increase in catalytic activity can be quantitatively measured and described as a percentage of the catalytic activity of an appropriate wild-type control. The catalytic activity exhibited by a genetically-modified polypeptide can be, for example, at least 110%, at least 125%, at least 150%, at least 175%, at least 200% (two-fold), at least 250%, at least 300% (three-fold), at least 400% (four-fold), at least 500% (five-fold), at least 600% (six-fold), at least 700% (seven-fold), at least 800% (eight-fold), at least 900% (nine-fold), at least 1000% (10-fold), at least 2000% (20-fold), at least 3000% (30-fold), at least 4000% (40-fold), at least 5000% (50-fold), at least 6000% (60-fold), at least 7000% (70-fold), at least 8000% (80-fold), at least 9000% (90-fold), at least 10,000% (100-fold), or at least 100,000% (1000-fold) of the activity of an appropriate wild-type control.
Alternatively, an increase in catalytic activity may be expressed as at an increase in kcat such as, for example, at least a two-fold increase, at least a three-fold increase, at least a four-fold increase, at least a five-fold increase, at least a six-fold increase, at least a seven-fold increase, at least an eight-fold increase, at least a nine-fold increase, at least a 10-fold increase, at least a 15-fold increase, or at least a 20-fold increase in the kcat value of the enzymatic conversion.
An increase in catalytic activity also may be expressed in terms of a decrease in Km such as, for example, at least a two-fold decrease, at least a three-fold decrease, at least a four-fold decrease, at least a five-fold decrease, at least a six-fold decrease, at least a seven-fold decrease, at least an eight-fold decrease, at least a nine-fold decrease, at least a 10-fold decrease, at least a 15-fold decrease, or at least a 20-fold decrease in the Km value of the enzymatic conversion.
A decrease in catalytic activity can be quantitatively measured and described as a percentage of the catalytic activity of an appropriate wild-type control. The catalytic activity exhibited by a genetically-modified polypeptide can be, for example, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% of the activity, or 0% of the activity of a suitable wild-type control.
Alternatively, a decrease in catalytic activity can be expressed as an appropriate change in a catalytic constant. For example, a decrease in catalytic activity may be expressed as at a decrease in kcat such as, for example, at least a two-fold decrease, at least a three-fold decrease, at least a four-fold decrease, at least a five-fold decrease, at least a six-fold decrease, at least a seven-fold decrease, at least an eight-fold decrease, at least a nine-fold decrease, at least a 10-fold decrease, at least a 15-fold decrease, or at least a 20-fold decrease in the kcat value of the enzymatic conversion.
A decrease in catalytic activity also may be expressed in terms of an increase in Km such as, for example, an increase in Km of at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least an eight-fold, at least nine-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 230-fold, at least 250-fold, at least 300-fold, at least 350-fold, or at least 400-fold.
Thus, in another aspect, we describe herein methods for biosynthesis of a TCA derivative. Generally, the methods includes incubating a recombinant cell as described herein in medium that includes a carbon source under conditions effective for the recombinant cell to produce the TCA derivative. The carbon source can include, for example, saccharides (e.g., xylose, arabinose, glucose, cellulose), a uronic acid (e.g., galacturonic acid or glucuronic acid), CO2, glycerol, or a native substrate of an enzyme that is part of the engineered metabolic pathway. Exemplary native substrates of exemplary enzymes are shown in Table 1 and include, for example, glucaric acid, galactaric acid, hydroxyproline, arabinonic acid, 2-oxo-4(S),5-dihydroxy-pentanoic acid, 2-oxo-4(R),5-dihydroxy-pentanoic acid, 2,5-dioxopentanoic acid, xylonolactone, xylonic acid, arabinonolactone, 4-deoxy-5-keto-D-glucaric acid, 4(R)-hydroxy-L-proline, 4(R)-hydroxy-D-proline, 2-carboxy-4(R)-hydroxy-pyrroline, 2,5-dioxopentanoic acid, succinaldehyde.
In yet another aspect, we describe herein methods for introducing a heterologous polynucleotide into cell so that the host cell exhibits an increased ability to convert a carbon source to a TCA derivative. The heterologous polynucleotide can encode a polypeptide operably linked to a promoter so that modified cell catalyzes conversion of the carbon source to the TCA derivative. In some of these embodiments, the carbon source can include xylose. The host cells for such methods can include, for example, any of the microbial species identified above with regard to the recombinant cells described herein.
In some embodiments, the heterologous polynucleotide may be inserted into a vector. A vector is a replicating polynucleotide such as, for example, a plasmid, phage, or cosmid, to which another polynucleotide may be inserted so as to bring about the replication of the inserted polynucleotide. Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989). A vector can permit, for example, further cloning—i.e., a cloning vector—or expression of the polypeptide encoded by the coding region—i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, or artificial chromosome vectors. In one embodiment, the vector is a plasmid. Selection of a vector can depend upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like.
An expression vector optionally includes regulatory sequences operably linked to the coding region. The polynucleotides described herein are not limited by the use of any particular promoter, and a wide variety of promoters are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3′ direction) coding region. The promoter used can be a constitutive or an inducible promoter. It can be, but need not be, heterologous with respect to the host cell. Exemplary promoters include, for example, trp, tac, and T7.
“Coding sequence” or “coding region” refers to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5′ end and a translation stop codon at its 3′ end. As used herein, the term “polypeptide” refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term “polypeptide” also includes molecules that contain more than one polypeptide joined by disulfide bonds, ionic bonds, or hydrophobic interactions, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms are used interchangeably. The term “polypeptide” does not connote a specific length of a polymer of amino acids, nor does it imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.
“Regulatory sequence” refers to a nucleotide sequence that regulates expression of a coding region to which it is operably linked. Nonlimiting examples of regulatory sequences include, for example, promoters, transcription initiation sites, translation start sites, translation stop sites, and terminators. “Operably linked” refers to a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. A regulatory sequence is “operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.
As used in the preceding description, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the term “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
EXAMPLES
Example 1
Bacterial Strains and Plasmids
All the primers were ordered from Eurofins MWG Operon and are listed in Table 1. The E. coli strains used in this study are listed in Table 2, which were all derived from E. coli K-12 strain BW25113.







TABLE 2







Strains, plasmids and primers used in this study









Name
Relevant genotype
Reference





Strains







BW25113
rrnBT14 ΔlaCZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78
A


SBDO-1
BW25113 ΔxylA ΔyjhH ΔyagE
This work


SBDO-2
SBDO-1 + pBDO-1
This work


SBDO-3
SBDO-1 + pBDO-1 and pBDO-2
This work


SBDO-4
SBDO-1 + pBDO-1 and pBDO-3
This work


SBDO-5
SBDO-1 + pBDO-1 and pBDO-4
This work





Plasmids





pIBA7
ColE1 ori, AmpR, PLlacO1::kivD padA
B


pBDO-1
p15A ori, KanR, PLlacO1::xylBCDX
This work


pBDO-2
ColE1 ori, AmpR, PLlacO1::xylA
This work


pBDO-3
ColE1 ori, AmpR, PLlacO1::BFD- yqhD
This work


pBDO-4
ColE1 ori, AmpR, PLlacO1::kivD- yqhD
This work





Primers

SEQ ID NO:





xylBAcc-F
GGGCCCggtaccatgtcctcagccatctatcccagcct
108


xylBHinNheBa-R
GGGCCCGCTCAGCAAGCTTGCTAGCggatcctTaacgccagccggcgtcgatccagt
109


xylCBamHI-F
GGGCCCggatccAGGAGAAATTAACTatgaccgctcaagtcacttgcgtatg
110


xylCHindNhe-R
GGGCCCAAGCTTgctagcttagacaaggcggacctcatgctggg
111


xylDNheI-F
GGGCCCgctagcAGGAGAAATTAACTatgaggtccgccttgtctaaccgcac
112


xylDHind-R
GGGCCCaagctttTagtggttgtggcggggcagcttgg
113


xylXHind-F
GGGCCCaagcttAGGAGAAATTAACTAtggtttgtcggcggcttctagcatg
114


xylXBIpRem-R
gcgcagctggcgttgttgtccttggccttTctgagcagcagggccgaacgaccttcgaa
115


XylXBIpI-R
GGGCCCGCTCAGCttagaggaggccgcggccggccaggt
116


pZEkivD-F
actgaccgaattcattaaagaggagaaaggtaccatgtatacagtaggagattacctatt
117


kivD-R
ttatgatttattttgttcagcaaata
118


YqhDkivD-F
ctgaacaaaataaatcataaAGGAGAAATTAACTATGAACAACTTTAATCTGCACACCCC
119


BFDpZE-F
actgaccgaattcattaaagaggagaaaggtaccatggcttcggtacacggcaccacata
120


BFD-R
tTacttcaccgggcttacggtgctta
121


CC0822Acc-F
GGGCCCggtaccatgaccgacaccctgcgccattacat
122


CC0822Xba-R
GGGCCCtctagattacgaccacgagtaggaggttttgg
123





A. Datsenko et al., 2000 Proc. Natl. Acad. Sci. U.S.A. 97: 6640-5.


B. Zhang et al., 2011 ChemSusChem 4: 1068-1070.






All cloning procedures were carried out in the E. coli strain XL10-gold (Stratagene, Agilent Technologies, Santa Clara, Calif.). To build the plasmid pBDO-1, the coding regions of xylB, xylC, xylD, and xylX were amplified by PCR with oligos of xylBAcc-F and xylBHinNheBa-R, xylCBamHI-F and xylCHindNhe-R, xylDNheI-F and xylDHind-R, xylXHind-F and xylXBlpRem-R, using genomic DNA of Caulobacter crescentus strain as template, and then these four coding regions of xylB, xylC, xylD, and xylX were inserted into the corresponding restriction sites of pZA vector after digestion.
To make the plasmid pBDO-2, the coding region of xylA was PCR amplified by oligos of CC0822Acc-F and CC0822Xba-R using genomic DNA of C. crescentus strain as template, and then this coding region was inserted into the site between Acc65I and XbaI of vector pZE after digestion.
To construct the plasmids pBDO-3 and pBDO-4, four coding regions of BFD (using Pseudomonas putida genomic DNA as template), yqhD-1 (using E. coli genomic DNA as template), KIVD (from Lactococcus lactis, using plasmid pIBA7 as template) and yqhD-2 (using E. coli genomic DNA as template), were PCR amplified with oligos of BFDpZE-F and BFD-R, yqhDBFD-F and yqhDpZE-R, pZEkivD-F and kivD-R, yqhDkivD-F and yqhDpZE-R, and then pBDO-3 and pBDO-4 were completed by Gibson cloning method (Gibson et al., 2009. Nat. Meth. 6:343-345). P1 phages of xylA, yjhH and yagE and were obtained from the Keio collection (Baba et al., 2006 Mol. Syst. Biol. 2:10.1038). The phages were used to transfect the BW25113 strain to construct triple knockout strains. All the knockout strains were then transformed with pCP20 plasmid to remove the kanamycin marker. The correct knockouts were verified by PCR.
Cell Cultivation and Shake Flask Fermentation
Unless otherwise stated, cells were grown in test tubes at 37° C. in 2× YT rich medium (16 g/L Bacto-tryptone, 10 g/L yeast extract, and 5 g/L NaCl) supplemented with 100 mg/L ampicillin and 50 mg/L kanamycin. 200 μL of overnight cultures incubated in 2× YT medium were transferred into 5 mL M9 minimal medium supplemented with 5 g/L yeast extract, 5 g/L glucose, 40 g/L xylose, 100 mg/L ampicillin, and 50 mg/L kanamycin in 125 mL conical flasks. Isopropyl-β-D-thiogalactoside (IPTG) was added at a concentration of 0.1 mM to induce protein expression. The fermentation broth was buffered by the presence of 0.5 g CaCO3.
Metabolite Analysis and Dry Cell Weight Determination
Fermentation products were analyzed using an Agilent 1260 Infinity HPLC equipped with an Aminex HPX 87H column (Bio-Rad Laboratories, Inc., Hercules, Calif.) and a refractive-index detector. The mobile phase was 5 mM H2SO4 with a flow rate 0.6 mL/min. The column temperature and detection temperature were 35° C. and 50° C., respectively. Cell dry weight was determined by filtering 5 mL culture through a 0.45 μm glass fiber filter (Pall Life Sciences, Ann Arbor, Mich.). After removal of medium, the filter was washed with 15 mL of MilliQ water (EMD Millipore Corp., Billerica, Mass.), dried in an oven and then weighed. Cell dry weight was determined in triplicate.
Example 2
To produce 1,4-butanediol from xylose in yeast, one artificial synthetic pathway was introduced into the wild type Saccharomyces cerevisiae strain W303. To generate the artificial pathway we cloned a polynucleotide that encodes enzymes that convert xylose into 2,5-dioxopentanoic acid into the host yeast cell. We also cloned a polynucleotide that encoded enzymes that convert 2,5-dioxopentanoic acid into 1,4-butanediol. These enzymes were cloned into plasmids YEplac195-xylBCDX and YEplac112-KivdDyqhD as described in more detail below.
The transformed yeast were grown under fermentation conditions as described in more detail below for two day. After the fermentation, 1,4-butanediol was accumulated to a concentration of 20 mg/L. (FIG. 14).
Plasmid Construction in the Yeast 1,4-Butanediol Synthetic Pathway
The construction of plasmid YEplac195-xylBCDX was finished by Gibson assembly. All of the primers are listed in Table 3.
The coding region for HXT7p was PCR amplified with the primer pair Hxt7p195-1F and Hxt7pXylB-R, using S. cerevisiae W303 genomic DNA as a template. Similarly, the PGK1p coding region was PCR amplified with the primer pair PGK1Phxt7t-F and PGK1PxylC-R; the ADH1p coding region was PCR amplified with the primer pair ADH1Ppgk1t-F and ADH1PxylD-R; the PDC1p coding region was PCR amplified with the primer pair PDC1PADH1T-F and PDC1PxylX-R; the HXT7t coding region was PCR amplified with the primer pair Hxt7tXylB-F and Hxt7tPGK1P-R; the PGK1t coding region was PCR amplified with the primer pair PGK1tXylC-F and PGK1tADH1p-R; the ADH1t coding region was PCR amplified with the primer pair ADH1TxylD-F and ADH1TPDC1P-R; and the PDC1t coding region was PCR amplified with primer pairs PDC1TxylX-F and PDC1T195-R, each by using S. cerevisiae W303 genomic DNA as template.
Caulobacter crescentus xylB coding region was PCR amplified with primer pair xylBhxt7p-F and xylBhxt7t-R using C. crescentus genomic DNA as template. Similarly, the xylC coding region was PCR amplified with primer pair xylCPGK1P-F and xylCPGK1t-R; the xylD coding region was PCR amplified with primer pair xylDADH1P-F/xylDADH1T-R; and the xylX coding region was PCR amplified with primer pair xylXPDC1P-F/xylXPDC1T-R; each using C. crescentus genomic DNA as template.
The combined fragment of HXT7p-xylB-HXT7t was amplified by overlapping PCR with the primer pair Hxt7p195-1F and Hxt7tPGK1P-R using the HXT7p/xylB/HXT7t DNA as a PCR template. The combined fragment PGK1p-xylC-PGK1t was amplified by overlapping PCR with the primer pair PGK1Phxt7t-F and PGK1tADH1p-R using the PGK1p/xylC/PGK1t DNA as a PCR template. The combined fragment ADH1p-xylD-ADH1t was amplified by overlapping PCR with the primer pair ADH1Ppgk1t-F and ADH1TPDC1P-R using the fragment ADH1p/xylD/ADH1t DNA as a PCR template. The combined fragment PDC1p-xylX-PDC1t was amplified by overlapping PCR with the primer pair PDC1PADH1T-F and PDC1T195-R using the PDC1p/xylX/PDC1t DNA as a PCR template.
The vector fragment YEp195v was amplified with primer pair 195HindIII-2F and 195EcoRI-2R by using YEplac195 as template. The fragments of YEp195v, HXT7p-xylB-HXT7t, PGK1p-xylC-PGK1t, ADH1p-xylD-ADH1t, and PDC1p-xylX-PDC1t were assembled by Gibson method to form the plasmid of YEplac195-xylBCDX (FIG. 12).
To build the plasmid of YEplac112-KivD-yqhD, the fragments of HXT7P2, HXT7T2, PGK1P2 and PGK1T2 were PCR amplified using S. cerevisiae W303 genmic DNA as a template. The HXT7P2 fragment was PCR amplified using the primer pair Hxt7p195-1F and HXT7PkivD-R; the HXT7T2 fragment was PCR amplified using the primer pair HXT7TKIVD-F and HXT7TPGK1P-R; the PGK1P2 fragment was PCR amplified using the primer pair PGK1PHXT7T-F/PGK1PyqhD-R; and the PGK1T2 fragment was PCR amplified using the primer pair PGK1TyqhD-F and PGK1T112-R.
The KIVD coding region from Lactococcus lactis was amplified with the primer pair KIVDHXT7P-F and KIVDHXT7T-R using L. lactis genomic DNA as template. The E. coli YqhD coding region was amplified with the primer pair yqhDPGK1P-F and yqhDPGK1T-R using E. coli genomic DNA as template.
The vector fragment YEp112v was amplified with primer pair 195HindIII-2F and 195EcoRI-2R by using YEplac112 as template. The fragments of YEp112v, HXT7P2, KIVD, HXT7T2, PGK1P2, yqhD and PGK1T2 were assembled by Gibson method to generate the plasmid of YEplac112-KivDyqhD. (FIG. 13).







TABLE 3







The used primers in this study









Primer

SEQ ID NO: 





195HindIII-2F
attgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctt
124


 


Hxt7p195-1F
cagctatgaccatgattacgccaagcttGGTACCtcgtaggaacaatttcgggcccctgc
125


 


Hxt7pXylB-R
cttcaggctgggatagatggctgaggacattttttgattaaaattaaaaaaactttttgt
126


 


XylBhxt7p-F
acaaaaagtttttttaattttaatcaaaaaatgtcctcagccatctatcccagcctgaag
127


 


XylBhxt7t-R
tgatcatgaattaataaaagtgttcgcaaatTaacgccagccggcgtcgatccagtattc
128


 


Hxt7tXylB-F
gaatactggatcgacgccggctggcgttAatttgcgaacacttttattaattcatgatca
129


 


Hxt7tPGK1P-R
actcacgagtaattcttgcaaatgcctCCTAGGagacactttttgaagcgggatacagaa
130


 


PGK1Phxt7t-F
ttctgtatcccgcttcaaaaagtgtctCCTAGGaggcatttgcaagaattactcgtgagt
131


 


PGK1PxylC-R
atcccatacgcaagtgacttgagcggtcattgttttatatttgttgtaaaaagtagataa
132


 


xylCPGKlP-F
ttatctactttttacaacaaatataaaacaatgaccgctcaagtcacttgcgtatgggat
133


 


XylCPGKlt-R
attgatctatcgatttcaattcaattcaatttagacaaggcggacctcatgctggggttg
134


 


PGK1tXylC-F
caaccccagcatgaggtccgccttgtctaaattgaattgaattgaaatcgatagatcaat
135


 


PGK1tADHlp-R
ccgatgtatgggtttggttgccagaaGCtgagcttggagcaggaagaatacactatactg
136


 


ADH1Ppgklt-F
cagtatagtgtattcttcctgctccaagctcaGCttctggcaaccaaacccatacatcgg
137


 


ADH1PxylD-R
gggcgtgcggttagacaaggcggacctcattgtatatgagatagttgattgtatgcttgg
138


 


xylDADH1P-F
ccaagcatacaatcaactatctcatatacaatgaggtccgccttgtctaaccgcacgccc
139


 


xylDADH1T-R
aataaaaatcataaatcataagaaattcgctTagtggttgtggcggggcagcttggccgc
140


 


ADH1Txy1D-F
gcggccaagctgccccgccacaaccactAagcgaatttcttatgatttatgatttttatt
141


 


ADH1TPDC1P-R
gaaggtatgggtgcagtgtgcttatctACTAGTtgtggaagaacgattacaacaggtgtt
142


 


PDC1PADH1T-F
aacacctgttgtaatcgttcttccacaACTAGTagataagcacactgcacccataccttc
143


 


PDC1PxylX-R
ggtccatgctagaagccgccgacaaaccaTtttgattgatttgactgtgttattttgcgt
144


 


xylXPDC1P-F
acgcaaaataacacagtcaaatcaatcaaaAtggtttgtcggcggcttctagcatggacc
145


 


xylXPDC1T-R
actttaactaataattagagattaaatcgcttagaggaggccgcggccggccaggttgcg
146


 


PDC1TxylX-F
cgcaacctggccggccgcggcctcctctaagcgatttaatctctaattattagttaaagt
147


 


PDC1T195-R
acgttgtaaaacgacggccagtgaattcTCTAGAgcttgtcttgagcaattgcagagtcg
148


 


195EcoRI-2R
agttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattc
149


 


HXT7PkivD-R
ctaataggtaatctcctactgtatacatGGATCCtttttgattaaaattaaaaaaacttt
150


 


KIVDHXT7P-F
aaagtttttttaattttaatcaaaaaGGATCCatgtatacagtaggagattacctattag
151


 


KIVDHXT7T-R
tcatgaattaataaaagtgttcgcaaaGGTACCttatgatttattttgttcagcaaatag
152


 


HXT7TKIVD-F
ctatttgctgaacaaaataaatcataaGGTACCtttgcgaacacttttattaattcatga
153


 


HXT7TPGK1P-R
cttactcacgagtaattcttgcaaatgcctagacactttttgaagcgggatacagaaaaa
154


 


PGK1PHXT7T-F
tttttctgtatcccgcttcaaaaagtgtctaggcatttgcaagaattactcgtgagtaag
155


 


PGK1PyqhD-R
TGGGGTGTGCAGATTAAAGTTGTTCATTCTAGAtgttttatatttgttgtaaaaagtaga
156


 


yqhDPGK1P-F
tctactttttacaacaaatataaaacaTCTAGAATGAACAACTTTAATCTGCACACCCCA
157


 


yqhDPGK1T-R
gatctatcgatttcaattcaattcaatCTCGAGTTAGCGGGCGGCTTCGTATATACGGCG
158


 


PGK1TyqhD-F
CGCCGTATATACGAAGCCGCCCGCTAACTCGAGattgaattgaattgaaatcgatagatc
159


 


PGK1T181-R
gtcacgacgttgtaaaacgacggccagtgaattctgagcttggagcaggaagaatacact
160









1,4-Butanediol Fermentation by Yeast in Shake Flask

The W303 yeast strain carrying plasmids of YEplac195-xylBCDX and YEplac112-KivDyqhD was cultured overnight in the Complete Minimal medium without uracil and tryptophan supplements at 30° C. with shaking at 200 rpm. The yeast cells were harvested and washed in the next day, and then inoculated into 10 mL fresh medium identical to the overnight culture medium except that it further contained 20 g/L xylose. The shake flask was then sealed with parafilm, and cultured for two days at 30° C. with shaking at 200 rpm. The fermentation broth was analyzed by gas chromatography to measure the amount of 1,4-butanediol. Results are shown in FIG. 14.
Exemplary Embodiments
Embodiment 1. A recombinant microbial cell modified to exhibit increased biosynthesis of a TCA derivative compared to a wild-type control.
Embodiment 2. The recombinant cell of Embodiment 1 wherein the TCA derivative comprises 1,4-butanediol.
Embodiment 3. The recombinant microbial cell any preceding Embodiment wherein the microbial cell is a fungal cell.
Embodiment 4. The recombinant cell of Embodiment 3 wherein the fungal cell is a member of the Saccharomycetaceae family.
Embodiment 5. The recombinant cell of Embodiment 3 wherein the fungal cell is Saccharomyces cerevisiae, Candida rugosa, or Candida albicans. 
Embodiment 6. The recombinant cell of Embodiment 1 or Embodiment 2 wherein the microbial cell is a bacterial cell.
Embodiment 7. The recombinant cell of Embodiment 6 wherein the bacterial cell is a member of the phylum Protobacteria.
Embodiment 8. The recombinant cell of Embodiment 7 wherein the bacterial cell is a member of the Enterobacteriaceae family.
Embodiment 9. The recombinant cell of Embodiment 8 wherein the bacterial cell is Escherichia coli. 
Embodiment 10. The recombinant cell of Embodiment 7 wherein the bacterial cell is a member of the Pseudomonaceae family.
Embodiment 11. The recombinant cell of Embodiment 10 wherein the bacterial cell is Pseudomonas putida. 
Embodiment 12. The recombinant cell of Embodiment 6 wherein the bacterial cell is a member of the phylum Firmicutes.
Embodiment 13. The recombinant cell of Embodiment 12 wherein the bacterial cell is a member of the Bacillaceae family.
Embodiment 14. The recombinant cell of Embodiment 13 wherein the bacterial cell is Bacillus subtilis. 
Embodiment 15. The recombinant cell of Embodiment 12 wherein the bacterial cell is a member of the Streptococcaceae family.
Embodiment 16. The recombinant cell of Embodiment 15 wherein the bacterial cell is Lactococcus lactis. 
Embodiment 17. The recombinant cell of Embodiment 12 wherein the bacterial cell is a member of the Clostridiaceae family.
Embodiment 18. The recombinant cell of Embodiment 17 wherein the bacterial cell is Clostridium cellulolyticum. 
Embodiment 19. The recombinant cell of Embodiment 6 wherein the bacterial cell is a member of the phylum Cyanobacteria.
Embodiment 20. The recombinant cell of any preceding Embodiment wherein the microbial cell is photosynthetic.
Embodiment 21. The recombinant cell of any preceding Embodiment wherein the microbial cell is cellulolytic.
Embodiment 22. The recombinant cell of any preceding Embodiment wherein the increased biosynthesis of the TCA derivative comprises an increase in xylose dehydrogenase activity, xylonolactonase activity, xylonate dehydratase activity, or 2-keto-3-deoxyaldonic acid dehydratase activity.
Embodiment 23. The recombinant cell of Embodiment 22 wherein the increased biosynthesis of the TCA derivative further comprises an increase in benzoylformate decarboxylase activity and an increase in alcohol dehydrogenase activity.
Embodiment 24. The recombinant cell of Embodiment 23 wherein the benzoylformate decarboxylase comprises BFD of Pseudomonas putida. 
Embodiment 25. The recombinant cell of Embodiment 23 wherein the alcohol dehydrogenase comprises yqhD of E. coli. 
Embodiment 26. The recombinant cell of Embodiment 22 wherein the increased biosynthesis of the TCA derivative further comprises an increase in decarboxylase activity and an increase in alcohol dehydrogenase activity.
Embodiment 27. The recombinant cell of Embodiment 26 wherein the decarboxylase comprises KIVD of Lactococcus lactis. 
Embodiment 28. The recombinant cell of Embodiment 26 wherein the alcohol dehydrogenase comprises yqhD of E. coli. 
Embodiment 29. The recombinant cell of preceding Embodiment wherein the increased biosynthesis of the TCA derivative comprises a decrease in α-ketoglutaric semialdehyde dehydrogenase activity.
Embodiment 30. The recombinant cell of preceding Embodiment wherein the increased biosynthesis of the TCA derivative comprises a decrease in aldolase activity.
Embodiment 31. The recombinant cell of preceding Embodiment wherein the increased biosynthesis of the TCA derivative comprises a decrease in 2-keto-3-deoxy gluconate aldolase activity.
Embodiment 32. The recombinant cell of any preceding Embodiment comprising an engineered metabolic pathway for converting 2,5-dioxopentanoic acid to 1,4-butanediol.
Embodiment 33. The recombinant cell of Embodiment 32 wherein the engineered metabolic pathway for converting 2,5-dioxopentanoic acid to 1,4-butanediol comprises an enzyme that converts 2,5-dioxopentonoic acid into succinaldehyde.
Embodiment 34. The recombinant cell of Embodiment 33 wherein the enzyme that converts 2,5-dioxopentonoic acid into succinaldehyde comprises a 2-ketoacid decarboxylase or a 2-oxoglutarate decarboxylase.
Embodiment 35. The recombinant cell of Embodiment 33 or 34 wherein the enzyme that converts 2,5-dioxopentonoic acid into succinaldehyde comprises KIVD, BFD, or IPDC.
Embodiment 36. The recombinant cell of any one of Embodiments 32-35 wherein the engineered metabolic pathway for converting 2,5-dioxopentanoic acid to 1,4-butanediol comprises an enzyme that converts succinaldehyde to 1,4-butanediol.
Embodiment 37. The recombinant cell of Embodiment 36 wherein the enzyme that converts succinaldehyde to 1,4-butanediol comprises an alcohol dehydrogenase.
Embodiment 38. The recombinant cell of Embodiment 36 or Embodiment 37 wherein the enzyme that converts succinaldehyde to 1,4-butanediol comprises YqhD, ADH6, YjgB, or YahK.
Embodiment 39. The recombinant cell of Embodiment 32 wherein the engineered metabolic pathway for converting 2,5-dioxopentanoic acid to 1,4-butanediol comprises an enzyme that converts 2,5-dioxopentonoic acid into 2-keto-5-hydroxy-pentanoic acid.
Embodiment 40. The recombinant cell of Embodiment 39 wherein the enzyme that converts 2,5-dioxopentonoic acid into 2-keto-5-hydroxy-pentanoic acid comprises an alcohol dehydrogenase.
Embodiment 41. The recombinant cell of Embodiment 39 or Embodiment 40 wherein the enzyme that converts 2,5-dioxopentonoic acid into 2-keto-5-hydroxy-pentanoic acid comprises YqhD, ADH6, YjgB, or YahK.
Embodiment 42. The recombinant cell of any one of Embodiments 39-41 wherein the engineered metabolic pathway for converting 2,5-dioxopentanoic acid to 1,4-butanediol comprises an enzyme that converts 2-keto-5-hydroxy-pentanoic acid to 4-hydroxy-1-butyraldehyde.
Embodiment 43. The recombinant cell of Embodiment 42 wherein the enzyme that converts 2-keto-5-hydroxy-pentanoic acid to 4-hydroxy-1-butyraldehyde comprises a 2-ketoacid decarboxylase or a 2-oxoglutarate decarboxylase.
Embodiment 44. The recombinant cell of Embodiment 42 or Embodiment 43 wherein the enzyme that converts 2-keto-5-hydroxy-pentanoic acid to 4-hydroxy-1-butyraldehyde comprises Kivd, BFD, or IPDC.
Embodiment 45. The recombinant cell of any one of Embodiments 42-44 wherein the engineered metabolic pathway for converting 2,5-dioxopentanoic acid to 1,4-butanediol comprises an enzyme that converts 4-hydroxy-1-butyraldehyde to 1,4-butanediol.
Embodiment 46. The recombinant cell of Embodiment 45 wherein the enzyme that converts 4-hydroxy-1-butyraldehyde to 1,4-butanediol comprises an alcohol dehydrogenase.
Embodiment 47. The recombinant cell of Embodiment 45 or Embodiment 46 wherein the enzyme that converts 4-hydroxy-1-butyraldehyde to 1,4-butanediol comprises YqhD, ADH6, YjgB, or YahK.
Embodiment 48. The recombinant cell of any preceding Embodiment comprising an engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid.
Embodiment 49. The recombinant cell of Embodiment 48 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts D-arabinose into D-arabinolactone.
Embodiment 50. The recombinant cell of Embodiment 49 wherein the enzyme that can convert D-arabinose into D-arabinolactone comprises a pentose dehydrogenase.
Embodiment 51. The recombinant cell of Embodiment 49 or Embodiment 50 wherein the enzyme that can convert D-arabinose into D-arabinonolactone comprises AraDH.
Embodiment 52. The recombinant cell of any one of Embodiments 49-51 wherein the recombinant cell exhibits conversion of D-arabinose into D-arabinonolactone at a level at least 110% of a wild-type control cell.
Embodiment 53. The recombinant cell of any one of Embodiments 49-52 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts D-arabinonic acid to 2-oxo-4(s),5-dihydroxy-pentanoic acid.
Embodiment 54. The recombinant cell of Embodiment 53 wherein the enzyme that converts D-arabinonic acid to 2-oxo-4(s),5-dihydroxy-pentanoic acid comprises an aldonic acid dehydratase.
Embodiment 55. The recombinant cell of Embodiment 53 or Embodiment 54 wherein the enzyme that converts D-arabinonic acid to 2-oxo-4(s),5-dihydroxy-pentanoic acid comprises AraD.
Embodiment 56. The recombinant cell of any one of Embodiments 53-55 wherein the recombinant cell exhibits conversion of D-arabinonic acid to 2-oxo-4(s),5-dihydroxy-pentanoic acid at a level at least 110% of a wild-type control cell.
Embodiment 57. The recombinant cell of any one of Embodiments 49-56 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts 2-oxo-4(s),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid.
Embodiment 58. The recombinant cell of Embodiment 57 wherein the enzyme that converts 2-oxo-4(s),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid comprises a 2-keto-3-deoxyaldonic acid dehydratase.
Embodiment 59. The recombinant cell of Embodiment 57 or Embodiment 58 wherein the enzyme that converts 2-oxo-4(s),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid comprises KdaD.
Embodiment 60. The recombinant cell of any one of Embodiments 57-59 wherein the recombinant cell exhibits conversion of 2-oxo-4(s),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid at a level at least 110% of a wild-type control cell.
Embodiment 61. The recombinant cell of Embodiment 48 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts D-xylose to D-xylonolactone.
Embodiment 62. The recombinant cell of Embodiment 61 wherein the enzyme that converts D-xylose to D-xylonolactone comprises a pentose dehydrogenase.
Embodiment 63. The recombinant cell of Embodiment 61 or Embodiment 62 wherein enzyme that converts D-xylose to D-xylonolactone comprises XylB or rrnAC3034.
Embodiment 64. The recombinant cell of any one of Embodiments 61-63 wherein the recombinant cell exhibits conversion of D-xylose to D-xylonolactone at a level at least 110% of a wild-type control.
Embodiment 65. The recombinant cell of any one of Embodiments 61-64 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts D-xylonolactone to D-xylonic acid.
Embodiment 66. The recombinant cell of Embodiment 65 wherein the enzyme that converts D-xylonolactone to D-xylonic acid comprises a pentonolactonase.
Embodiment 67. The recombinant cell of Embodiment 65 or Embodiment 66 wherein the enzyme that converts D-xylonolactone to D-xylonic acid comprises XylC or rrnAC3033.
Embodiment 68. The recombinant cell of any one of Embodiments 65-67 wherein the recombinant cell exhibits conversion of D-xylonolactone to D-xylonic acid at a level at least 110% of a wild-type control.
Embodiment 69. The recombinant cell of any one of Embodiments 61-68 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts D-xylonic acid to 2-oxo-4(S),5-dihydroxy-pentanoic acid.
Embodiment 70. The recombinant cell of Embodiment 69 wherein the enzyme that converts D-xylonic acid to 2-oxo-4(S),5-dihydroxy-pentanoic acid comprises an aldonic acid dehydratase.
Embodiment 71. The recombinant cell of Embodiment 69 or Embodiment 70 wherein the enzyme that converts D-xylonic acid to 2-oxo-4(S),5-dihydroxy-pentanoic acid comprises XylD or rrnAC3032.
Embodiment 72. The recombinant cell of any one of Embodiments 69-71 wherein the recombinant cell exhibits conversion of D-xylonic acid to 2-oxo-4(S),5-dihydroxy-pentanoic acid at a level at least 110% of a wild-type control.
Embodiment 73. The recombinant cell of any one of Embodiments 61-72 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts 2-oxo-4(S),5-dihydroxy-pentanoic acid to 2,5-dioxopenatnoic acid.
Embodiment 74. The recombinant cell of Embodiment 73 wherein the enzyme that converts 2-oxo-4(S),5-dihydroxy-pentanoic acid to 2,5-dioxopenatnoic acid comprises a 2-keto-3-deoxyaldonic acid dehydratase.
Embodiment 75. The recombinant cell of Embodiment 73 or Embodiment 74 wherein the enzyme that converts 2-oxo-4(S),5-dihydroxy-pentanoic acid to 2,5-dioxopenatnoic acid comprises XylX or rrnAC3039.
Embodiment 76. The recombinant cell of any one of Embodiments 73-75 wherein the recombinant cell exhibits conversion of 2-oxo-4(S),5-dihydroxy-pentanoic acid to 2,5-dioxopenatnoic acid at a level at least 110% of a wild-type control.
Embodiment 77. The recombinant cell of Embodiment 48 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts L-arabinose to L-arabinolactone.
Embodiment 78. The recombinant cell of Embodiment 77 wherein the enzyme that converts L-arabinose to L-arabinolactone comprises a pentose dehydrogenase.
Embodiment 79. The recombinant cell of Embodiment 77 or Embodiment 78 wherein the enzyme that converts L-arabinose to L-arabinolactone comprises AraE.
Embodiment 80. The recombinant cell of any one of Embodiments 77-79 wherein the recombinant cell exhibits conversion of L-arabinose to L-arabinolactone at a level at least 110% of a wild-type control.
Embodiment 81. The recombinant cell of any one of Embodiments 77-80 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts L-arabinolactone to L-arabinonic acid.
Embodiment 82. The recombinant cell of Embodiment 81 wherein the enzyme that converts L-arabinolactone to L-arabinonic acid comprises a pentonolactonase.
Embodiment 83. The recombinant cell of Embodiment 81 or Embodiment 82 wherein the enzyme that converts L-arabinolactone to L-arabinonic acid comprises AraI.
Embodiment 84. The recombinant cell of any one of Embodiments 81-83 wherein the recombinant cell exhibits conversion of L-arabinolactone to L-arabinonic acid at a level at least 110% of a wild-type control.
Embodiment 85. The recombinant cell of any one of Embodiments 77-84 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts L-arabinonic acid to 2-oxo-4(R),5-dihydroxy-pentanoic acid.
Embodiment 86. The recombinant cell of Embodiment 85 wherein the enzyme that converts L-arabinonic acid to 2-oxo-4(R),5-dihydroxy-pentanoic acid comprises an aldonic acid dehydratase.
Embodiment 87. The recombinant cell of Embodiment 85 or Embodiment 86 wherein the enzyme that converts L-arabinonic acid to 2-oxo-4(R),5-dihydroxy-pentanoic acid comprises AraB.
Embodiment 88. The recombinant cell of any one of Embodiments 81-87 wherein the recombinant cell exhibits conversion of L-arabinonic acid to 2-oxo-4(R),5-dihydroxy-pentanoic acid at a level at least 110% of a wild-type control.
Embodiment 89. The recombinant cell of any one of Embodiments 77-88 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts 2-oxo-4(R),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid.
Embodiment 90. The recombinant cell of Embodiments 89 wherein the enzyme that converts 2-oxo-4(R),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid comprises a 2-keto-3-deoxyaldonic acid dehydratase.
Embodiment 91. The recombinant cell of Embodiments 89 wherein the enzyme that converts 2-oxo-4(R),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid comprises AraD.
Embodiment 92. The recombinant cell of any one of Embodiments 89-90 wherein the recombinant cell exhibits conversion of 2-oxo-4(R),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid at a level at least 110% of a wild-type control.
Embodiment 93. The recombinant cell of Embodiment 48 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts D-glucaric acid to 4-deoxy-5-keto-D-glucaric acid.
Embodiment 94. The recombinant cell of Embodiment 93 wherein the enzyme that converts D-glucaric acid to 4-deoxy-5-keto-D-glucaric acid comprises an aldonic acid dehydratase.
Embodiment 95. The recombinant cell of Embodiment 93 or Embodiment 94 wherein the enzyme that converts D-glucaric acid to 4-deoxy-5-keto-D-glucaric acid comprises YcbF.
Embodiment 96. The recombinant cell of any one of Embodiments 93-95 wherein the recombinant cell exhibits conversion of D-glucaric acid to 4-deoxy-5-keto-D-glucaric acid at a level at least 110% of a wild-type control.
Embodiment 97. The recombinant cell of any one of Embodiments 93-96 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid.
Embodiment 98. The recombinant cell of Embodiment 97 wherein the enzyme that converts 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid comprises a 2-keto-3-deoxyaldonic acid dehydratase.
Embodiment 99. The recombinant cell of Embodiment 97 or Embodiment 98 wherein the enzyme that converts 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid comprises YcbC.
Embodiment 100. The recombinant cell of Embodiment 48 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts D-galactaric acid to 4-deoxy-5-keto-D-glucaric acid.
Embodiment 101. The recombinant cell of Embodiment 100 wherein the enzyme that converts D-galactaric acid to 4-deoxy-5-keto-D-glucaric acid comprises an aldonic acid dehydratase.
Embodiment 102. The recombinant cell of Embodiment 100 or Embodiment 101 wherein the enzyme that converts D-galactaric acid to 4-deoxy-5-keto-D-glucaric acid comprises YcbH.
Embodiment 103. The recombinant cell of any one of Embodiments 100-102 wherein the recombinant cell exhibits conversion of D-galactaric acid to 4-deoxy-5-keto-D-glucaric acid at a level at least 110% of a wild-type control.
Embodiment 104. The recombinant cell of any one of Embodiments 100-103 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid.
Embodiment 105. The recombinant cell of Embodiment 104 wherein the enzyme that converts 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid comprises a 2-keto-3-deoxyaldonic acid dehydratase.
Embodiment 106. The recombinant cell of Embodiment 104 or Embodiment 105 wherein the enzyme that converts 4-deoxy-5-keto-D-glucaric acid to 2,5-dioxopentanoic acid comprises YcbC.
Embodiment 107. The recombinant cell of Embodiment 48 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts 4(R)-hydroxy-L-proline to 4(R)-hydroxy-D-proline.
Embodiment 108. The recombinant cell of Embodiment 107 wherein the enzyme that converts 4(R)-hydroxy-L-proline to 4(R)-hydroxy-D-proline comprises an amino acid transporter.
Embodiment 109. The recombinant cell of Embodiment 107 or Embodiment 108 wherein the enzyme that converts 4(R)-hydroxy-L-proline to 4(R)-hydroxy-D-proline comprises LysE or HypE.
Embodiment 110. The recombinant cell of any one of Embodiments 107-109 wherein the recombinant cell exhibits conversion of 4(R)-hydroxy-L-proline to 4(R)-hydroxy-D-proline at a level at least 110% of a wild-type control.
Embodiment 111. The recombinant cell of any one of Embodiments 107-110 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts 4(R)-hydroxy-D-proline to 2-carboxy-4(R)-hydroxy-δ-pyrroline.
Embodiment 112. The recombinant cell of Embodiment 111 wherein the enzyme that converts 4(R)-hydroxy-D-proline to 2-carboxy-4(R)-hydroxy-δ-pyrroline comprises HypOX.
Embodiment 113. The recombinant cell of Embodiment 111 or Embodiment 112 wherein the recombinant cell exhibits conversion of 4(R)-hydroxy-D-proline to 2-carboxy-4(R)-hydroxy-δ-pyrroline at a level at least 110% of a wild-type control.
Embodiment 114. The recombinant cell of any one of Embodiments 107-113 wherein the engineered metabolic pathway for converting a carbon source to 2,5-dioxopentanoic acid comprises an enzyme that converts 2-oxo-4(R),5-hydroxy-5-aminopentanoic acid to 2,5-dioxopentanoic acid.
Embodiment 115. The recombinant cell of Embodiment 114 wherein the enzyme that converts 2-oxo-4(R),5-hydroxy-5-aminopentanoic acid to 2,5-dioxopentanoic acid comprises a 2-keto-3-deoxyaldonic acid dehydratase.
Embodiment 116. The recombinant cell of Embodiment 114 or Embodiment 115 wherein the enzyme that converts 2-oxo-4(R),5-hydroxy-5-aminopentanoic acid to 2,5-dioxopentanoic acid comprises PP1247.
Embodiment 117. The recombinant cell of any one of Embodiments 114-116 wherein the recombinant cell exhibits conversion of 2-oxo-4(R),5-hydroxy-5-aminopentanoic acid to 2,5-dioxopentanoic acid at a level at least 110% of a wild-type control.
Embodiment 118. The recombinant cell of any one of Embodiments 48-117 modified to exhibit increased α-ketoglutaric semialdehyde dehydrogenase activity compared to a wild-type control.
Embodiment 119. The recombinant cell of Embodiment 118 exhibiting increased conversion of 2,5-dioxopentanoic acid to a TCA derivative compared to a wild-type control.
Embodiment 120. The recombinant cell of Embodiment 119 wherein the TCA derivative comprises succinate, fumarate, malate, glutamate, lysine, threonine, or 4-hydroxybutyrate.
Embodiment 121. The recombinant cell of any preceding Embodiment genetically modified to increase consumption of xylose, arabinose, glucaric acid, galactaric acid, or hydroxyproline compared to a wild-type control.
Embodiment 122. The recombinant cell of any preceding Embodiment genetically modified to in crease consumption of a uronic acid compared to a wild-type control.
Embodiment 123. The recombinant cell of Embodiment 122 wherein the urnic acid comprises galacturonic acid or glucuronic acid.
Embodiment 124. The recombinant cell of Embodiment 122 or Embodiment 123 genetically modified to increase conversion of the uronic acid to an aldonic acid compared to a wild-type control.
Embodiment 125. The recombinant cell of any one of Embodiments 122-124 wherein the recombinant cell comprises an exogenous urinate dehydrogenase.
Embodiment 126. A method comprising:
incubating a recombinant cell of any preceding Embodiment in medium that comprises a carbon source under conditions effective for the recombinant cell to produce a TCA derivative.
Embodiment 127. The method of Embodiment 126 wherein the TCA derivative comprises 1,4-butanediol.
Embodiment 128. The method of Embodiment 126 wherein the carbon source comprises xylose, arabinose, glucaric acid, galactaric acid, or hydroxyproline.
Embodiment 129. The method of any one of Embodiments 126-128 wherein the increased biosynthesis of the TCA derivative comprises an increase in pentose dehydrogenase activity, pentonolactonase activity, aldonic acid dehydratase activity, or 2-keto-3-deoxyaldonic acid dehydratase activity.
Embodiment 130. The method of any one of Embodiments 126-129 wherein the increased biosynthesis of the TCA derivative comprises an increase in hexic acid dehydratase activity or 5-dehydro-4-deoxyglucarate dehydratase activity.
Embodiment 131. A method comprising:
introducing into a host cell a heterologous polynucleotide encoding at least one polypeptide that catalyzes conversion of a carbon source to a TCA derivative, wherein the at least one polypeptide is operably linked to a promoter so that the modified host cell catalyzes conversion of the carbon source to TCA derivative.
Embodiment 132. The method of Embodiment 131 wherein the TCA derivative comprises 1,4-butanediol.
Embodiment 133. The method of Embodiment 131 wherein the carbon source comprises xylose.
Embodiment 134. The method of Embodiment 131 wherein the TCA derivative comprises succinate, fumarate, malate, glutamate, lysine, threonine, 4-hydroxybutyrate.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.










Sequence Listing Free Text







D-arabinose dehydrogenase (AraDH)


SEQ ID NO: 1 (NP_342747.1; GI: 15898142; Zinc-containing alcohol dehydrogenase


(Sulfolobus solfataricus P2))








1
menvnmvksk aallkkfsep lsiedvnipe pqgeevliri ggagvcrtdl



rvwkgveakq


61
gfrlpiilgh enagtivevg elakvkkgdn vvvyatwgdl tcrycregkf



nicknqiipg


121
qttnggfsey mlvkssrwlv klnslspvea apladagtts mgairqalpf



iskfaepvvi


181
vngigglavy tiqilkalmk nitivgisrs kkhrdfalel gadyvsemkd



aeslinkltd


241
glgasiaidl vgteettynl gkllaqegai ilvgmegkrv sleafdtavw



nkkllgsnyg


301
slndledvvr lsesgkikpy iikvplddin kaftnldegr vdgrqvit


 







SEQ ID NO: 2 (Chain A, D-arabinose dehydrogenase (Sulfolobus solfataricus))








1
mvkskaallk kfseplsied vnipepqgee vliriggagv crtdlrvwkg



veakqgfrlp


61
iilghenagt ivevgelakv kkgdnvvvya twgdltcryc regkfnickn



qiipgqttng


121
gfseymlvks srwlvklnsl spveaaplad agttsmgair qalpfiskfa



epvvivngig


181
glavytiqil kalmknitiv gisrskkhrd falelgadyv semkdaesli



nkltdglgas


241
iaidlvgtee ttynlgklla qegaiilvgm egkrvsleaf dtavwnkkll



gsnygslndl


301
edvvrlsesg kikpyiikvp lddinkaftn ldegrvdgrq vitp


 







SEQ ID NO: 3 (Alcohol dehydrogenase GroES domain-containing protein (Sulfolobus


islandicus M.14.25))








1
mfgitfysam rknismvksk aallkkfsep lsiedveipe pkgeevlvri



ggagvcrtdl


61
rvwkgveakq gfrlpiilgh enagtvvevg elakakkgdn vvvyatwgdm



tcrycregkf


121
nicknqvipg qttnggfsey mlvksyrwlv kldslspvda spladagtts



mgairqalpf


181
mnkfaepvvi vngigglavy tiqilkalmk nivivgisrs kkhrdlalel



gadyavemke


241
aesliskltd glgasvaidl vgteetsynl gkllaqegai ilvgmegkrv



sleafdtavw


301
nkkllgsnyg slndledvvr lsesgkikpy vvkipldein kafkdldegr vegrqvitp


 







SEQ ID NO: 4 (Alcohol dehydrogenase GroES domain-containing protein (Sulfolobus


islandicus M.16.27))








1
mfgitfysam rknismvksk aallkkfsep lsiedveipe pkgeevlvri



ggagvcrtdl


61
rvwkgveakq gfrlpiilgh enagtvvevg elakakkgdn vvvyatwgdm



tcrycregkf


121
nicknqvipg qttnggfsey mlvksyrwlv kldslspvda spladagtts



mgairqalpf


181
mnkfaepvvi vngigglavy tiqilkalmk nivivgisrs rkhrdlalel



gadyavemke


241
aesliskltd glgasvaidl vgteetsynl gkllaqegai ilvgmegkrv



sleafdtavw


301
nkkllgsnyg slndledvvr lsesgkikpy vvkipldein kafkdldegr vegrqvitp


 







SEQ ID NO: 5 (Alcohol dehydrogenase GroES domain-containing protein (Sulfolobus


islandicus L.S.2.15))








1
mfgitfysam rknismvksk aallkkfsep lsiedveipe pkgeevlvri



ggagvcrtdl


61
rvwkgveakq gfrlpiilgh enagtvvevg elakakkgdn vvvyatwgdm



tcrycregkf


121
nicknqvipg qttnggfsey mlvksyrwlv kldslspvda spladagtts



mgairqalpf


181
mnkfaepvvi vngigglavy tiqilkalmk nivivgisrs kkhrdlalel



gadhavemke


241
aesliskltd glgasvaidl vgteetsynl gkllaqegai ilvgmegkrv



sleafdtavw


301
nkkllgsnyg slndledvvr lsesgkikpy vvkipldein kafkdldegr vegrqvitp


 







Arabinonate dehydratase (AraD)


SEQ ID NO: 6 (NP_344435.1; GI: 15899830; Mandelate racemase/muconate lactonizing


family protein (Sulfolobus solfataricus P2))








1
mikdirtykl cyeginderd alaikglaeh pmeivateie tsdgyvgyge



slaygcsdav


61
qvtiekilkp lllkedeeli eylwdkmyka tlrfgrrgia iagisgvdta



lwdimgkkak


121
kpiykllggs krkvrayitg gyysekkdle klrdeeayyv



kmgfkgikvk igaksmeedi


181
erlkairevv gedvkiavda nnvytfeeal emgrrleklg iwffeepiqt



dyldlsarla


241
eelevpiagy etaytrwefy eimrkravdi vqtdvmwtgg isemmkignm



akvmgyplip


301
hysaggisli gnlhvaaaln spwiemhlrk ndlrdkifke sieidnghlv



vpdrpglgyt


361
irdgvfeeyk cks


 







SEQ ID NO: 7 (Mandelate racemase/muconate lactonizing protein (Sulfolobus islandicus


Y.G.57.14))








1
mikdirtykl cyeginderd alaikglaeh pmeivvteie tsdgyvgyge



slaygcsdav


61
qvtiekilkp lllkedeeli eylwdkmyka tlrfgrrgia iagisgvdta



lwdimgkkak


121
kpiykllggs krkvrayitg gyysekkdle klrdeeayyv kmgfkgikvk



igaksmeedi


181
erlkairevv gedvkiavda nnvytfeeal emgrrleklg iwffeepiqt



dyldlsarla


241
eelevpiagy etaytrwefy eimrkravdi vqtdvmwtgg isemmkignm



akvmgyplip


301
hysaggisli gnlhvaaaln spwiemhlrk ndlrdkifke sieidnghlv



vpdrpglgyt


361
irdgvfeeyk cks


 







SEQ ID NO: 8 (Mandelate racemase/muconate lactonizing domain-containing protein


(Sulfolobus islandicus L.D.8.5))








1
mikdirtykl cyeginderd alaikglaeh pmeivvteie tsdgyvgyge



slaygcsdav


61
qvtiekilkp lllkedeefi eylwdkmyka tlrfgrrgia iagisgvdta



lwdimgkkak


121
kpiykllggs krkvrayitg gyysekkdle klrdeeayyv kmgfkgikvk



igaksmeedi


181
erlkairevv gedvkiavda nnvytfeeal emgrrleklg iwffeepiqt



dyldlsarla


241
eelevpiagy etaytrwefy eimrkravdi vqtdvmwtgg isemmkignm



akvmgyslip


301
hysaggisli gnlhvaaaln spwiemhlrk ndlrdkifke sieidnghlv



vpdrpglgyt


361
irdgvfeeyk cks


 







SEQ ID NO: 9 (Mandelate racemase/muconate lactonizing protein (Sulfolobus islandicus


M.14.25))








1
mikdirtykl cyeginderd alaikglaeh pmeivvteie tsdgyvgyge



slaygcsdav


61
qvtiekilkp lllkedeeli eylwdkmyka tlrfgrrgia iagisgvdtg



lwdimgkkak


121
kpiykllggs krkvrayitg gyysekkdle klrdeeayyv kmgfkgikvk



igaksmeedi


181
erlkairevv gedvkiavda nnvytfeeal emgrrleklg iwffeepiqt



dyldlsarla


241
eelevpiagy etaytrwefy eimrkravdi vqtdvmwtgg isenmkignm



akvmgyplip


301
hysaggisli gnlhvaaaln spwiemhlrk ndlrdkifke sieidnghlv



vpdrpglgyt


361
irdgvfeeyk cks


 







SEQ ID NO: 10 (Mandelate racemase/muconate lactonizing protein (Sulfolobus islandicus


L.S.2.15))








1
mikdirtykl cyeginderd alaikglaeh pmeivvteie tsdgyvgyge



slaygcsdav


61
qvtiekilkp lllkedeeli eylwdkmyka tlrfgrrgia iagisgvdta



lwdimgkkak


121
kpiykllggs krkvrayitg gyysekkdle klrdeeayyv kmgfkgikik



igaksmeedi


181
erlkairevv gedvkiavda nnvytfeeal emgrrleklg iwffeepiqt



dyldlsarla


241
eelevpiagy etaytrwefy eimrkravdi vqtdvmwtgg isemmkignm



akvmgyplip


301
hysaggisli gnlhvaaaln spwiemhlrk ndlrdkifke sieidnghlv



vpdrpglgyt


361
irdgvfeeyk cks


 







2-Keto-3-deoxy-D-arabinonate Dehydratase (KdaD)


SEQ ID NO: 11 (NP_344431.1; GI: 15899826; Hypothetical protein SSO3118 (Sulfolobus


solfataricus P2)








1
mhfimmklfr vvkrgyyisy aildnstiir ldedpikalm rysenkevlg



drvtgidyqs


61
llksfqindi ritkpidppe vwgsgisyem areryseenv akilgktiye



kvydavrpei


121
ffkatpnrcv ghgeaiavrs dsewtlpepe lavvldsngk ilgytimddv



sardleaenp


181
lylpqskiya gccafgpviv tsdeiknpys lditlkivre grvffegsvn



tnkmrrkiee


241
qiqylirdnp ipdgtilttg taivpgrdkg lkdediveit isnigtlitp vkkrrkit


 







SEQ ID NO: 12 (Fumarylacetoacetate (FAA) hydro lase (Sulfolobus islandicus Y.N.15.51))








1
mltcllptll yakcifimmk lfrvvkrgyy isyaildnst iirldedpik



almrysenke


61
vlgdrvtgid yqsllksfqi ndiritkpid ppevwgsgis yemareryse



envakilgkt


121
iyekvydavr peiffkatpn rcvghgeaia vrsdsewtlp epelavvlds



ngkilgytim


181
ddvsardlea enplylpqsk iyagccafgp vivtsdeikn pyslditlki



vregrvffeg


241
svntnkmrrk ieegiqylir dnpipdgtil ttgtaivpgr dkglkdediv



eitisnigtl


301
itpvkkrrki t


 







SEQ ID NO: 13 (Fumarylacetoacetate (FAA) hydrolase (Sulfolobus solfataricus 98/2))








1
mmklfrvvkr gyyisyaild nstiirlded pikalmryse nkevlgdrvt



gidyqsllks


61
fqindiritk pidppevwgs gisyemarer yseenvakil gktiyekvyd



avrpeiffka


121
tpnrcvghge aiavrsdsew tlpepelavv ldsngkilgy timddvsard



leaenplylp


181
qskiyagcca fgpvivtsde iknpysldit lkivregrvf fegsvntnkm



rrkieeqiqy


241
lirdnpipdg tilttgtaiv pgrdkglkde diveitisni gtlitpvkkr rkit


 







SEQ ID NO: 14 (Chain in X, 2-keto-3-deoxy-D-arabinonate, dehydratase)








1
mklfrvvkrg yyisyaildn stiirldedp ikalmrysen kevlgdrvtg



idyqsllksf


61
qindiritkp idppevwgsg isyemarery seenvakilg ktiyekvyda



vrpeiffkat


121
pnrcvghgea iavrsdsewt lpepelavvl dsngkilgyt imddvsardl



eaenplylpq


181
skiyagccaf gpvivtsdei knpyslditl kivregrvff egsvntnkmr



rkieeqiqyl


241
irdnpipdgt ilttgtaivp grdkglkded iveitisnig tlitpvkkrr kit


 







SEQ ID NO: 15 (Fumarylacetoacetate (FAA) hydrolase (Sulfolobus islandicus HVE10/4))








1
mmklfrvvkr gyyisyaild nstiirlded pikalmryse nkevlgdrvt



gidyqsllks


61
fqindiritk pidppevwgs gisyemarer yseenvakil gktiyekvyd



avrpeiffka


121
tpnrcvghge aiavrsdsew tlpepelavv ldsngkilgy timddvsard



leaenplylp


181
qskiyagcca fgpvivtsde iknpysldit lkivrkdrvf fegsvntnkm



rrkieeqiqy


241
lirdnpipdg tilttgtaiv pgrdkglkde diveitisni gtlitpvkkr rkit


 







2,5-dioxopentanoate dehydrogenase (DopDH)


SEQ ID NO: 16 (NP_344430.1; GI: 15899825; Aldehyde dehydrogenase (Sulfolobus


solfataricus P2)








1
mksyqgladk wikgsgeeyl dinpadkdhv lakirlytkd dvkeainkav



akfdewsrtp


61
apkrgsillk agelmeqeaq efallmtlee gktlkdsmfe vtrsynllkf



ygalafkisg


121
ktlpsadpnt riftvkeplg vvalitpwnf plsipvwkla palaagntav



ikpatktplm


181
vaklvevlsk aglpegvvnl vvgkgsevgd tivsddniaa vsftgstevg



kriyklvgnk


241
nrmtriqlel ggknalyvdk sadltlaael avrggfgltg qsctatsrli



inkdvytqfk


301
qrllervkkw rvgpgtedvd mgpvvdegqf kkdleyieyg knvgakliyg



gniipgkgyf


361
leptifegvt sdmrlfkeei fgpvlsvtea kdldeairlv navdyghtag



ivasdikain


421
efvsrveagv ikvnkptvgl elqapfggfk nsgattwkem gedalefylk ektvyegw


 







SEQ ID NO: 17 (Aldehyde dehydrogenase (Sulfolobus islandicus HVE10/4))








1
mksyqgladk wikgsgeeyl dinpadkdhv lakirlytkd dvkeainkav



akfdewsrtp


61
apkrgsillk agelmeqeaq efallmtlee gktlkdsmfe vtrsynllkf



ygalgfkisg


121
ktlpsadpnt riftvkeplg vvalitpwnf plsipvwkla palaagntav



ikpatktplm


181
vaklvevlsk aglpegvvnl vvgkgsevgd tivsddniaa vsftgstevg



kriyklvgnk


241
nrmtriqlel ggknalyvdk sadltlaael avrggfgltg qsctatsrli



ihkdvytqfk


301
qrllervkkw rvgpgtedvd mgpvvdegqf kkdleyieyg knagakliyg



gniipgkgyf


361
leptifegvt shmrlfkeei fgpvlsvtea kdldeairlv navdyghtag



ivasdikain


421
efvsrveagv ikvnkptvgl elqapfggfk nsgattwkem gedalefylk ektvyegw


 







SEQ ID NO: 18 (Aldehyde dehydrogenase (Sulfolobus islandicus Y.G.57.14))








1
mksyqgladk wikgsgeeyl dinpadkdhv lakirlytkd dvkeainkav



akfdewsrtp


61
apkrgsillk agelmeqeaq efallmtlee gktlkdsmfe vtrsynllkf



ygalafkisg


121
ktlpsadpnt riftvkeplg vvalitpwnf plsipvwkla palaagntav



ikpatktplm


181
vaklvevlsk aglpegvvnl vvgkgsevgd tivsddniaa vsftgstevg



kriyklvgnk


241
nrmtriqlel ggknalyvdk sadltlaael avrggfgltg qsctatsrli



inkdvytqfk


301
qrllervkkw rvgpgtedvd mgpvvdegqf kkdleyieyg knvgakliyg



gniipgkgyf


361
leptifegvt sdmrlfkeei fgpvlsvtea kdldeairlv navdyghtag



ivasdinain


421
efvsrveagv ikvnkptvgl elqapfggfk nsgattwkem gedalefylk ektvyegw


 







SEQ ID NO: 19 (Aldehyde dehydrogenase (Sulfolobus islandicus Y.N.15.51))








1
mksyqgladk wikgsgeeyl dinpadkdhv lakirlytkd dvkeainkav



akfdewsrtp


61
apkrgsillk agelmeqeaq efallmtlee gktlkdsmfe vtrsynllkf



ygalafkisg


121
ktlpsadpnt riftvkeplg vvalitpwnf plsipvwkla palaagntai



ikpatktplm


181
vaklvevlsk aglpegvvnl vvgkgsevgd tivsddniaa vsftgstevg



kriyklvgnk


241
nrmtriqlel ggknalyvdk sadltlaael avrggfgltg qsctatsrli



inkdvytqfk


301
qrllervkkw rvgpgtedvd mgpvvdegqf kkdleyieyg knvgakliyg



gniipgkgyf


361
leptifegvt sdmrlfkeei fgpvlsvtea kdldeairlv navdyghtag



ivasdikain


421
efvsrveagv ikvnkptvgl elqapfggfk nsgattwkem gedalefylk ektvyegw


 







SEQ ID NO: 20 (Aldehyde dehydrogenase (Sulfolobus islandicus L.S.2.15))








1
mksyqgladk wikgsgeeyl dinpadkdhv lakirlytkd dvkeainkav



akfdewsrtp


61
apkrgsillk agelmeqeaq efallmtlee gktlkdsmfe vtrsynllkf



ygalafkisg


121
ktlpsadpnt riftvkeplg vvalitpwnf plsipvwkla palaagntav



ikpatktplm


181
vaklvevlsk aglpegvvnl vvgkgsevgd tivsddniaa vsftgstevg



kriyklvgnk


241
nrmtriqlel ggknalyvdk sadltlaael airggfgltg qsctatsrli



inkdvytqfk


301
qrllervkkw rvgpgtedvd mgpvvdegqf kkdleyieyg knvgakliyg



gniipgkgyf


361
leptifegvt sdmrlfkeei fgpvlsvtea kdldeairlv navdyghtag



ivasdikain


421
efvsrveagv ikvnkptvgl elqapfggfk nsgattwkem gedalefylk ektvyegw


 







2,5-dioxovalerate dehydrogenase (YcbD)


SEQ ID NO: 21 (NP_388129.1; GI: 16077316; 2,5-dioxovalerate dehydrogenase (Bacillus


subtilis subsp. subtilis str. 168))








1
msviteqnty lnfingewvk sqsgdmvkve npadvndivg yvqnstaedv



eravtaanea


61
ktawrkltga ergqylykta dimeqrleei aacatremgk tlpeakgeta



rgiailryya


121
gegmrktgdv ipstdkdalm fttrvplgvv gvispwnfpv aipiwkmapa



lvygntvvik


181
patetavtca kiiacfeeag lpagvinlvt gpgsvvgqgl aehdgvnavt



ftgsnqvgki


241
igqaalarga kyqlemggkn pvivaddadl eaaaeavitg afrstgqkqe



atsrvivqsg


301
iyerfkekll qrtkditigd slkedvwmgp iasknqldnc lsyiekgkqe



gaslliggek


361
lengkyqngy yvqpaifdnv tsemtiaqee ifgpvialik vdsieealni



andvkfglsa


421
siftenigrm lsfideidag lvrinaesag velqapfggm kqssshsreq



geaakdffta


481
iktvfvkp


 







SEQ ID NO: 22 (Aldehyde dehydrogenase, thermostable (Bacillus subtilis subsp. subtilis str.


ISO-NN-1))








1
msviteqnty lnfingewvk sqsgdmvkve npadvndivg yvqnstaedv



eravaaanea


61
ktawrkltga ergqylykta dimeqrleei aacatremgk tlpeakgeta



rgiailryya


121
gegmrktgdv ipstdkdalm fttrvplgvv gvispwnfpv aipiwkmapa



lvygntvvik


181
patetavtca kiiacfeeag lpagvinlvt gpgsvvgqgl aehegvnavt



ftgsnqvgki


241
igqaalarga kyqlemggkn pvivaddadl eaaaeavitg afrstgqkct



atsraivqsg


301
iyerfkekll qrtkditigd slkedvwmgp iasknqldnc lsyiekgkqe



gaslliggek


361
lengkyqngy yvqpaifdnv tsemtiaqee ifgpvialik vdsmeealni



andvkfglsa


421
siftenigrm lsfideidag lvrinaesag velqapfggm kgssshsreq



geaakdffta


481
iktvfvkp


 







SEQ ID NO: 23 (Hypothetical protein BSNT_00439 (Bacillus subtilis subsp. natto BEST195))








1
msviteqnty lnfikgewvk sqsgdmvkve npadvndivg yvqnstaedv



eravaaanea


61
ktawrkltga ergqylykta dimeqrleei aacatremgk tlpeakgeta



rgiailryya


121
gegmrktgdv ipstdkaalm fttrvplgvv gvispwnfpv aipiwkmapa



lvygntvvik


181
patetavtca kiiacfeeag lpagvinlvt gpgsvvgqgl aehdgvnavt



ftgsnqvgki


241
igqaalarga kyqlemggkn pvivaddadl eaaaeavitg afrstgqkct



atsrvivqse


301
iyerfkekll qrtkditigd slkedvwmgp iasknqldnc lsyiekgkqe



gaslliggek


361
lengkyqngy yvqpaifdnv tsemtiaqee ifgpvialik vdsmeealni



andvkfglsa


421
siftenigrm lsfideidag lvrinaesag velqapfggm kqssshsreq



geaakdffta


481
iktvfvkp


 







SEQ ID NO: 24 (Aldehyde dehydrogenase (Bacillus subtilis subsp. spizizenii TU-B-10))








1
msviteqnty lnfingewvk sqsgdmvkve npadvndivg yvqnstaddv



eravaaanea


61
ktawrkltga ergqylykta dimeqrleei aacatremgk tlpeakgeta



rgiailryya


121
gegmrktgdv ipstdkdalm fttrvplgvv gvispwnfpv aipiwkmapa



lvygntvvik


181
patetavtca kiiacfeeag lpagvinlvt gpgsvvgqgl aehegvnait



ftgsnqvgki


241
igqaalarga kyqlemggkn pvivaddadl eaaaeavitg afrstgqkct



atsrvivqsg


301
iydrfkekll qrtkdikigd slkedvwmgp iasknqldnc lsyiekgkqe



gaslliggek


361
ledgkyqngy yvqpaifdnv tsemtiaqee ifgpvialik vdsmeealdi



andvkfglsa


421
siftqnigrm lsfvdeidag lvrinaesag velqapfggm kqssshsreq



geaakdffta


481
iktvfvkp


 







SEQ ID NO: 25 (Aldehyde dehydrogenase (Bacillus sp. JS))








1
msviteqnty lnfingewvq sqsgdmvkve npadvndivg yvqnstaedv



eravaaanka


61
ktawrkltga ergqylykta dimerrleei aacatremgk tlpeakgeta



rgiailryya


121
gegmrktgdv ipstdkdalm fttrvplgvv gvispwnfpv aipiwkmapa



lvygntvvik


181
patetavtca kiiacfeeag lpagvinlvt gpgsvvgqgl aehdsvnavt



ftgsnqvgki


241
igqaalarga kyqlemggkn pvivaddadl eaaaeavitg afrstgqkct



atsrvivqsg


301
iyerfkekll qrtkditigd slkedvwmgp iasknqldnc lsyiekgkre



gasllmggek


361
lenekyqngy yvqpaifdnv tsemtiaqee ifgpvialik vdsmeealdi



andvkfglsa


421
siftenigkm lsfideidag lvrvnaesag velqapfggm kgssshsreq



geaakdffta


481
iktvfvkp


 







Xylose dehydrogenase (xylB)


SEQ ID NO: 26 (YP_002516237.1; GI: 221233801; Xylose dehydrogenase xylB (Caulobacter


crescentus NA1000))








1
mssaiypslk gkrvvitggg sgigagltag farqgaevif ldiadedsra



leaelagspi


61
ppvykrcdlm nleaikavfa eigdvdvlvn nagnddrhkl advtgaywde



rinvnlrhml


121
fctqavapgm kkrgggavin fgsiswhlgl edlvlyetak agiegmtral



arelgpddir


181
vtcvvpgnvk tkrqekwytp egeaqivaaq clkgrivpen vaalvlflas



ddaslctghe


241
ywidagwr


 







SEQ ID NO: 27 (Oxidoreductase, short-chain dehydrogenase/reductase (Phenylobacterium


zucineum HLK1))








1
mgvtsaiyps lkgkrvvvtg ggsgigaglv eafvrqgaev hfldvletes



rvletslaga


61
evppvfhrcd ltdagaiegc fakigpvqvl vnnagnddrh tldevtpayf



ddriavnlrh


121
mvfcakavvp amkaagegai infgsiswhl glpdlvlyet akagiegmtr



alarelgpfg


181
irvtcvapgn vktlrqmkwy tpegeaeiva qqclksriep advaalvlfl



asddarmctg


241
heywidagwr


 







SEQ ID NO: 28 (Dehydrogenase of unknown specificity, short-chain alcohol dehydrogenase


(Caulobacter sp. AP07))








1
mssaiypslq gkrvvvtggg sgigagivaa farqgaevif ldvvdadsea



laaklsdspi


61
aptymrcdlt dleamaetfa rigpidvlvn nagnddrhgl aeitpaywdq



rmavnlrhml


121
fatqavapgm kargggavin fgsiswhlgl pdlvlyetak agiegmtral



arelgpddir


181
vtcvvpgnvk tkrqekwytp egeaeivaaq alkgrlvpdh vaslvlflas



ddaalctghe


241
ywidagwr


 







SEQ ID NO: 29 (Short-chain dehydrogenase/reductase SDR (Caulobacter sp. K31))








1
mnievkrpqv stssaiypsl kgkrvvvtgg gsgigagiva gfarqgsevi



fldvadqdsk


61
alaeqlsgae iapvylrcdl tdldavaktf adigpvdvlv nnagnddrhg



laqitpaywd


121
ermsvnlrhm lfatqavapg mkargggaii nfgsiswhlg lpdlvlyeta



kagiegmtra


181
larelgpddi rvtcvvpgni ktkrqekwyt pegeaeivaa galkgrlvpd



hvaslvmfla


241
sddaslctgh eywidagwr


 







SEQ ID NO: 30 (Short-chain dehydrogenase/reductase SDR (Caulobacter segnis ATCC


21756))








1
mssaiypslk gkrvvitggg sgigaglvag fvrqgaevif ldivdadsqa



lvaelskdav


61
iapvykrcdl mdidalkatf aeigdvdvlv nnagnddrhs ladltpaywd



nrigvnlrhm


121
vfaaqavagg mkkrgggaii nfgsiswhlg ledlvlyeta kagiegmtra



larelgpddi


181
rvtcvvpgnv ktkrqekwyt pegeaeivka qclkgrilpd hvaslvlfla



sddaslctgh


241
eywidagwr


 







Xylonolactonase (xylC)


SEQ ID NO: 31 (YP_002516236.1; GI: 221233800; Xylonolactonase xylC (Caulobacter


crescentus NA1000))








1
mtaqvtcvwd lkatlgegpi whgdtlwfvd ikqrkihnyh patgerfsfd



apdqvtflap


61
ivgatgfvvg lktgihrfhp atgfslllev edaalnnrpn datvdaqgrl



wfgtmhdgee


121
nnsgslyrmd ltgvarmdrd icitngpcvs pdgktfyhtd tlektiyafd



laedgllsnk


181
rvfvqfalgd dvypdgsvvd segylwtalw ggfgavrfsp qgdavtriel



papnvtkpcf


241
ggpdlktlyf ttarkglsde tlaqyplagg vfavpvdvag qpqhevrlv


 







SEQ ID NO: 32 (S MP-30/gluconolaconase/LRE domain-containing protein (Caulobacter


segnis ATCC 21756))








1
mtaevtcvwd lkatlgegpi whgdalwfvd ikqrkihnyk pttgehfsfd



apdqvtflap


61
iadaggfvvg lktgihrfhp itgfrlliev edsaldnrpn datvdangrl



wfgtmhdgee


121
aksgslyrmd aegvarmdkd icitngpcvs pdgktfyhtd tlektvwayd



laedgtlsnk


181
rafvhvklgd diypdgtvvd segclwialw ggfgvirvsp ageivgriev



papnvtkvcf


241
ggpdlktlfl ttarkglsde tlaqyplagg lfaigvniag qpqhevrlv


 







SEQ ID NO: 33 (Gluconolactonase (Caulobacter sp. AP07))








1
mpepicvwdl katlgegpiw iaaeqalwfv dikshkvhrf hpesgetksf



dapdqvtfla


61
pragggfvag lksglhhfhp etgfaylgei epadlnnrpn datvdaegrl



wfgtmhdgee


121
tptgalyrlg adgqpvqqdq gvcitngpcv spdgktfyht dtlekviway



dlgadgelsn


181
krqffrleid dawpdgsvvd aegyvwaalw gghgairisp agelvdrvtl



painvtkpcf


241
ggpdlktlyf ttarkglgde qlaayplcgg vfalpvavag qpgyevrldl p


 







SEQ ID NO: 34 (SMP-30/gluconolaconase/LRE domain-containing protein (Caulobacter sp.


K31))








1
mpepicvwdl katlgegpiw saeeqavwfv dikghkvhrf hpasgatasf



dapdqvtfla


61
phaggggfva glksglhrfd pttgafvfla qieppelnnr pndatvdaeg



rlwfgtmhdg


121
emtptgalyr lsadgkpiqq degvcitngp caspdgktfy htdtlekviw



aydlgadgsl


181
snkreffrle iadawpdgsv vdsegfvwta lwgghgalrl spageivdrv



ilpainvtkp


241
cfggpdlktv yftsarkgls deqlaaypqc gglfalpvav aggpqyevrl dlr


 







SEQ ID NO: 35 (Gluconolactonase (Phenylobacteriurn zucineum HLK1))








1
mkvlsepdcv lradaelgeg pvwradddav wfvdikgrri hryepvtgaa



wswaapaqpg


61
fiapvagggw vaglktglhr feprggrfel itavedpsld nrlndgfvda



kgrlwfgsmh


121
dgetaltgal yrlderglqr cdtgycitng paaspdgrtl yhtdtlqkti



yafdlspage


181
lsnkrvfari eegggypdgp avdaegcvwt glfagwhvrr yspkgellak



vgfpvanitk


241
lafggddlts vyattawkgl saderekqpl agglfrfevd vpglpqnqma ha


 







D-xylonate dehydratase (xylD)


SEQ ID NO: 36 (NP_419636.1; GI: 16125072; Dihydroxy-acid dehydratase)Caulobacter


crescentus CB15))








1
mrsalsnrtp rrfrsrdwfd npdhidmtal ylerfmnygi tpeelrsgkp



iigiaqtgsd


61
ispcnrihld lvqrvrdgir daggipmefp vhpifencrr ptaaldrnls



ylglvetlhg


121
ypidavvltt gcdkttpagi maattvnipa ivlsggpmld gwhenelvgs



gtviwrsrrk


181
laageiteee fidraassap saghcntmgt astmnavaea lglsltgcaa



ipapyrergq


241
mayktgqriv dlayddvkpl diltkqafen aialvaaagg stnaqphiva



marhagveit


301
addwraaydi plivnmqpag kylgerfhra ggapavlwel lqqgrlhgdv



ltvtgktmse


361
nlqgretsdr evifpyhepl aekagflvlk gnlfdfaimk ssvigeefrk



rylsqpgqeg


421
vfearaivfd gsddyhkrin dpaleiderc ilvirgagpi gwpgsaevvn



mqppdhllkk


481
gimslptlgd grqsgtadsp silnaspesa iggglswlrt gdtiridlnt



grcdalvdea


541
tiaarkqdgi pavpatmtpw qeiyrahasq ldtggvlefa vkyqdlaakl prhnh


 







SEQ ID NO: 37 (Dihydroxy-acid dehydratase (Caulobacter sp. K31))








1
mtsantpsgr pprrfrsrdw fdnpdhidmt alylerfmny gitpeelrsg



kpiigiaqtg


61
sdispcnrih ldlvtrirdg irdaggipme fpvhpifenc rrptaaldrn



lsylglvevl


121
hgypidavvl ttgcdkttpa gimaattvni paivlsggpm ldgwhdgelv



gsgtviwrsr


181
rklaageine eefiqrasds apsaghcntm gtastmnava ealglsltgc



aaipapyrer


241
gqmayktgqr ivdlayedvk pldiltkkaf enaialvaaa ggstnaqphi



vamarhagld


301
itaddwraay diplilnmqp agkylgerfh raggapavlw ellqagrlhg



dvmtvtgktm


361
genlegretk drevvfpygq pmseragflv lkgnlfdfai mktsvisqef



rqrylsepgk


421
edsfearavv fdgsddyhar indpslnide rtilvirgag pigwpgsaev



vnmqppdall


481
krgimslptl gdgrqsgtad spsilnaspe saiggglswl rtgdmiridl



ntgrcdalvd


541
eatiaerrke gvppvpatmt pwqeiyraht gqletggvle favkyqdlas klprhnh


 







SEQ ID NO: 38 (Dihydroxyacid dehydratase/phosphogluconate dehydratase (Caulobacter sp.


AP07))








1
mtspnrtprr frsrdwfdnp dhidmtalyl erfmnygitp eelrsgkpii



giaqtgsdis


61
pcnrihldlv trirdgirda ggipmefpvh pifencrrpt aaldrnlsyl



glvetlhgyp


121
idavvlttgc dkttpagima attvnipaiv lsggpmldgw hdgelvgsgt



viwrsrrkla


181
ageiteeefi qrasdsapsa ghcntmgtas tmnavaealg lsltgcaaip



apyrergqma


241
yrtggrivd1 ayedikpkdi ltkqafenai alvaaaggst naqphivama



rhagldvtad


301
dwraaydipl ilnmqpagky lgerfhragg apavlwellq agrlhgdamt



vtgktmaenl


361
egretrdrev vfpyaapmse ragflvlkgn lfdfaimkts visqefrdry



lsepgqegaf


421
earavvfdgs gdyharindp slgidertil virgagpigw pgsaevvnmq



ppdallkkgi


481
mslptlgdgr qsgtadspsi lnaspesavg gglswlrtgd viridlntgr



cdalvdeati


541
aarkleglpp vpetmtpwqe iyrahtgqle tggvlefavk yqdlaaklpr hnh


 







SEQ ID NO: 39 (Dihydroxy-acid dehydratase (Caulobacter segnis ATCC 21756))








1
msertprrfr srdwfdnpdh idmtalyler fmnygitpee lrsgkpiigi



aqtgsdispc


61
nrihldlvtr irdgirdagg ipmefpvhpi fencrrptaa ldrnlsylgl



vetlhgypid


121
avvlttgcdk ttpagimaat tvnipaivls ggpmldgwhe gelvgsgtvi



wrsrrklaag


181
eiteeefidr aassapsagh cntmgtastm navaealgls ltgcaaipap



yrergqmayk


241
tgqrivdlay edvkpldilt kkafqnaial vaaaggstna qphivamarh



agveitaddw


301
raaydipliv nmqpagkylg erfhraggap avlwellqqg rlhgdvltvt



gktmgenlqg


361
retsdrevif pyhqplaeka gflvlkgnlf dfaimkssvi geefrkryls



epgkegvfea


421
raivfdgsdd yhkrindpal eidercilvi rgagpigwpg saevvnmqpp



dhllkkgims


481
1ptlgdgrqs gtadspsiln aspesaiggg lswlrtgdti ridintgrcd



alvdeatiae


541
rkkegipavp atmtpwqeiy rahtgqlesg gvlefavkyq dlasklprhn h


 







SEQ ID NO: 40 (Dihydroxy-acid dehydratase (Caulobacter crescentus NA1000))








1
msnrtprrfr srdwfdnpdh idmtalyler fmnygitpee lrsgkpiigi



aqtgsdispc


61
nrihldlvqr vrdgirdagg ipmefpvhpi fencrrptaa ldrnlsylgl



vetlhgypid


121
avvlttgcdk ttpagimaat tvnipaivls ggpmldgwhe nelvgsgtvi



wrsrrklaag


181
eiteeefidr aassapsagh cntmgtastm navaealgls ltgcaaipap



yrergqmayk


241
tgqrivdlay ddvkpldilt kqafenaial vaaaggstna qphivamarh



agveitaddw


301
raaydipliv nmqpagkylg erfhraggap avlwellqqg rlhgdvltvt



gktmsenlqg


361
retsdrevif pyheplaeka gflvlkgnlf dfaimkssvi geefrkryls



qpgqegvfea


421
raivfdgsdd yhkrindpal eidercilvi rgagpigwpg saevvnmqpp



dhllkkgims


481
lptlgdgrqs gtadspsiln aspesaiggg lswlrtgdti ridlntgrcd



alvdeatiaa


541
rkqdgipavp atmtpwqeiy rahasqldtg gvlefavkyq dlaaklprhn h


 







2-Keto-3-deoxy-D-arabinonate Dehydratase (xylX)


SEQ ID NO: 41 (NP_419640.1; GI: 16125076; Hypothetical protein CC_0823 (Caulobacter


crescentus CB15))








1
mvcrrllawt arareaedfa lvrqptcrph mlalpsader apptvsalqt



lefwgddavg


61
vseflpedwk aatllgridf gegptpvlvr ggrvedvski aptvadlmna



fqpgaviprg


121
edkgpleald irpvwedpdg aapvkllapv dlqclkaagv tfavstlerv



ieerargdag


181
ealkirtlla ermggdlksv epgsqgaqrl kdaliadglw sqylevaigp



daeiftkgpt


241
lssmgwgdqv gvrydshwnn pepevvllcd gsglirgaal gndvnlrdfe



grsalllska


301
kdnnascaig pffrlfdetf glddvrsaev elkitgrdnf vldgksnmsl



isrdpavlag


361
qaygkqhqyp dgfalflgtm fapiqdrdtp gqgfthkvgd rvrvstpklg



vlenevttcd


421
kakpwtfgis alirnlagrg ll


 







SEQ ID NO: 42 (Fumarylacetoacetate hydrolase family protein (Caulobacter crescentus


NA1000))








1
mgvseflped wkaatllgri dfgegptpvl vrggrvedvs kiaptvadlm



nafqpgavip


61
rgedkgplea ldirpvwedp dgaapvklla pvdlqclkaa gvtfaystle



rvieerargd


121
agealkirtl laermggdlk svepgsqgaq rlkdaliadg lwsqylevai



gpdaeiftkg


181
ptlssmgwgd qvgvrydshw nnpepevvll cdgsglirga algndvnlrd



fegrsallls


241
kakdnnasca igpffrlfde tfglddvrsa evelkitgrd nfvldgksnm



slisrdpavl


301
agqaygkqhq ypdgfalflg tmfapiqdrd tpgqgfthkv gdrvrvstpk



lgvlenevtt


361
cdkakpwtfg isalirnlag rgll


 







SEQ ID NO: 43 (Fumarylacetoacetate (FAA) hydrolase (Caulobacter segnis ATCC 21756))








1
mgvseflpdd wknatllgri dfgegptpvl vrggrvedms kvaptvadlm



nafgpgaaip


61
rgedkgples ldirpvwedp dgaapvklla pvd1qclkaa gvtfavstle



rvieerargd


121
aaaalkireq lsasmggdlr svnpgsegae rlkqtlikdg lwsqylevai



gpdaeiftkg


181
ptlssmgwgd hvgvrydshw nnpepevvll cdgagqirga slgndvnlrd



fegrsallls


241
kakdnnasca igpffrlfde tfalddvrsa evelkitgrd nfvldgksnm



slisrdpavl


301
agqaygkqhq ypdgfalflg tmfapiqdrd tpgqgfthkv gdrvrvstpk



lgvlenevtt


361
cdkakpwtfg isalirnlag rgll


 







SEQ ID NO: 44 (Hypothetical protein Caul_4000 (Caulobacter sp. K31))








1
malsdflpdd wrdatllgri dfgqgptpvl irggriedvs kiapttsdlm



nafapgaaip


61
rgedlgplea ldvravwenp qgaaakllap vdlqvlkaag vtfavstler



vieerargda


121
aealkiraql adsmggdlrs vnpgsdgaer lkqtlikdgl wsqylevaig



pdaeiftkgp


181
tlssmgwgdh vgvrsdshwn npepevvllc dgsgqirgaa lgndvnlrdf



egrsalllsk


241
akdnnascai gpffrlfddg fslddvrsae vtlkitgrdn fvldghsnms



lisrdpavla


301
gqafgkqhqy pdgfalflgt mfapiqdrda agqgfthkvg drvrvatpkl



gvlenevttc


361
dlaapwtfgv salirnlagr gll


 







SEQ ID NO: 45 (Fumarylacetoacetate (FAA) hydrolase family protein (Caulobacter sp.


AP07))








1
malsdflpdd wrdatllgrv dfgdgptpvl vrggriedvs riapttsdlm



nafapgaaip


61
agadlgplea ldvrpvwenp dgaaakllap vdlqvlkaag vtfavstler



vieerargda


121
aealkiraql adsmggdlrg vnpgsegaar lketlikggl wsqylevaig



pdaeiftkgp


181
tlssmgwgdq vgvrsdshwn npepevvllc dgsgrirgas lgndvnlrdf



egrsalllsk


241
akdnnascai gpffrlfddg fglddvrsae vtlkitgrdn fvldghsnms



lisrdpavla


301
gqafgkqhqy pdgfvlflgt mfapiqdrdt agqgfthkvg drvrvatpkl



gvlenevttc


361
dvappwtfgv salirnlagr gll


 







L-arabinose dehydrogenase (AraE)


SEQ ID NO: 46 (YP_439823.1; GI: 83716868; Dehydrogenase (Burkholderia thailandensis


E264))








1
mnsvytlglv gigkiardqh lpaiaaepgf dllacasrha qvrgvrnypd



idallaaepa


61
ldavslaapp qvryaqaraa lgagkhvmle kppgatagei aalralarer



grtlfaawhs


121
rhasavepar awlatrtira vqarwkedvr rwhpgqqwiw epgglgvfdp



ginalsivtr


181
ilprelvlra atlvvpanah tpiaaeldcv dtagvpvrae fdwrhgpveq



wdiavdtdgg


241
vlsigaggar lsiagepval ppereypsly arfraligeg asdvddrplr



lvadafmigr


301
riaadpfqr


 







SEQ ID NO: 47 (Dehydrogenase (Burkholderia thailandensis TXDOH))








1
mnsvytlglv gigkiardqh lpaiaaepgf dllacasrha qvrgvrnypd



idallaaepa


61
ldavslaapp qvryaqaraa lgagkhvmle kppgatagei aalhalarer



grtlfaawhs


121
rhasavepar awlatrtira vqvrwkedvr rwhpgqqwiw epgglgvfdp



ginalsivtr


181
ilprelvlra atlvvpanah tpiaaeldcv dtagvpvrae fdwrhgpveq



wdiavdtdgg


241
vlaigaggar lsiagepval ppereypsly arfraligeg asdvddrplr



lvadafmigr


301
riaadpfqr


 







SEQ ID NO: 48 (Galactose 1-dehydrogenase (Burkholderia ambifaria IOP40-10))








1
mskvislgvi gigkiardqh lpaiaaepgf altacasrha evngvrnype



lgallaaepe


61
leavslcapp qvryaqaraa leagkhvmle kppgatlgev aaldalarer



gltlfatwhs


121
rcasavepar awlatrtira vqvrwkedvr rwhpgqqwiw epgglgvfdp



ginalsivtr


181
ilprelvlre atlyvpsdvq tpiaaeldca dtdgvpvhae fdwrhgpveq



weiavdtsdg


241
vlaisrggaq lsiggepvei gpqreypaly ahfraliarg esdvdvrplr



lvadaflfgr


301
rvgtdafgr


 







SEQ ID NO: 49 (Galactose 1-dehydrogenase (Burkholderia ambifaria MC40-6))








1
mskvislgvi gigkiardqh lpaiaaepgf altacasrha evngvrnype



lgallaaepe


61
leavslcapp qvryaqaraa leagkhvmle kppgatlgev aaldalarer



gltlfatwhs


121
rcasavepar awlatrtira vqvrwkedvr rwhpgqqwiw epgglgvfdp



ginalsivtr


181
ilprelvlre atlyvpsdvq tpiaaeldca dtdgvpvhae fdwrhgpveq



weiavdtsdg


241
vlaisrggaq lsiagepvei gpqreypaly ahfraliarg esdvdvrplr



lvadaflfgr


301
rvgtdafgr


 







SEQ ID NO: 50 (Dehydrogenase (Burkholderia thailandensis MSMB43))








1
mntvytlglv gigkiardqh lpaiaaepgf dlracasrha evrgvrnhpd



igallaaepa


61
ldavslaapp qvryaqaraa ldagkhvmle kppgatvgei aalralarer



grtlfaswhs


121
rharavepar awlatrtira vqvrwkedvr rwhpgqqwiw epgglgvfdp



ginalsivtr


181
ilprelvlra atlvvpanvh tpiaaefdcv dtagvpvrae fdwrhgpveq



wdiavdtdgg


241
vlaigaggar lsiagepval ppeceypsly arfhaliaar esdvddrplr



lvadafmvgr


301
riaadpfhr


 







L-arabinonolactonase (AraI)


SEQ ID NO: 51 (YP_439819.1; GI: 83717359; Senescence marker protein-30 family protein


(Burkholderia thailandensis E264))








1
messnrpart gaasaatlrv dcrnalgega twcdatraly wvdiegarlw



rwraagaqgg


61
aatdswempe rigcfaltdd pdvllvglas rlaffdarrr aftpivdvep



dlptrlndgr


121
cdragafvfg mkdegggspr avggyyrlnp dlslqrlalp laaiangitf



spdgsamyfc


181
dsptreiqvc dyrpggdvdr irsfvrladd cgepdgsavd adggvwnaqw



ggarivryda


241
qgveteriav ptpqpscval ddggrlyvts arvglddgal arspgaggvf



vadtrhagla


301
tsrfalarna


 







SEQ ID NO: 52 (Senescence marker protein-30 family protein (Burkholderia thailandensis


TXDOH))








1
messsrpart gaasaatlrv dcrnalgega twcdatraly wvdiegarlw



rwraagaqgg


61
aatdswempe rigcfaltdd pdvllvglas rlaffdarrr aftpivdvep



dlptrlndgr


121
cdragafvfg mkdegggspr avggyyrlnp dlslqrlalp paaiangiaf



spdgsamyfc


181
dsptreiqvc dyrpggdvdr irpfvrladd cgepdgstvd adggvwsaqw



ggarivryda


241
qgveteriav ptpqpscval ddggrlyvts arvglddgal arspgaggvf



vadtrhagla


301
tsrfalarna


 







SEQ ID NO: 53 (Senescence marker protein-30 family protein (Burkholderia thailandensis


Bt4)








1
messnrpart gaasaatlrv dcrnalgega twcdatraly wvdiegarlw



rwraagaqgg


61
aatdswempe rigcfaltdd pdvllvglas rlaffdarrr aftpivdvep



dlptrlndgr


121
cdragafvfg mkdegggspr avggyyrlnp dlslqrlalp laaiangiaf



spdgsamyfc


181
dsptreiqvc dyrpggdvdr irsfvrladd cgepdgsavd adggvwnaqw



ggarivryda


241
qgveteriav ptpqpscval ddggrlyvts arvglddgal arspgaggvf



vadtrhagla


 







SEQ ID NO: 54 (Hypothetical protein BPSS0776 (Burkholderia pseudomallei K96243))








1
messnrpart heasaatllv dcrnalgega twcdaahaly wvdiegarlw



rwraagahgg


61
ercdswempe riacfaltgd pdvllvglas rlaffdtrrr altpivdvep



drptrlndgr


121
cdragafvfg tkdesggasp raiggyyrln adlslqrlal ppaaiangia



fspdgsamyf


181
cdsptreiqv cdyrpggdvd rvrsfvrlad ahgepdgstv dasggvwnaq



wggarvvryd


241
aqgvetdria vptpqpscvt ldaagrlyvt sarvglddga lagnpgaggv



fvahtrhsgs


301
atprfalarh a


 







SEQ ID NO: 55 (Gluconolactonase (Burkholderia pseudomallei NCTC 13177))








1
messnrpart heasaatllv dcrnalgega twcdaahaly wvdiegarlw



rwraagahgg


61
ercdswempe riacfaltgd pdvllvglas rlaffdtrrr altpivdvep



drptrlndgr


121
cdragafvfg tkdesggasp raiggyyrln adlslqrlal ppaaiangia



fspdgsamyf


181
cdsptreiqv cdyrpggdvd rvrsfvrlad ehgepdgstv dasggvwnaq



wggarvvryd


241
aqgvetdria vptpqpscvt ldaagrlyvt sarvglddga lagnpgaggv



fvahtrhpgg


301
atprfalarh a


 







L-arabinonate dehydratase (AraB)


SEQ ID NO: 56 (YP_439826.1; GI: 83718062; Dihydroxy- cid dehydratase (Burkholderia


thailandensis E264))








1
msaskpklrs aqwfgthdkn gfmyrswmkn qgipdhefdg rpivgicntw



seltpcnahf


61
rklaehvkrg vyeaggfpve fpvfsngesn lrpsamltrn lasmdveeai



rgnpidavvl


121
lagcdkttpa llmgaascdv paivvsggpm lngkldgkni gsgtavwqlh



ealkageidl


181
hrflsaeagm srsagtcntm gtastmacla ealgvalphn aaipavdarr



yvlahmsgmr


241
ivgmaheglv lskiltraaf enairvnaai ggstnavihl kaiagrlgvp



leledwlrlg


301
rgtptivdlm psgrflmeef yyagglpavl rrlgeanllp hpgaltvngq



slwdnvrdap


361
shddevirpl drpliadggi rilrgnlapr gavlkpsaas pellkhrgra



vvfenfehyk


421
atiddealdv dansvlvlkn cgprgypgma evgnmglppk llrqgvkdmv



risdarmsgt


481
aygtvvlhva peaaaggpla avrngdwiel dgeagtltld vsddelarrl



sdhdpasapg


541
vaehaagggy arlyvdhvlq adegcdldfl vgrrgaavpr hsh


 







SEQ ID NO: 57 (Dihydroxy-acid dehydratase (Burkholderia thailandensis TXDOH))








1
msaskpklrs aqwfgthdkn gfmyrswmkn qgipdhefdg rpivgicntw



seltpcnahf


61
rklaehvkrg vyeaggfpve fpvfsngesn lrpsamltrn lasmdveeai



rgnpidavvl


121
lagcdkttpa llmgaascdv paivvsggpm lngkldgrni gsgtavwqlh



ealkageidl


181
hrflsaeagm srsagtcntm gtastmacla ealgvalphn aaipavdarr



yvlahmsgmr


241
ivgmaheglv lskiltraaf enairvnaai ggstnavihl kaiagrlgvp



leledwlrlg


301
rgtptivdlm psgrflmeef yyagglpavl rrlgeanllp hpgaltvngq



slwdnvrdap


361
shddevirpl drpliadggi rilrgnlapr gavlkpsaas pellkhrgra



vvfenfehyk


421
atiddealev dansvlvlkn cgprgypgma evgnmglppk llrqgvkdmv



risdarmsgt


481
aygtvvlhva peaaaggpla avrngdwiel dceagtltld vsddelarrl



sdhdpasapg


541
vaehaagggy arlyvdhvlq adegcdldfl vgrrgaavpr hsh


 







SEQ ID NO: 58 (Dihydroxy-acid dehydratase (Burkholderia multivorans ATCC 17616))








1
msatkprlrs aqwfgtndkn gfmyrswmkn qgipdhefdg rpiigicntw



seltpcnahf


61
rklaehvkrg ifeaggfpve fpvfsngesn lrpsamltrn lasmdveeai



rgnpidavvl


121
lagcdkttpa llmgaascdv paivvsggpm lngklegkni gsgtavwqlh



ealkageidl


181
hhflsaeagm srsagtcntm gtastmacma ealgvalphn aaipavdsrr



yvlahmsgir


241
ivemaleglv lskvltraaf enairvnaai ggstnavihl kaiagrigvp



leledwmrig


301
rdtptivdlm psgrflmeef yyagglpavl rrlgeggllp hpdaltvngk



tlwdnvreap


361
nyddevirpl drpliadggi rilrgnlapr gavlkpsaas pellkhrgra



vvfenfdhyk


421
atindesldv dansvlvlkn cgprgypgma evgnmglppk llrqgvkdmv



risdarmsgt


481
aygtvvlhva peaaaggpla avrngdwiel dceagtlhld ipddelqrrl



sdvdpaaapg


541
vagqagkggy arlyldhvlq adegcdldfl vgtrgaevps hsh


 







SEQ ID NO: 59 (Dihydroxy-acid dehydratase (Burkholderia multivorans CGD2M))








1
msatkprlrs aqwfgtndkn gfmyrswmkn qgipdhefdg rpiigicntw



seltpcnahf


61
rklaehvkrg ifeaggfpve fpvfsngesn lrpsamltrn lasmdveeai



rgnpidavvl


121
lagcdkttpa llmgaascdv paivvsggpm lngklegkni gsgtavwqlh



ealkageidl


181
hhflsaeagm srsagtcntm gtastmacma ealgvalphn aaipavdsrr



yvlahmsgir


241
ivemaleglv lskvltraaf enairvnaai ggstnavihl kaiagrigvp



leledwmrig


301
rdtptivdlm psgrflmeef yyagglpavl rrlgeggllp hpdaltvngk



tlwdnvrdap


361
nyddevirpl drpliadggi rilrgnlapr gavlkpsaas pellkhrgra



vvfenfdhyk


421
atindealdv dansvlvlkn cgprgypgma evgnmglppk llrqgvkdmv



risdarmsgt


481
aygtvvlhva peaaaggpla avrngdwiel dceagtlhld ipddelqrrl



sdvdpaaapg


541
vagqagkggy arlyldhvlq adegcdldfl vgtrgaevps hsh


 







SEQ ID NO: 60 (Dihydroxy-acid dehydratase (Burkholderia thailandensis MSMB43))








1
msaskpklrs aqwfgthdkn gfmyrswmkn qgipdhefdg rpivgicntw



seltpcnahf


61
rklaehvkrg vyeaggfpve fpvfsngesn lrpsamltrn lasmdveeai



rgnpidavvl


121
lagcdkttpa llmgaascdv paivvsggpm lngkldgkni gsgtavwqlh



ealkageidl


181
hrflsaeagm srsagtcntm gtastmacla ealgvalphn aaipavdarr



yvlahlsgar


241
ivemahegla lstiltraaf enairanaai ggstnavihl kaiagrlgvp



leledwmrig


301
rdtptivdlm psgrflmeef yyagglpavl rrlgeanllp hpgaltvngk



slwenvrdap


361
nhddevirpl arpliadggi rvlrgnlapr gavlkpsaas pellrhrgra



vvfenfehyk


421
atiddealdv dassvlvlkn cgprgypgma evgnmglppk llrqgvkdmv



risdarmsgt


481
aygtvvlhva peaaaggpla avrngdwial dceagtltld vsddelarrl



sdldpasapg


541
aagqagsggy arlyvdhvlq adegcdldfl vgrrgaavpr hsh


 







2-Keto-3-deoxy-L-arabinonate Dehydratase (AraD)


SEQ ID NO: 61 (YP_439824.1; GI: 83717217; Dihydrodipicolinate synthase (Burkholderia


thailandensis E264))








1
mntsrspryr gvfpvvpttf aeageldlps qkravdfmid agseglcila



nfseqfalad


61
derdvltrti lehvagrvpv ivttthystq vcaarsrraq elgaamvmam



ppyhgatfrv


121
pdtqihafya rlsdaldipi miqdapasgt vlsapflarm areieqvsyf



kietpgaank


181
lrelirlggd aiegpwdgee aitlladlna gatgamtgga ypdgirpive



ahregradda


241
falyqrwlpl inhenrqtgl laakalmreg gviacerprh plppihpdsr



aeliaiarrl


301
dplvlrwar


 







SEQ ID NO: 62 (Dihydrodipicolinate synthase, putative (Burkholderia thailandensis


TXDOH))








1
mntsrspryr gvfpvvpttf teageldlps qkravdfmid agseglcila



nfseqfalad


61
derdvltrti lehvagrvpv ivttthystq vcaarsrraq elgaamvmam



ppyhgatfrv


121
pdtqihafya rlsdaldipi miqdapasgt vlsapflarm areieqvsyf



kietpgaank


181
lrelirlggd aiegpwdgee aitlladlna gatgamtgga ypdgirpive



ahregradda


241
falyqrwlpl inhenrqtgl laakalmreg gviacerprh plppihpdsr



aeliaiarrl


301
dplvlrwar


 







SEQ ID NO: 63 (Dihydrodipicolinate synthase, putative (Burkholderia thailandensis


MSMB43))








1
mntsrspryr gvfpvvpttf tetgeldlps qmravdfmid agseglcila



nfseqfalad


61
derdvltrti lehvagrvpv ivttthystr vcaarsrraq elgaamvmam



ppyhgatfry


121
pdtqihafya rlsdaldipi miqdapasgt vlsapflarm areieqvsyf



kietpgaank


181
lrelirlggd aiegpwdgee aitlladlna gatgamtgga ypdgirpivd



ahrdgradda


241
falyqrwlpl inhenrqtgl vaakalmreg gviacerprh plppihpdsr



aelieiarrl


301
dplvlrwar


 







SEQ ID NO: 64 (Dihydrodipicolinate synthase/N-acetylneuraminate lyase (Burkholderia


dolosa AUO158))








1
mtssrtpryr gifpvvpttf tdtgeldlas qkravdfmid agsdglcila



nfseqfaitd


61
derdvltrti lehvagrvpv ivttthystq vcaarslraq qlgaamvmam



ppyhgatfrv


121
peaqiydfya rvsdaidipi miqdapasgt vlsapllarm areieqvsyf



kietpgaank


181
lrelirlggd avegpwdgee aitlladlna gatgamtgga ypdgirpile



ahregrhdda


241
fahygrwlpl inhenrqsgi lsakalmreg gviacerprh pmpelhpdtr



aeliaiarrl


301
dplvlrwar


 







SEQ ID NO: 65 (Dihydrodipicolinate synthetase family protein (Burkhoideria multivorans


ATCC BAA-247))








1
mtssrtpryr gifpvvpttf tetgeldlas qkravdfmid agsdglcila



nfseqfalad


61
derdvltrti lehvagrvpv ivttshystq tciarsvraq qlgaamvmvm



ppyhgatfrv


121
peaqihafya rlsdalsipi miqdapasgt vlsapflaql areiehvayf



kietpgaank


181
lrelirlggd aiegpwdgee aitlladlha gatgamtgga ypdgirpile



ahregrhdda


241
faryqtwlpl inhenrqsgi ltakalmreg gviaceaprh pmpalhpdtr



aeliaiarrl


301
dplvlrwar


 







D-glucarate dehydratase (YcbF)


SEQ ID NO: 66 (NP_388131.2; GI: 255767063; Glucarate dehydratase (Bacillus subtilis


subsp. subtilis str.168))








1
msspiqeqvq kekrsnipsi semkvipvag hdsmllnlsg ahspfftrni



viltdssgnq


61
gvgevpggeh irrtlelsep lvvgksigay qailqtvrkq fgdqdrggrg



nqtfdlrttv


121
havtaleaal ldllgkflqe pvaallgegk qrdevkmlgy lfyigdrnrt



tlpyqsdeqs


181
dcawfrlrhe ealtpeaivr laesaqeryg fqdfklkggv lrgeeeieav



talskrfpea


241
ritldpngaw sleeaialck gkqdvlayae dpcgdengys arevmaefrr



atglptatnm


301
iatdwremgh aiqlhavdip ladphfwtmq gsvrvaqmch dwgltwgshs



nnhfdislam


361
fthvaaaapg ritaidthwi wqdgqrltkq pfeissgcvk vpdkpglgvd



idmeqvekah


421
eiyrkmnlga rndaipmqfl isnwefdrkr pclvr


 







SEQ ID NO: 67 (Glucarate dehydratase (Bacillus subtilis))








1
msspiqeqvq kekrsnipsi semkvipvag hdsmllnlsg ahspfftrni



viltdssgnq


61
gvgevpggeh irrtlelsep lvvgksigay qailqtvrkq fgdqdrggrg



nqtfdlrttv


121
havtaleaal ldflgkflqe pvaallgegk qrdevkmlgy lfyigdrnrt



tlpyqsdeqs


181
dcawfrlrhe ealtpeaivr laesaqeryg fqdfklkggv lrgeeeieav



talskrfpea


241
ritldpngaw sleeaialck gkqdvlayae dpcgdengys arevmaefrr



atglptatnm


301
iatdwremgh aiqlhavdip ladphfwtmq gsvrvaqmch dwgltwgshs



nnhfdislam


361
fthvaaaapg ritaidthwi wqdgqrltkq pfeissgcvk vpdkpglgvd



idmeqvekah


421
eiyrkmnlga rndaipmqfl isnwefdrkr pclvr


 







SEQ ID NO: 68 (Hypothetical protein BSNT_00441 (Bacillus subtilis subsp. natto BEST195))








1
msspiqeqvq kekrsnipsi temkvipvag hdsmllnlsg ahspfftrni



viltdssgnq


61
gvgevpggeh irrtlelsep lvvgksigay qailqtvrkq fgdqdrggrg



nqtfdlrttv


121
havtaleaal ldllgkflqe pvaallgegk qrdevkmlgy lfyigdrkrt



tlpyqsdeqs


181
dcawfrlrhe ealtpeaivr laesaqeryg fqdfklkggv lqgeeeieav



talskrfpea


241
ritldpngaw sleeaialck gkqdvlayae dpcgdengys arevmaefrr



atglptatnm


301
iatdwremgh aiqlhavdip ladphfwtmq gsvrvaqmch dwgltwgshs



nnhfdislam


361
fthvaaaapg ritaidthwi wqdgqrltkq pfeissgcvk vpdkpglgid



idmegvekah


421
eiyrkmnlga rndaipmqfl isnwefdrkr pclvr


 







SEQ ID NO: 69 (Glucarate dehydratase (Bacillus subtilis subsp. spizizenii TU-B-10))








1
msspiqeqvq kekrsnipsi cemkvipvag hdsmllnlsg ahspfftrni



viltdssgnq


61
gvgevpggeq irrtlelaep lvvgksigay qsilqtvrkg fadqdrggrg



iqtfdlrttv


121
havtaleaal ldllgkflqe pvaallgegk qrdevkmlgy lfyigdrkqt



tlpyqsdeqs


181
dcgwfrlrhe ealtpeaivr laesaqeryg fqdfklkggv lrgedeieav



talakrfpea


241
ritldpngaw sleeaialck gkhdvlayae dpcgdengys arevmaefrr



atglptatnm


301
iatdwremgh aiqlhavdip ladphfwtmq gsvrvaqmch dwgltwgshs



nnhfdislam


361
fthvaaaapg ritaidthwi wqdgqrltkq pfeisegcvk vpnkpglgid



idmeqvekah


421
elyrkmnlga rndavpmqfl isnwefdrkr pclvr


 







SEQ ID NO: 70 (Glucarate dehydratase (Bacillus subtilis subsp. subtilis str. RO-NN-1))








1
msspmqeqiq kekrsnvpsi semkvipvag hdsmllnlsg ahspfftrni



viltdssgnq


61
gvgevpggeh irrtlelsep lvvgksigay qailqtvrkq fgdqdrggrg



nqtfdlrttv


121
havtaleaal ldllgkflqe pvaallgegk qrdevkmlgy lfyigdrkrt



tlpyqsdeqs


181
ycawfrlrhe ealtpeaivr laesaqeryg fqdfklkggv lrgeeeieav



talskrfpea


241
ritldpngaw sleeaialck gkqdvlayae dpcgdengys arevmaefrr



atglptatnm


301
iatdwremgh aiqlhavdip ladphfwtmq gsvrvaqmcn dwgltwgshs



nnhfdislam


361
fthvaaaapg ritaidthwi wqdgqrltkq pfeissgcvk vpdkpglgvd



idmeqvekah


421
eiyrkmnlga rndaipmqsl isnwefdrkr pclvr


 







D-galactarate dehydratase (YcbH)


SEQ ID NO: 71 (NP_388133.2; GI: 255767065; D-galactarate dehydratase (Bacillus subtilis


subsp. subtilis str.168))








1
mamnlrknqa plyikvheid ntaiivndgg lpkgtvfscg lvleedvpqg



hkvaltdlnq


61
gdeivrygev igfadetikr gswirealvr mpappalddl planrvpqpr



pplegytfeg


121
yrnadgsagt knilgittsv qcvvgvldya vkrikeellp kypnvddvvp



lhhqygcgva


181
inapdavipi rtiqnlakhp nfggevmvig lgcekllper iasendddil



slqdhrgfaa


241
miqsilemae erlirinsrt rvscpvsdlv iglqcggsda fsgvtanpav



gyaadllvra


301
gatvlfsevt evrdaihllt prayseevgq slikemkwyd sylrrgdadr



sanpspgnkk


361
gglsnvveka lgsvaksgts pisgvlgpge rakqkgllfa atpasdfvcg



tlqlaagmnl


421
qvfttgrgtp yglaaapvlk vstrhslseh wadlidinag riatgeasie



dvgweifrti


481
ldvasgrkqt wadrwglhnd lclfnpapvt


 







SEQ ID NO: 72 (Hypothetical protein BSNT 00443 (Bacillus subtilis subsp. natto BEST195))








1
mamnlrknqa plyikvheid ntaiivndgg lpkgtvfscg lvleedvpqg



hkvaltdlnq


61
gdeivrygev igfadetikr gswirealvr mpappalddl planrvpqpr



pplegytfeg


121
yrnadgsagt knilgittsv qcvvgvldya vkrikeellp kypnvddvvp



lhhqygcgva


181
inapdavipi rtiqnlakhp nfggevmvig lgcekllper iasendddil



slqdhrgfaa


241
miqsilemae erlirinsrt rvscpvsdlv iglqcggsda fsgvtanpav



gyaadllvra


301
gatvlfsevt evrdaihllt prayseevgq slikemkwyd sylrrgdadr



sanpspgnkk


361
gglsnvveka lgsvaksgts pisgvlgpge raeqkgllfa atpasdfvcg



tlqlaagmnl


421
qvfttgrgtp yglaaapvlk vstrhslseh wadlidinag riatgeasie



dvgweifrti


481
ldvasgrkqt wadrwglhnd lclfnpapvt


 







SEQ ID NO: 73 (D-galactarate dehydratase (Bacillus subtilis subsp. subtilis str. RO-NN-1))








1
mamnlrknqa plyikvheid ntaiivndgg lpkgtvfscg lvleedvpqg



hkvaltdlnq


61
gdeivrygev igfadetikr gswirealvr mpappalddl planrvpqpr



pplegytfeg


121
yrnadgsagt knilgittsv qcvvgvldya vkrikeellp kypnvddvvp



lhhqygcgva


181
inapdavipi rtiqnlakhp nfggevmvig lgcekllper iasendddil



slqdhrgfaa


241
miqsilemae erlirinsrt rvscpvsdlv iglqcggsda fsgvtanpav



gyaadllvra


301
gatvlfsevt evrdaihllt prayseevgq slieemkwyd sylrrgdadr



sanpspgnkk


361
gglsnvveka lgsvaksgts pisgvlgpge raeqkgllfa atpasdfvcg



tlqlaagmnl


421
qvfttgrgtp yglaaapvlk vstrhslseh wadlidinag riatgeasie



dvgweifrti


481
ldvasgrkqt wadrwglhnd lclfnpapvt


 







SEQ ID NO: 74 (Hypothetical protein BSSC8_40810 (Bacillus subtilis subsp. subtilis SC-


8))








1
mamnlrknqa plyikvheid ntaiivnegg lpkgtvfscg lvleedvpqg



hkvaltdlnq


61
gdeivrygev igfadetikr gswirealvr mpappalddl plenrvpqpr



pplegytfeg


121
yrnadgsagt knilgittsv qcvvgvldya vkrikeellp kypnvddvvp



lhhqygcgva


181
inapdavipi rtiqnlakhp nfggevmvig lgcekllper iasendddil



slqdhrgfaa


241
miqsilemae erlirinsrt rvscpvsdlv iglqcggsda fsgvtanpav



gyaadllvra


301
gatvlfsevt evrdaihllt prayseevgq slikemkwyd sylrrgdadr



sanpspgnkk


361
gglsnvveka lgsvaksgts pisgvlgpge raeqkgllfa atpasdfvcg



tlqlaagmnl


421
qvfttgrgtp yglaaapvlk vstrhslseh wadlidinag qiatgeasie



dvgweifrti


481
ldvasgrkqt wadrwglhnd lclfnpapvt


 







SEQ ID NO: 75 (Galactarate dehydratase (Bacillus subtilis BSn15))








1
mamnlrknqa plyikvheid ntaiivndgg lpkgtvfscg lvleedvpqg



hkvaltdlnq


61
gdeivrygev igfadetikr gswiredlvr mpappalddl planrvpqpr



pslegytfeg


121
yrnadgstgt knilgittsv qcvvgvldya vkrikeellp kypnvddvvp



lhhqygcgva


181
inapdavipi rtiqnlakhp nfggevmvig lgcekllper iasengddil



slqdhrgfaa


241
miqsilemae erlirinsrt rvscpvsdlv iglqcggsda fsgvtanpav



gyaadllvra


301
gatvlfsevt evrdaihllt prayseevgq slikemkwyd sylrrgdadr



sanpspgnkk


361
gglsnvveka lgsvaksgts pisgvlgpge rakqkgllfa atpasdfvcg



tlqlaagmnl


421
qvfttgrgtp yglaaapvlk vstrhslseh wadlidinag riatgeasie



dvgweifrti


481
ldvasgrkqt wadrwglhnd lclfnpapvt


 







5-dehydro-4-deoxyglucarate dehydratase (YcbC)


SEQ ID NO: 76 (NP_388128.2; GI: 255767061; 5-dehydro-4-deoxyglucarate dehydratase


(Bacillus subtilis subsp. subtilis str.168))








1
msrirkapag ilgfpvapfn tqgkleeeal fqniefllne gleaifiacg



sgefqslsqk


61
eyeqmvevav saaggkvpvy tgvggnlsta ldwaqlsekk gadgylilpp



ylvhgeqegl


121
yqyaktiies tdlnailyqr dnavlsveqi krlteceqlv gvkdgvgnmd



lninlvytig


181
drlgwlngmp maevtmpayl pigfhsyssa isnyiphisr mfydalkngn



delvkelyrh


241
vilpindirk qrkgyavsli kagmeimgln vrntarppvg pvekdhyqql



eailkqaadr


301
fpkkaatv


 







SEQ ID NO: 77 (Putative 5-dehydro-4-deoxyglucarate dehydratase (Bacillus subtilis subsp.


subtilis str. RO-NN-1))








1
msrirkapag ilgfpvapfn tqgkleeeal fqniefllne gleaifiacg



sgefqslsqk


61
eyeqmvevav saaggkvpvy tgvggnlsta lewaqlsekk gadgylilpp



ylvhgeqegl


121
yqyaktiies tdlnailyqr dnavlsveqi krlteceqlv gvkdgvgnmd



lninlvytig


181
drlgwlngmp maevtmpayl pigfhsyssa isnyiphisr mfydalkngn



delvkelyrh


241
vilpindirk qrkgyavsli kagmeimgln vrntarppvg pvekdhyqql



eailkqaadr


301
fpkkaatv


 







SEQ ID NO: 78 (5-dehydro-4-deoxyglucarate dehydratase (Bacillus vallismortis DV1-F-3))








1
mnrirkaptg ilgfpvapfn tqgqleeeal fqniefllee gleaifiacg



sgefqslsqk


61
eyeqmvevav saaegkvpvy tgvggnlsta lewarlsekk gadgylilpp



ylvhgeqegl


121
yqyaktiies tdlnailyqr dnavlsleqi krlteceqlv gvkdgvgnmd



lninlvytlg


181
drlgwlngmp maevtmpayl pigfhsyssa isnyiphisr mfydalkngn



delvkelyqh


241
vilpindirk qrkgyavsli kagmeimgln vrntarppvg pvekehyrql



eailkqaadr


301
fpkkaatv


 







SEQ ID NO: 79 (5-dehydro-4-deoxyglucarate dehydratase (Bacillus subtilis subsp. spizizenii


TU-B-10))








1
msrirkapag ilgfpvapfn tqgkleeeal fqniefllee gleaifiacg



sgefqslsqk


61
eyeqmvevai saaggkvpvy tgvggnlsta lewaqlsekk gadgylilpp



ylvhgeqegl


121
yqyaktiies tdlnailyqr dnavlsveqi krltefeqlv gvkdgvgnmd



lninlvytlg


181
drlgwlngmp maevtmpayl pigfhsyssa isnyiphisr mfydalkngd



delvkelyqh


241
vilpindirk qrkgyavsli kagmeimgln vrntarppvg pvekdhyqql



eailkqaadr


301
fpkkaatv


 







SEQ ID NO: 80 (ycbC (Bacillus subtilis))








1
msrirkapag ilgfpvapfn tqgtleeeal fqniefllne gleaifiacg



sgefqslsqk


61
eyeqmvevav saaggkvpvy tgvggnlsta ldwaqlsekk gadgylilpp



ylvhgeqegl


121
yqyaktiies tdlnailyqr dnavlsveqi krlteceqlv gvkdgvgnmd



lninlvytig


181
drlgwlngmp maevtmpayl pigfhsyssa isnyiphisr mfydalkngn



delvkelyrh


241
vilpindirk qrkgyavsli kagmeimgln vrntarppvg pvekdhyqql



eailkqpadr


301
fpkkaatv


 







Amino acid transporter LysE (HypE)


SEQ ID NO: 81 (NP_743408.1; GI: 26987983; Amino acid transporter LysE (Pseudomonas


putida KT2440))








1
maaesyrlqa ldpsrawhrf fatvqqqvek rafgddsseh clrnaqqelt



mlgvtdygaf


61
viaflillai pgpgnfalit atgkggikag laatcgvivg dqvllwlava



gvatllatyp


121
aafhmvqwag aaylaylglr mllskpggaa htcrmdngqy lrqtmmitll



npkaimfyma


181
ffplfvdpvk hqglvtfgfm aatvavvtfl ygliavvlth qlaermrasp



rianmferla


241
gaclvgfgik laamr


 







SEQ ID NO: 82 (Amino acid transporter LysE (Pseudomonas putida BIRD-1))








1
mqqqvekraf gddssahclr naqqeltmlg vtdygafvia flillaipgp



gnfalitatg


61
kggikaglaa tcgvivgdqv llwlavagva tllatypaaf hvvqwagaay



laylglrmll


121
skpggaahtc rmdngqylrq tmmitllnpk aimfymaffp lfvdpvkhqg



lvtfgfmaat


181
vavvtflygl iavvlthqla ermraspria nmferlagac lvgfgiklaa mr


 







SEQ ID NO: 83 (Amino acid transporter LysE (Pseudomonas putida ND6))








1
mqqqvekrav gddssahclr naqqeltmlg vtdygafvia flillaipgp



gnfalitatg


61
kggikaglaa tcgvivgdqv llwlavagva tllatypaaf hmvqwagaay



laylglrmll


121
skpggaahtc rmdngqylrq tmmitllnpk aimfymaffp lfvdpvkhqg



lvtfgfmaat


181
vavvtflygl iavvlthgla ermranpria nmferlagac lvgfgiklaa mr


 







SEQ ID NO: 84 (Lysine exporter protein LysE/YggA (Pseudomonas putida F1))








1
mlgvtdygaf viaflillai pgpgnfalit atgkggikag laatcgvivg



dqvllwlava


61
gvatllatyp aafhmvqwag aaylaylglr mllskpggaa htcrmdngqy



lrqtmmitll


121
npkaimfyma ffplfvdpvk hqglvtfgfm aatvavvtfl ygliavvlth



qlaermranp


181
rianmferla gaclvgfgik laamr


 







SEQ ID NO: 85 (Unknown (Pseudomonas putida))








1
mlgvtdygaf viafiillai pgpgnfalit atgkggikag laatcgvivg



dqvllwlava


61
gvatllatyp aafhivqwag aaylaylglr mllskpgdap rtsrmdngqy



lrqtmlitll


121
npkaimfyma ffplfidpvk hqglvtfgfm aatvavitfl ygliavvlth



rlaermranp


181
ritnmferla gaclvgfgik laamr


 







PP_1245


SEQ ID NO: 86 (NP_743405.1; GI: 26987980; Hypothetical protein PP_1245 (Pseudomonas


putida KT2440))








1
mrptengvlh lrkkfvasll avaiasttac aqlgiskeqa gtvigglagv



aigstmgsgn


61
gkiaaaliag gigayvgnri ghmldekdqq alalrtqevl sqqqttasaq



pvtwksdhsg


121
ataqivpgke ytktkqvevk rapkiqavps mklinepyvt isdnlnvraa



pngagekvgs


181
lknhteftav gstgdwilvg rkgvtvgyvh knyvepkaqa vakrvtpavn



ldeldvaask


241
etqgfdldsv qslptqtvaa eaacrpvtvs lksgsgqteq eqntfckqan gtweli


 







SEQ ID NO: 87 (SH3 type 3 domain-containing protein (Pseudomonas putida W619))








1
mrkkfvasll avaiatttac aqlgiskeqa gtvigglagv aigstmgsgn



gkiaaaliag


61
gigayvgnri ghmldekdqq alalrtqevl sqsatasaqp vtwksdhsga



taqitpgkey


121
tqtkkvevkr apkiqavpsm klinepyvti sdnlnvraap nttgekvgsl



kshteftavg


181
stgdwilvgr kgvtvgyvhk nyvepkaqai akraapavnl ddldvaanke



tqgfdldsiq


241
slptetvaae aacrpvtvsl ksqsgqteqe qntfckqang tweli


 







SEQ ID NO: 88 (Hypothetical protein G1E_03180 (Pseudomonas sp. TJI-51))








1
mrkkfvasll avaiasttac aqlgiskeqa gtvigglagv aigstlgsgn



gkiaaaliag


61
gigayvgnri gnmldekdqq alalrtqevl sqqqatasaq pvtwksdhsg



asaqivpgke


121
ytktkqvevk rapkiqavps mklinepyvt tsdnlnvraa pnasgekvgs



lknhteftav


181
gatgdwilvg rkgvtvgyvh kdyvepkaqa vakrvtpavn ldeldvaask



etqafdldsl


241
qslptqtvaa eaacrpvtvs lkaqngkteq eqntfckqan gtweli


 







SEQ ID NO: 89 (SH3 type 3 domain-containing protein (Pseudomonas putida GB-1))








1
mrkkfvasll avaiasttac aqlgiskeqa gtvigglagv aigstmgsgn



gkiaaaliag


61
gigayvgnri ghmldekdqq alalrtqevl sqqqatasaq pvtwksdhsg



ataqivpgke


121
ytqtkkvevk rapkiqavps mklinepyvt vsdnlnvraa pnqsgekvgs



lknhteftav


181
gstgdwilvg rkgvtvgyvh knyvepkaqa vakrvtpavn ldeldvaask



etqgfdldsv


241
qslptetvaa eaacrpvtvs lksqsgqteq eqntfckqan gtweli


 







SEQ ID NO: 90 (SH3 type 3 domain-containing protein (Pseudomonas putida F1))








1
mrkkfvasll avaiasttac aqlgiskeqa gtvigglagv aigstmgsgn



gkiaaaliag


61
gigayvgnri ghmldekdqq alalrtqevl sqqqttasaq pvtwksdhsg



ataqivpgke


121
ytktkqvevk rapkiqavps mklinepyvt isdnlnvraa pnqagekvgs



lknhteftav


181
gstgdwilvg rkgvtvgyvh knyvepkaqa vakrvtpavn ldeldvaask



etqgfdldsv


241
qslptqtvaa eaacrpvtvs lksqsgqteq eqntfckqan gtweli


 







PP_1247


SEQ ID NO: 91 (NP_743407.1; GI: 26987982; Hypothetical protein PP_1247 (Pseudomonas


putida KT2440))








1
mpicssgwrg lawwdsasnw rrcadpkpds vrarltatlk kppathgsrg



lvhsaitqsi


61
gfqliglahe qrrkqalafl egvllferav fdqllpdgaf rvavvlglga



kvtaprrqpn


121
llaegcelcl gdlllvfaes lfqrfeaava hrvvldlgla gkaahrfsqh



rlagvravra


181
nqhraqgtle lgfdivqfrq rlevglandf phlgavvavg dherhrafai



agaldgevqv


241
drgtkvtgaa dqkragywla hrhvgapgev rrggptiggq lgtwldfvad



irhqhdfgpl


301
ggnvrvahlh aqqldmnaai laysvmgqlq rislqvhpgh iaadielvlg



parqaffsrt


361
tlyglhqarq aahellgaig lrrrhadlrv gyrqvagkrr vgnvplrqhi



lkeiallevv


421
vvgqrsllar agdhriatte hqhrcghtan qqlllvhlfd hgvcltgpwr



krcssrsrtv


481
grprgss


 







SEQ ID NO: 92 (Uncharacterized protein LOC100789425 (Glycine max))








1
msniafrsti vfllfsavls tppedpikca tsenttctit nsygafpdrs



ickaaqvlyp


61
tteqelvsvv asatrnktkm kvatrfshsi pklvcpegen gllistkyln



kilkvdvetr


121
tmtvesgvtl qqlineaakv glalpyapyw wgltigglmg tgahgstlrg



kgsavhdyvv


181
elrivrpagp edgyamvenl neqhedlnaa kvslgvlgvi sqitlklepl



fkrsityvak


241
ddsdlggqvv afgdahefad itwypsqhka iyrvddrvpi ntsgnglydf



ipfrptpsla


301
svfirtteei qestndangk civastasnt litaaygltn ngiifagypi



igfqnrlqss


361
gscldslqda littcawdpr mkglffhqtt fsirlsfvks fiedvqklve



lepkglcvlg


421
lyngmlmryv tassaylghq enaldidity yrskdpmtpr lyedileeve



qlgifkyggl


481
phwgknrnla fegaikkyks aeyflkvkek ydldglfsst wtdqvlglkd



gvtilkdgca


541
leglciclqd shcnpskgyy crpgkvykea rvctnlk


 







PP_1246


SEQ ID NO: 93 (NP_743406.1; GI: 26987981; Hypothetical protein PP_1246 (Pseudomonas


putida KT2440))








1
mkkhalalav igacglvpqa fahelafskk dnikvevpgd atswckpqvd



ltitrpawdn


61
qellaglltk lpfvfakdcs takvswkavd akgnlyasgs gnasnlglvt



laaapataap


121
apaaaptptp apapapapap aaaaapavve aapaqakpap apapapapav



aaepapapea


181
paaapvvppa papatavaaa ptsdfgrsvv lenrnlmqvt dgtgckwvls



tsiigdgdtl


241
sfgttpampc pasgfgegsf dkiswkavgt yrgdnwtrvy ahpsglifnk



nlepavkdka


301
vsyltpqadq aaflvgeipg rqmkvyltft rssygvlrpf ssdpyyvavt



pdesfaldat


361
kykeaaleif dlikttsptt tdvanlfivk dlsaisnniw gndaqkitrn



riginrqglf


421
fdvrdganwa vqreqqrvre qrqrqqelar vhtrvleryq qlqdgmsdfk



gretealaqm


481
agikvrfasp leqqnpatsa svvpmmvhvt gkkgdfysid fpsngrlvad



eeysegwyvt


541
qvanatpyyp lddgravpty raysagepea ckqdhcadrv sfgavlakef



pnagidfswt


601
pevsqqyvnd wnnasamvq


 







SEQ ID NO: 94 (Hypothetical protein T1E_4663 (Pseudomonas putida DOT-T1E))








1
mvlenrnlmq vtdgtgckwv lstsiigdgd tlsfgttpam pcpasgfgeg



sfdkiswkav


61
gtyrgdnwtr vyahpsglif nkhlepavkd kaysyltpqa dqaaflvgei



pgrqmkvylt


121
ftrssygvlr pfgsdpyyva vtpdesfald atkykeaale ifdlikttsp



tttdvanlfi


181
vkdlsaisnn iwgndaqkit rnriginrqg lffdvrdgan wavqreqqrv



reqrqrqqel


241
arvhtrvler yqqlqdgmsd fkgreteala qmagikvrfa spleqqnpat



sasvvpmmvh


301
vtgkkgdfys idfpsngrlv adeeysegwy vtqvanatpy yplddgravp



tyraysagep


361
eackqdhcad rvsfgavlak efpnagidfs wtpevsqqyv ndwnnasamv q


 







SEQ ID NO: 95 (Hypothetical protein YSA_07676 (Pseudomonas putida ND6))








1
mkkhalalav igacglvpqa fahelafskk dnikvevpgd attwckpqvd



ltitrpawdn


61
qellsglltk lpfvfakdcs takvswkavd akgnlyasgs gnasnlglvt



laaapataap


121
apaaavapap apaqpeapaa aaptpapapa papapaaaaa pavveaapaq



akpapapapa


181
pavaaepapt peapaaapvv ppapapatav aaaptsdfgr svvlenrnlm



qvtdgtgckw


241
vlstsiigdg dtlsfgttpa mpcpasgfge gsfdkiswka vgtyrgdnwt



rvyahpsgli


301
fnkhlepavk dkaysyltpq adqaaflvge ipgrqmkvyl tftrssygvl



rpfgsdpyyv


361
avtpdesfal datkykeaal eifdliktts ptttdvanlf ivkdlsaisn



niwgndaqki


421
trnriginrq glffdvrdga nwavqreqqr vreqrqrqqe larvhtrvle



ryqqlqdgms


481
dfkgreteal aqmagikvrf aspleqqnpa tsasvvpmmv hvtgkkgdfy



sidfpsngrl


541
vadeeysegw yvtqvanatp yyplddgrav ptyraysage peackqdhca



drvsfgavla


601
kefpnagidf swtpevsqqy vndwnnasam vq


 







SEQ ID NO: 96 (Hypothetical protein Pput_1275 (Pseudomonas putida F1))








1
mkkhalalav igacglvpqa fahelafskk dnikvevpgd attwckpqvd



ltitrpawdn


61
qellsglltk 1pfvfakdcs takvswkavd akgnlyasgs gnasnlglvt



laaapapapa


121
papapapaaa apapaaavap apapaqpeap aaaaptpapa papapaaaaa



pavveaaaaq


181
akpapapapa pavaaepapt peapaaapvv ppapapatav aaaptsdfgr



svvlenrnlm


241
qvtdgtgckw vlstsiigdg dtlsfgttpa mpcpasgfge gsfdkiswka



vgtyrgdnwt


301
rvyahpsgli fnknlepavk dkavsyltpq adqaaflvge ipgrqmkvyl



tftrssygvl


361
rpfgsdpyyv avtpdesfal datkykeaal eifdliktts ptttdvanlf



ivkdlsaisn


421
niwgndaqki trnriginrq glffdvrdga nwavqreqqr vreqrqrqqe



larvhtrvle


481
ryqqlqdgms dfkgreteal aqmagikvrf aspleqqnpa tsasvvpmmv



hvtgkkgdfy


541
sidfpsngrl vadeeysegw yvtqvanatp yyplddgrav ptyraysage



peackqdhca


601
drvsfgavla kefpnagidf swtpevsqqy vndwnnasam vq


 







SEQ ID NO: 97 (Hypothetical protein PputGB1_4145 (Pseudomonas putida GB-1))








1
mkkhalalav vgacglvpqa fahelafskk enikvevpgd aatwckpeve



ltitrpawdk


61
qellsglltk lpfvfakdca takvswkavd akgnlyasgs gnatnlglvt



lavapaaasa


121
apapapapap apapapapap avaalapaap avpapaeapa avaaapapav



vepapakaev


181
apapvvaaep apapvaetpv aapvappvpa padavaaapt sdfgravvlq



nrnlmqvtdg


241
tgckwvlsts iisdgdtlsf gttpvmpcpa sgfgegsfek iswkavgtyr



gdnwtrvyah


301
psglifnknl esavkdkavs yltadadqaa flvgeipsrq mkvyltftrs



sygvlrpfss


361
dpyyvavtpd esfaldaaky keaaleifdl ikatsptttd vanlfivkdi



saitnsmwgn


421
daqkitrnri gitrqglffd vreganwavq reqqrvreer grqqelarvh



trvleryqql


481
qdgmsdfkgr etealaqmag ikvrfaspla qqdpatsarv apmmvhvtgk



kgdfytldfp


541
skgrlvadee ysegwyvtqv anatpyypld dgravptyra ysagepeacq



qdhcadrvsf


601
gavlakefpn agidfswtpe vsqkyvndwn nasamvq


 







Alpha-ketoisovalerate decarboxylase


SEQ ID NO: 98 (YP_003353820.1; GI: 281491840; Alpha-ketoisovalerate decarboxylase


(Lactococcus lactis subsp. lactis KF147))








1
mytvgdylld rlhelgieei fgvpgdynlq fldqiisrkd mkwvgnanel



nasymadgya


61
rtkkaaaflt tfgvgelsav nglagsyaen lpvveivgsp tskvqnegkf



vhhtladgdf


121
khfmkmhepv taartlltae natveidrvl sallkerkpv yinlpvdvaa



akaekpslpl


181
kkenptsnts dqeilnkiqe slknakkpiv itgheiisfg lentvtqfis



ktklpittln


241
fgkssvdetl psflgiyngk lsepnlkefv esadfilmlg vkltdsstga



fthhlnenkm


301
islnidegki fnesiqnfdf eslisslldl sgieykgkyi dkkqedfvps



nallsqdrlw


361
qavenltqsn etivaeqgts ffgassiflk pkshfigqpl wgsigytfpa



algsqiadke


421
srhllfigdg slqltvqelg lairekinpi cfiinndgyt vereihgpnq



syndipmwny


481
sklpesfgat eervvskivr tenefvsvmk eaqadpnrmy wielvlaked



apkvlkkmgk


541
lfaeqnks


 







SEQ ID NO: 99 (Indole-3-pyruvate decarboxylase (Lactococcus lactis subsp. lactis IO-1))








1
mytvgdylld rlhelgieei fgvpgdynlq fldqiisrkd mkwvgnanel



nasymadgya


61
rtkkaaaflt tfgvgelsav nglagsyaen lpvveivgsp tskvqnegkf



vhhtladgdf


121
khfvkmhepv taartlltae natveidrvl svllkerkpv yinlpvdvaa



akaekpslpl


181
kkenpnsnts dqeilnkiqe slknakkpiv itgheiisfg lektvtqfis



ktklpittln


241
fgkssvdeal psflgiyngk lsepnlkefv esadfilmlg vkltdsstga



fthhlnenkm


301
islninegki fsesiqnfdf eslisslldl sgieykgkyi dkkqenfvps



nallsqdrlw


361
qavenitqsn etivaeqgts ffgassiflk pkshfigqpl wgsigftfpa



algsqiadke


421
srhllfigdg slqltvqelg lairekinpi cfiinndgyt vereihgpnq



syndipmwny


481
sklpesfgat edrvvskivr tenefvsvmk eaqadpnrmy wielvlaked



apkvlkkmgk


541
lfaeqnks


 







SEQ ID NO: 100 (Branched-chain alpha-ketoacid decarboxylase (Lactococcus lactis))








1
mytvgdylld rlhelgieei fgvpgdynlq fldqiisred mkwignanel



nasymadgya


61
rtkkaaaflt tfgvgelsai nglagsyaen lpvveivgsp tskvqndgkf



vhhtladgdf


121
khfmkmhepv taartlltae natyeidrvl sqllkerkpv yinlpvdvaa



akaekpalsl


181
ekessttntt eqvilskiee slknaqkpvv iaghevisfg lektvtqfvs



etklpittln


241
fgksavdesl psflgiyngk lseislknfv esadfilmlg vkltdsstga



fthhldenkm


301
islnidegii fnkvvedfdf ravvsslsel kgieyegqyi dkqyeefips



saplsqdrlw


361
qavesltqsn etivaeqgts ffgastiflk snsrfigqpl wgsigytfpa



algsqiadke


421
srhllfigdg slqltvqelg lsireklnpi cfiinndgyt vereihgptq



syndipmwny


481
sklpetfgat edrvvskivr tenefvsvmk eaqadvnrmy wielvleked



apkllkkmgk


541
lfaeqnk


 







SEQ ID NO: 101 (Chain A, branched-chain ketoacid decarboxylase (Kdca)(Lactococcus


Lactis))








1
mgsshhhhhh ssglvprgsh masmytvgdy lldrlhelgi eeifgvpgdy



nlqfldqiis


61
redmkwigna nelnasymad gyartkkaaa flttfgvgel sainglagsy



aenlpvveiv


121
gsptskvqnd gkfvhhtlad gdfkhfmkmh epvtaartll taenatyeid



rvlsqllker


181
kpvyinlpvd vaaakaekpa lslekesstt ntteqvilsk ieeslknaqk



pvviaghevi


241
sfglektvtq fvsetklpit tlnfgksavd eslpsflgiy ngklseislk



nfvesadfil


301
mlgvkltdss tgafthhlde nkmislnide giifnkvved fdfravvssl



selkgieyeg


361
qyidkqyeef ipssaplsqd rlwqaveslt qsnetivaeq gtsffgasti



flksnsrfig


421
qplwgsigyt fpaalgsqia dkesrhllfi gdgslqltvq elglsirekl



npicfiinnd


481
gytvereihg ptqsyndipm wnysklpetf gatedrvvsk ivrtenefvs



vmkeaqadvn


541
rmywielvle kedapkllkk mgklfaeqnk


 







SEQ ID NO: 102 (Indole-3-pyruvate decarboxylase (Lactococcus lactis subsp. lactis II1403))








1
mytvgdylld rlhelgieei fgvpgdynlq fldqiisrkd mkwvgnanel



nasymadgya


61
rtkkaaaflt tfgvgelsav nglagsyaen lpvveivgsp tskvqnegkf



vhhtladgdf


121
khfmkmhepv taartlltae natveidrvl sallkerkpv yinlpvdvaa



akaekpslpl


181
kkenptsnts dqeilnkiqe slknakkpiv itgheiisfg lektvtqfis



ktklpittln


241
fgkssvdetl psflgiyngk lsepnlkefv esadfilmlg vkltdsstga



fthhlnenkm


301
islninegki fneriqnfdf eslisslldl sgieykgkyi dkkqedfvps



nallsqdrlw


361
qavenltqsn etivaeqgts ffgassiflk pkshfigqpl wgsigytfpa



algsqiadke


421
srhllfigdg slqltvqerk lqvqvsqpss shmnsys


 







Alcohol dehydrogenase yqhD


SEQ ID NO: 103 (YP_001459806.1; GI: 157162488; Alcohol dehydrogenase yqhD


(Escherichia coli HS))








1
mnnfnlhtpt rilfgkgaia glreqiphda rvlitygggs vkktgvldqv



ldalkgmdvl


61
efggiepnpa yetlmnavkl vreqkvtfll avgggsvldg tkfiaaaany



penidpwhil


121
qtggkeiksa ipmgcvltlp atgsesnaga visrkttgdk qafhsahvqp



vfavldpvyt


181
ytlpprqvan gvvdafvhtv eqyvtkpvda kiqdrfaegi lltliedgpk



alkepenydv


241
ranvmwaatq alngligagv pqdwathmlg heltamhgld haqtlaivlp



alwnekrdtk


301
rakllqyaer vwnitegsdd eridaaiaat rnffeqlgvp thlsdygldg



ssipallkkl


361
eehgmtqlge nhditldvsr riyeaar


 







SEQ ID NO: 104 (Alcohol dehydrogenase, iron-dependent (Escherichia coli 97.0259))








1
mnnfnlhtpt rilfgkgaia glreqiphda rvlitygggs vkktgvldqv



ldalkgmdvl


61
efggiepnpa yetlmnavkl vreqkvtfll avgggsvldg tkfiaaaany



penidpwhil


121
qtggkeiksa ipmgcvltlp atgsesnaga visrkttgdk qafhsahvqp



vfavldpvyt


181
ytlpprqvan gvvdafvhtv eqyvtkpvda kiqdrfaegi lltliedgpk



alkepenydv


241
ranvmwaatq alngligagv pqdwathmlg heltamhgld haqtlaivlp



alwnekrdtk


301
rakllqyaer iwnitegsdd eridaaiaat rnffeqlgvp thlsdygldg



ssipallkkl


361
eehgmtqlge nhditldvsr riyeaar


 







SEQ ID NO: 105 (Alcohol dehydrogenase (Escherichia coli MS 200-1))








1
mnnfnlhtpt rilfgkgaia glreqiphda rvlitygggs vkktgvldqv



lnalkgmdvl


61
efggiepnpa yetlmnavkl vreqkvtfll avgggsvldg tkfiaaaany



penidpwhil


121
qtggkeiksa ipmgcvltlp atgsesnaga visrkttgdk qafhsahvqp



vfavldpvyt


181
ytlpprqvan gvvdafvhtv eqyvtkpvda kiqdrfaegi lltliedgpk



alkepenydv


241
ranvmwaatq alngligagv pqdwathmlg heltamhgld haqtlaivlp



alwnekrdtk


301
rakllqyaer vwnitegsdd eridaaiaat rnffeqlgvp thlsdygldg



ssipallkkl


361
eehgmtqlge nhditldvsr riyeaar


 







SEQ ID NO: 106 (Alcohol dehydrogenase yqhD (Escherichia coli B7A))








1
mnnfnlhtpt rilfgkgaia glreqiphda rvlitygggs vkktgvldqv



ldalkgmdvl


61
efggiepnpa yetlmnavkl vreqkvtfll avgggsvldg tkfiaaaany



penidpwhil


121
qtggkeiksa ipmgcvltlp atgsesnaga visrkttgdk qafhsahvqp



vfavldpvyt


181
ytlpprqvan gvvdafvhtv eqyvtkpvda kiqdrfaegi lltliedgpk



alkepenydv


241
ranvmwaatq alngligagv pqdwathmlg heltamhgld haqtlaivlp



alwnekretk


301
rakllqyaer vwnitegsdd eridaaiaat rnffeqlgvp thlsdygldg



ssipallkkl


361
eehgmtqlge nhditldvsr riyeaar


 







SEQ ID NO: 107 (Alcohol dehydrogenase (Escherichia coli MS 196-1))








1
mnnfnlhtpt rilfgkgaia glreqiphda rvlitygggs vkktgvldqv



ldalkgmdvl


61
efggiepnpa yetlmnavkl vreqkvtfll avgggsvldg tkfiaaaany



penidpwhil


121
qtggkeiksa ipmgcvltlp atgsesnaga visrkttgdk qafhsahvqp



vfavldpvyt


181
ytlpprqvan gvvdafvhtv eqyvtkpvda kiqdrfaegi lltliedgpk



alkepenydv


241
ranvmwaatq alngligagv pqdwathmlg hkltamhgld haqtlaivlp



alwnekrdtk


301
rakllqyaer vwnitegsdd eridaaiaat rnffeqlgvp thlsdygldg



ssipallkkl


361
eehgmtqlge nhditldvsr riyeaar

## Claims

1. A recombinant microbial cell modified to exhibit increased biosynthesis of 1,4-butanediol from D-arabinose compared to a wild-type control, the cell comprising an engineered metabolic pathway comprising:
an enzyme that converts D-arabinose to D-arabinolactone comprising the amino acid sequence of SEQ ID NO:1;
an enzyme that converts D-arabinonic acid to 2-oxo-4(s),5-dihydroxy-pentanoic acid comprising the amino acid sequence of SEQ ID NO:6;
an enzyme that converts 2-oxo-4(s),5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid comprising the amino acid sequence of SEQ ID NO:11;
an enzymatic pathway that converts 2,5-dioxopentanoic acid to 1,4-butanediol.

2. The recombinant cell of claim 1 wherein the recombinant cell exhibits conversion of D-arabinose into D-arabinonolactone at a level at least 110% of a wild-type control cell.

3. The recombinant cell of claim 1 wherein the recombinant cell exhibits conversion of D-arabinonic acid to 2-oxo-4(s),5-dihydroxy-pentanoic acid at a level at least 110% of a wild-type control cell.

4. The recombinant cell of claim 1 wherein the recombinant cell exhibits conversion of 2-oxo-4(s), 5-dihydroxy-pentanoic acid to 2,5-dioxopentanoic acid at a level at least 110% of a wild-type control cell.

5. The recombinant cell of claim 1 wherein the enzymatic pathway that converts 2,5-dioxopentanoic acid to 1,4-butanediol comprises:
an enzyme that converts 2,5-dioxopentanoic acid to succinaldehyde; and
an enzyme that converts succinaldehyde to 1,4-butanediol.

6. The recombinant cell of claim 5 wherein the enzyme that converts 2,5-dioxopentanoic acid to succinaldehyde is 2-ketoacid decarboxylase or 2-oxoglutarate decarboxylase.

7. The recombinant cell of claim 5 wherein the enzyme that converts succinaldehyde to 1,4-butanediol is an alcohol dehydrogenase.

8. The recombinant cell of claim 1 wherein the enzymatic pathway that converts 2,5-dioxopentanoic acid to 1,4-butanediol comprises:
an enzyme that converts 2,5-dioxopentanoic acid to 2-keto-5-hydroxy-pentonate;
an enzyme that converts 2-keto-5-hydroxy-pentonate to 4-hydroxy-1-butyraldehyde; and
an enzyme that converts 4-hydroxy-1-butyraldehyde to 1,4-butanediol.

9. The recombinant cell of claim 8 wherein the enzyme that converts 2,5-dioxopentanoic acid to 2-keto-5-hydroxy-pentonate is an alcohol dehydrogenase.

10. The recombinant cell of claim 8 wherein the enzyme that converts 2-keto-5-hydroxy-pentonate to 4-hydroxy-1-butyraldehyde is a 2-ketoacid decarboxylase or a 2-oxoglutarate decarboxylase.

11. The recombinant cell of claim 8 wherein the enzyme that converts 4-hydroxy-1-butyraldehyde to 1,4-butanediol is an alcohol dehydrogenase.