# Patent

## Title
Fermentive production of four carbon alcohols

## Abstract

Methods for the fermentive production of four carbon alcohols are provided. Specifically, butanol, preferably 2-butanol is produced by the fermentive growth of a recombinant bacteria expressing a 2-butanol biosynthetic pathway. The recombinant microorganisms and methods of the invention can also be adapted to produce 2-butanone, an intermediate in the 2-butanol biosynthetic pathways disclosed herein. Specifically disclosed herein are the use of coenzyme B12-independent butanediol dehydratases that catalyzes the substrate to product conversion of 2,3-butanediol to 2-butanone in the process of producing 2-butanol and 2-butanone.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a Continuation in Part claiming priority to U.S. application Ser. No. 11/741,916 filed May 2, 2007.


FIELD OF THE INVENTION
The invention relates to the field of industrial microbiology and the production of alcohols. More specifically, 2-butanol is produced via industrial fermentation of a recombinant microorganism. The recombinant microorganisms and methods of the invention can also be adapted to produce 2-butanone, an intermediate in the 2-butanol biosynthetic pathways disclosed herein.
BACKGROUND OF THE INVENTION
Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase. 2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant and activator of oxidative reactions.
Methods for the chemical synthesis of 2-butanone are known, such as by dehydrogenation of 2-butanol, or in a process where liquid butane is catalytically oxidized giving 2-butanone and acetic acid (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 727-732). 2-Butanone may also be converted chemically to 2-butanol by hydrogenation (Breen et al., J. or Catalysis 236: 270-281 (2005)). Methods for the chemical synthesis of 2-butanol are known, such as n-butene hydration (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). These processes use starting materials derived from petrochemicals and are generally expensive, and are not environmentally friendly. The production of 2-butanone and 2-butanol from plant-derived raw materials would minimize greenhouse gas emissions and would represent an advance in the art.
Methods for producing 2-butanol by biotransformation of other organic chemicals are also known. For example, Stampfer et al. (WO 03/078615) describe the production of secondary alcohols, such as 2-butanol, by the reduction of ketones which is catalyzed by an alcohol dehydrogenase enzyme obtained from Rhodococcus ruber. Similarly, Kojima et al. (EP 0645453) describe a method for preparing secondary alcohols, such as 2-butanol, by reduction of ketones which is catalyzed by a secondary alcohol dehydrogenase enzyme obtained from Candida parapsilosis. Additionally, Kuehnle et al. (EP 1149918) describe a process that produces both 1-butanol and 2-butanol by the oxidation of hydrocarbons by various strains of Rhodococcus ruber. The process favored 1-butanol production with a selectivity of 93.8%.
The production of 2-butanol by certain strains of Lactobacilli is also known (Speranza et. al. J. Agric. Food Chem. (1997) 45:3476-3480). The 2-butanol is produced by the transformation of meso-2,3-butanediol. The production of 2-butanol from acetolactate and acetoin by these Lactobacilli strains was also demonstrated. However, there have been no reports of a recombinant microorganism designed to produce 2-butanol.
There is a need, therefore, for environmentally responsible, cost-effective processes for the production of 2-butanol and 2-butanone. The present invention addresses this need through the discovery of recombinant microbial production hosts expressing 2-butanol and 2-butanone biosynthetic pathways.
SUMMARY OF THE INVENTION
The invention provides a recombinant microorganism having an engineered 2-butanol biosynthetic pathway. Also provided is a recombinant microorganism having an engineered 2-butanone biosynthetic pathway, which is the same as the 2-butanol biosynthetic pathway with omission of the last step. The engineered microorganisms may be used for the commercial production of 2-butanol or 2-butanone. Accordingly, the invention provides a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

    
    
        i) pyruvate to alpha-acetolactate;
        ii) alpha-acetolactate to acetoin;
        iii) acetoin to 2,3-butanediol;
        iv) 2,3-butanediol to 2-butanone; and
        v) 2-butanone to 2-butanol;

wherein the at least one DNA molecule is heterologous to said microbial host cell and wherein said microbial host cell produces 2-butanol.

    
    


In another embodiment the invention provides a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

    
    
        i) pyruvate to alpha-acetolactate;
        ii) alpha-acetolactate to acetoin;
        iii) acetoin to 2,3-butanediol; and
        iv) 2,3-butanediol to 2-butanone;

wherein the at least one DNA molecule is heterologous to said microbial host cell and wherein said microbial host cell produces 2-butanone.

    
    


In another embodiment the invention provides a method for the production of 2-butanol comprising:

    
    
        1) providing a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:
        
            i) pyruvate to alpha-acetolactate;
            ii) alpha-acetolactate to acetoin,
            iii) acetoin to 2,3-butanediol;
            iv) 2,3-butanediol to 2-butanone; and
            v) 2-butanone to 2-butanol;
        
        
        wherein the at least one DNA molecule is heterologous to said microbial host cell; and
        2) contacting the host cell of (1) with a fermentable carbon substrate in a fermentation medium under conditions whereby 2-butanol is produced.
    
    


Similarly the invention provides A method for the production of 2-butanone comprising:

    
    
        1) providing a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:
        
            i) pyruvate to alpha-acetolactate;
            ii) alpha-acetolactate to acetoin;
            iii) acetoin to 2,3-butanediol; and
            iv) 2,3-butanediol to 2-butanone;
        
        
        wherein the at least one DNA molecule is heterologous to said microbial host cell; and
        2) contacting the host cell of (1) with a fermentable carbon substrate in a fermentation medium under conditions whereby 2-butanone is produced.
    
    


In another embodiment the invention provides a recombinant microbial host cell comprising at least one DNA molecule encoding a coenzyme B12-independent butanediol dehydratase that catalyzes the substrate to product conversion of 2,3-butanediol to 2-butanone wherein the at least one DNA molecule is heterologous to said microbial host cell and wherein said microbial host cell produces 2-butanone or 2-butanol.
In addition, the invention provides a method for the production of 2-butanone comprising:

    
    
        a) providing a recombinant microbial host cell comprising at least one DNA molecule encoding a B12-independent butanediol dehydratase that catalyzes the substrate to product conversion of 2,3-butanediol to 2-butanone and a source of 2,3-butanediol; wherein the at least one DNA molecule is heterologous to said microbial host cell; and
        b) growing the microbial host cell of (a) under conditions whereby the isolated nucleic acid molecule is expressed and the 2,3-butanediol is converted to 2-butanone; and
        c) optionally recovering the 2-butanone.
        Further, the invention provides a method for the production of 2-butanol comprising:
        a) providing a recombinant microbial host cell comprising at least one DNA molecule encoding a B12-independent butanediol dehydratase that catalyzes the substrate to product conversion of 2,3-butanediol to 2-butanone, butanol dehydrogenase activity and a source of 2,3-butanediol;
        wherein the at least one DNA molecule is heterologous to said microbial host cell; and
        b) growing the microbial host cell of (a) under conditions whereby the isolated nucleic acid molecule is expressed and the 2,3-butanediol is converted to 2-butanone, and the 2-butanone is converted to 2-butanol; and
        c) optionally recovering the 2-butanol.
    
    





BRIEF DESCRIPTION OF THE FIGURES, TABLES AND SEQUENCE DESCRIPTIONS
The invention can be more fully understood from the following detailed description, figure, and the accompanying sequence descriptions, which form a part of this application.
FIG. 1 shows four different pathways for biosynthesis of 2-butanone and 2-butanol.
FIG. 2 shows a phylogenetic tree of full length large subunits of diol/glycerol dehydratases, with >95% identical sequences removed (except that all experimentally verified function sequences were retained), and a key listing the identity of each sequence in the tree. Sequences with experimentally determined function as diol or glycerol dehydratases are highlighted in dark or light grey, respectively.
FIG. 3 shows a phylogenetic tree of full length medium subunits of diol/glycerol dehydratases, with >95% identical sequences removed, and a key listing the identity of each sequence in the tree. Sequences with experimentally determined function as diol or glycerol dehydratases are highlighted in dark or light grey, respectively.
FIG. 4 shows a phylogenetic tree of full length small subunits of diol/glycerol dehydratases, with >95% identical sequences removed, and a key listing the identity of each sequence in the tree. Sequences with experimentally determined function as diol or glycerol dehydratases are highlighted in dark or light grey, respectively.



Table 12 is a table of the alpha large subunit Profile HMM for the diol/glycerol dehydratase enzyme. Table 12 is submitted herewith electronically and is incorporated herein by reference.
Table 13 is a table of the beta medium subunit Profile HMM for the diol/glycerol dehydratase enzyme. Table 13 is submitted herewith electronically and is incorporated herein by reference.
Table 14 is a table of the gamma small subunit Profile HMM for the diol/glycerol dehydratase enzyme. Table 14 is submitted herewith electronically and is incorporated herein by reference.
The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.







TABLE 1







Summary of Nucleic Acid and Protein SEQ ID Numbers










SEQ ID




Nucleic
SEQ ID


Description
acid
Protein












budA, acetolactate decarboxylase from Klebsiella
1
2


pneumoniae ATCC 25955




alsD, acetolactate decarboxylase from Bacillus
80
81


subtilis




budA, acetolactate decarboxylase from Klebsiella
82
83


terrigena




budB, acetolactate synthase from Klebsiella
3
4


pneumoniae ATCC 25955




alsS, acetolactate synthase from Bacillus subtilis
76
77


budB, acetolactate synthase from Klebsiella
78
79


terrigena




budC butanediol dehydrogenase from Klebsiella
5
6


pneumoniae IAM1063




butanediol dehydrogenase from Bacillus cereus
84
85


butanediol dehydrogenase from Bacillus cereus
86
87


butB, butanediol dehydrogenase from Lactococcus
88
89


lactis




pddA, butanediol dehydratase alpha subunit from
7
8


Klebsiella oxytoca ATCC 8724




pddB, butanediol dehydratase beta subunit from
9
10


Klebsiella oxytoca ATCC 8724




pddC, butanediol dehydratase gamma subunit from
11
12


Klebsiella oxytoca ATCC 8724




pduC, B12 dependent diol dehydratase large
92
93


subunit from Salmonella typhimurium




pduD, B12 dependent diol dehydratase medium
94
95


subunit from Salmonella typhimurium




pduE, B12 dependent diol dehydratase small
96
97


subunit from Salmonella typhimurium




pduC, B12 dependent diol dehydratase large
98
99


subunit from Lactobacillus collinoides




pduD, B12 dependent diol dehydratase medium
100
101


subunit from Lactobacillus collinoides




pduE, B12 dependent diol dehydratase small
102
103


subunit from Lactobacillus collinoides




pddC, adenosylcobalamin-dependent diol
104
105


dehydratase alpha subunit from Klebsiella




pneumoniae




pddD, adenosylcobalamin-dependent diol
106
107


dehydratase beta subunit from Klebsiella




pneumoniae




pddD, adenosylcobalamin-dependent diol
108
109


dehydratase gamma subunit from Klebsiella




pneumoniae




ddrA, diol dehydratase reactivating factor large
110
111


subunit from Klebsiella oxytoca




ddrB, diol dehydratase reactivating factor small
112
113


subunit from Klebsiella oxytoca




pduG, diol dehydratase reactivating factor large
114
115


subunit from Salmonella typhimurium




pduH, diol dehydratase reactivating factor small
116
117


subunit from Salmonella typhimurium




pduG, diol dehydratase reactivating factor large
118
119


subunit from Lactobacillus collinoides




pduH, diol dehydratase reactivating factor small
120
121


subunit from Lactobacillus collinoides




sadH, butanol dehydrogenase from Rhodococcus
13
14


ruber 219




adhA, butanol dehydrogenase from Pyrococcus
90
91


furiosus




chnA, cyclohexanol dehydrogenase from
71
72


Acinteobacter sp.




yqhD, butanol dehydrogenase from Escherichia coli
74
75


amine: pyruvate transaminase from Vibrio fluvialis
144
122


(an acetoin aminase)
codon




opt.



amino alcohol kinase from Erwinia carotovora
123
124


subsp. atroseptica




amino alcohol O-phosphate lyase from Erwinia
125
126


carotovora subsp. atroseptica




budC, acetoin reductase (butanediol
133
134


dehydrogenase) from Klebsiella terrigena (now




Raoultella terrigena)




glycerol dehydratase alpha subunit from Klebsiella
145
146


pneumoniae




glycerol dehydratase beta subunit from Klebsiella
147
148


pneumoniae




glycerol dehydratase gamma subunit from
149
150


Klebsiella pneumoniae




glycerol dehydratase reactivase large subunit from
151
152


Klebsiella pneumoniae




glycerol dehydratase reactivase small subunit from
153
154


Klebsiella pneumoniae




rdhtA B-12 independent butanediol dehydratase
277
276


from Roseburia inulinivorans




rdhtB B-12 independent butanediol dehydratase
279
278


reactivase from Roseburia inulinivorans




B12-independent glycerol dehydratase from
281
280


Clostridium butyricum




B-12 independent butanediol dehydratase
283
282


reactivase from Clostridium butyricum




sadB butanol dehydrogenase from Achromobacter
290
289


xylosoxidans









SEQ ID NOs:15-65 are the nucleotide sequences of oligonucleotide PCR, cloning, screening, and sequencing primers used in the Examples.
SEQ ID NO:66 is nucleotide sequence of the deleted region of the yqhD gene in E. coli strain MG1655 ΔyqhCD, described in Example 11.
SEQ ID NO:67 is the nucleotide sequence of a variant of the glucose isomerase promoter 1.6GI.
SEQ ID NO:68 is the nucleotide sequence of the 1.5GI promoter.
SEQ ID NO:69 is the nucleotide sequence of the diol dehydratase operon from Klebsiella oxytoca. 
SEQ ID NO:70 is the nucleotide sequence of the diol dehydratase reactivating factor operon from Klebsiella oxytoca. 
SEQ ID NO:73 is the nucleotide sequence of pDCQ2, which is described in Example 9.
SEQ ID NOs:127-132 are the nucleotide sequences of additional oligonucleotide PCR and cloning primers used in the Examples.
SEQ ID NO: 55 is a codon optimized coding region for the amino alcohol kinase of Erwinia carotovora subsp. atroseptica. 
SEQ ID NO:156 is a codon optimized coding region for the amino alcohol O-phosphate lyase of Erwinia carotovora subsp. atroseptica. 
SEQ ID NOs:157-163 are the nucleotide sequences of additional oligonucleotide PCR and cloning primers used in the Examples.
SEQ ID NO:275 is the nucleotide sequence of an operon from Erwinia carotovora subsp. atroseptica.







TABLE 2







Additional glycerol and diol dehydratase large,


medium and small subunits











pro-




tein



bsub-
SEQ


aDescription
unit
ID





Corresponding subunits from same organismc




Glycerol dehydratase alpha subunit from Clostridium
L
135


pasteurianum




Glycerol dehydratase beta subunit from Clostridium
M
136


pasteurianum




Glycerol dehydratase gamma subunit from Clostridium
S
137


pasteurianum




Glycerol dehydratase alpha subunit from Escherichia
L
138


blattae




Glycerol dehydratase beta subunit from Escherichia
M
139


blattae




Glycerol dehydratase gamma subunit from Escherichia
S
140


blattae




Glycerol dehydratase alpha subunit from Citrobacter
L
141


freundii




Glycerol dehydratase beta subunit from Citrobacter
M
142


freundii




Glycerol dehydratase gamma subunit from Citrobacter
S
143


freundii




Diol dehydratase alpha subunit from Lactobacillus brevis
L
164


Diol dehydratase beta subunit from Lactobacillus brevis
M
165


Diol dehydratase gamma subunit from Lactobacillus brevis
S
166


Diol dehydratase alpha subunit from Salmonella enterica
L
167


subsp. enterica serovar Choleraesuis str. SC-B67




Diol dehydratase beta subunit from Salmonella enterica
M
168


subsp. enterica serovar Choleraesuis str. SC-B67




Diol dehydratase gamma subunit from Salmonella enterica
S
169


subsp. enterica serovar Choleraesuis str. SC-B67




Propanediol dehydratase, large subunit from Escherichia
L
170


coli E24377A




Diol/Glyderol Dehydratase medium subunit from
M
171


Escherichia coli E24377A




Propanediol dehydratase, small subunit from Escherichia
S
172


coli E24377A




diol dehydratase large subunit from Shigella sonnei Ss046
L
173


diol dehydratase medium subunit from Shigella sonnei
M
174


Ss046




diol dehydratase small subunit from Shigella sonnei Ss046
S
175


Propanediol dehydratase large subunit from Yersinia
L
176


bercovieri ATCC 43970




hypothetical protein YberA_01000484 from Yersinia
M
177


bercovieri ATCC 43970




Propanediol dehydratase small subunit from Yersinia
S
178


bercovieri ATCC 43970




Propanediol dehydratase large subunit from Yersinia
L
179


mollaretii ATCC 43969




hypothetical protein YmolA_01001292 from Yersinia
M
180


mollaretii ATCC 43969




Propanediol dehydratase small subunit from Yersinia
S
181


mollaretii ATCC 43969




Diol dehydratase large subunit from Yersinia enterocolitica
L
182


subsp. enterocolitica 8081




Diol dehydratase medium subunit from Yersinia
M
183


enterocolitica subsp. enterocolitica 8081




diol dehydratase small subunit from Yersinia enterocolitica
S
184


subsp. enterocolitica 8081




Propanediol dehydratase large subunit from Yersinia
L
185


intermedia ATCC 29909




diol/glycerol dehydratase medium subunit from Yersinia
M
186


intermedia ATCC 29909




Propanediol dehydratase small subunit from Yersinia
S
187


intermedia ATCC 29909




glycerol dehydratase large subunit from Listeria welshimeri
L
188


serovar 6b str. SLCC5334




propanediol utilization dehydratase medium subunit from
M
189


Listeria welshimeri serovar 6b str. SLCC5334




propanediol utilization dehydratase small subunit from
S
190


Listeria welshimeri serovar 6b str. SLCC5334




hypothetical protein lin1117 from Listeria innocua
L
191


Clip11262




hypothetical protein lin1118 from Listeria innocua
M
192


Clip11262




hypothetical protein lin1119 from Listeria innocua
S
193


Clip11262




hypothetical protein lmo1153 from Listeria monocytogenes
L
194


EGD-e




hypothetical protein lmo1154 from Listeria monocytogenes
M
195


EGD-e




hypothetical protein lmo1155 from Listeria monocytogenes
S
196


EGD-e




glycerol dehydratase large subunit from Salmonella
L
197


enterica subsp. enterica serovar Typhi str. CT18




diol dehydratase medium subunit from Salmonella enterica
M
198


subsp. enterica serovar Typhi str. CT18




diol dehydratase small subunit from Salmonella enterica
S
199


subsp. enterica serovar Typhi str. CT18




putative glycerol dehydratase large subunit [from
L
200


Escherichia coli




putative diol dehydratase medium subunit from
M
201


Escherichia coli




putative diol dehydratase small subunit from Escherichia
S
202


coli




glycerol dehydratase large subunit from Listeria
L
203


monocytogenes str. 4b F2365




propanediol utilization: dehydratase, medium subunit from
M
204


Listeria monocytogenes str. 4b F2365




propanediol utilization: dehydratase, small subunit from
S
205


Listeria monocytogenes str. 4b F2365




Glycerol dehydratase large subunit pduC, putative from
L
206


Streptococcus sanguinis SK36




Propanediol utilization: dehydratase medium subunit,
M
207


putative from Streptococcus sanguinis SK36




B12-dependent diol dehydratase small subunit, putative
S
208


from Streptococcus sanguinis SK36




DhaB from Escherichia blattae
L
209


DhaC from Escherichia blattae
M
210


DhaE from Escherichia blattae
S
211


coenzyme B12-dependent glycerol dehydrogenase large
L
212


subunit from Clostridium perfringens str. 13




coenzyme B12-dependent glycerol dehydrogenase
M
213


medium subunit from Clostridium perfringens str. 13




coenzyme B12-dependent glycerol dehydrogenase small
S
214


subunit from Clostridium perfringens str. 13




Propanediol dehydratase large subunit from Yersinia
L
215


frederiksenii ATCC 33641




hypothetical protein YfreA_01000478 from Yersinia
M
216


frederiksenii ATCC 33641]




Propanediol dehydratase, small subunit from Yersinia
S
217


frederiksenii ATCC 33641




Glycerol dehydratase from Thermoanaerobacter
L
218


ethanolicus X514




dehydratase medium subunit from Thermoanaerobacter
M
219


ethanolicus X514




dehydratase small subunit from Thermoanaerobacter
S
220


ethanolicus X514




glycerol dehydratase large subunit GldC from Lactobacillus
L
221


hilgardii




_glycerol dehydratase medium subunit GldD from
M
222


Lactobacillus hilgardii




glycerol dehydratase small subunit GldE from
S
223


Lactobacillus hilgardii




Glycerol dehydratase from Lactobacillus reuteri JCM 1112
L
224


similar to diol dehydratase gamma subunit from
M
225


Lactobacillus reuteri JCM 1112




Propanediol utilization: dehydratase small subunit from
S
226


Lactobacillus reuteri JCM 1112




glycerol dehydratase large subunit GldC from Lactobacillus
L
227


diolivorans




glycerol dehydratase medium subunit GldD from
M
228


Lactobacillus diolivorans




glycerol dehydratase small subunit GldE from
S
229


Lactobacillus diolivorans




propanediol dehydratase large subunit from Lactobacillus
L
230


reuteri




propanediol dehydratase medium subunit from
M
231


Lactobacillus reuteri




propanediol dehydratase small subunit from Lactobacillus
S
232


reuteri




glycerol dehydratase large subunit from Mesorhizobium loti
L +
233


MAFF303099
M



glycerol dehydratase small subunit from Mesorhizobium
S
234


loti MAFF303099




Glycerol dehydratase from Mycobacterium vanbaalenii
L +
235


PYR-1
M



propanediol utilization: dehydratase small subunit from
S
236


Mycobacterium vanbaalenii PYR-1




Glycerol dehydratase from Mycobacterium sp. MCS
L +
237



M



dehydratase small subunit Mycobacterium sp. MCS
S
238


Dehydratase large subunit: Dehydratase medium subunit
L +
239


from Mycobacterium flavescens PYR-GCK
M



propanediol utilization: dehydratase, small subunit from
S
240


Mycobacterium flavescens PYR-GCK




Glycerol dehydratase from Mycobacterium sp. JLS
L +
241



M



dehydratase small subunit from Mycobacterium sp. JLS
S
242


glycerol dehydratase large subunit from Mycobacterium
L
243


smegmatis str. MC2 155




dehydratase medium subunit from Mycobacterium
M
244


smegmatis str. MC2 155




diol dehydrase gamma subunit from Mycobacterium
S
245


smegmatis str. MC2 155




Additional subunits




glycerol dehydratase large subunit from Mycobacterium
L +
246


smegmatis str. MC2 155
M



glycerol dehydratase large subunit from Mycobacterium
L +
247


smegmatis str. MC2 155
M



coenzyme B12-dependent glycerol dehydrogenase small
S
248


subunit from Mycobacterium smegmatis str. MC2 155




coenzyme B12-dependent glycerol dehydrogenase small
S
249


subunit from Mycobacterium smegmatis str. MC2 155




diol dehydratase medium subunit from Salmonella enterica
M
250


subsp. enterica serovar Paratyphi A str. ATCC 9150




diol dehydratase small subunit from Salmonella enterica
S
251


subsp. enterica serovar Paratyphi A str. ATCC 9150




glycerol dehydratase, beta subunit from Clostridium
M
252


perfringens SM101




glycerol dehydrase, gamma subunit from Clostridium
S
253


perfringens SM101




PduC from Salmonella enterica subsp. enterica serovar
L
254


Typhimurium




glycerol dehydratase large subunit from Listeria
L
255


monocytogenes str. 4b H7858




DhaB from Escherichia blattae
L
256


DhaB from uncultured bacterium
L
257


DhaB from uncultured bacterium
L
258


glycerol dehydratase large subunit GldC from Lactobacillus
L
259


collinoides




PduD from uncultured bacterium
M
260


PduD from uncultured bacterium
M
261


DhaC from uncultured bacterium
M
262


DhaC from uncultured bacterium
M
263


DhaC from uncultured bacterium
M
264


coenzyme B12-dependent glycerol dehydratase, medium
M
265


subunit from Clostridium perfringens ATCC 13124




unknown
M
266


glycerol dehydratase beta subunit from Escherichia blattae
M
267


PduE from uncultured bacterium
S
268


PduE from uncultured bacterium
S
269


dehydratase, small subunit from Listeria monocytogenes
S
270


str. 1/2a F6854




DhaE from uncultured bacterium
S
271


DhaE from uncultured bacterium
S
272


DhaE from uncultured bacterium
S
273


dehydratase small subunit from Listeria monocytogenes
S
274


FSL N1-017





aDescription: from the Genbank annotation of the sequence and may not be correct including the glycerol or diol designation, or may not include subunit information.


bSubunit: identified by sequence homology to the large, medium, or small subunit.of the Klebsiella oxytoca enzyme.


cSubunts are listed together that are from the same organism and have annotations as the same enzyme, or have Genbank numbers close together indicating proximity in the genome.






SEQ ID NO:284 is the sequence of a synthetic fragment containing coding regions for Roseburia inulinivorans B12-independent diol dehydratase and reactivase.
SEQ ID NOs:285-296, 302-307, 311-316, 319, 320, 322, 323 are the nucleotide sequences of additional oligonucleotide PCR and cloning primers used in the Examples.
SEQ ID NO:297 is the dual terminator.
SEQ ID NO:298 is the ADH1 terminator.
SEQ ID NO:299 is the CYC1 terminator.
SEQ ID NO:300 is the FBA promoter.
SEQ ID NO:301 is the GPM promoter.
SEQ ID NO:308 is the CUP1 promoter.
SEQ ID NO:309 is the alsS coding region from Bacillus subtilis. 
SEQ ID NO:310 is the ILV3 coding region from S. cerevisiae. 
SEQ ID NO:317 is the GPD promoter.
SEQ ID NO:318 is the coding region of the ILV5 gene of S. cerevisiae. 
SEQ ID NO:321 is the kivD coding region from Lactococcus lactis. 
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods for the production of 2-butanol using recombinant microorganisms. The present invention meets a number of commercial and industrial needs. Butanol is an important industrial commodity chemical with a variety of applications, where its potential as a fuel or fuel additive is particularly significant. Although only a four-carbon alcohol, butanol has an energy content similar to that of gasoline and can be blended with any fossil fuel. Butanol is favored as a fuel or fuel additive as it yields only CO2 and little or no SOx or NOx when burned in the standard internal combustion engine. Additionally butanol is less corrosive than ethanol, the most preferred fuel additive to date.
In addition to its utility as a biofuel or fuel additive, butanol has the potential of impacting hydrogen distribution problems in the emerging fuel cell industry. Fuel cells today are plagued by safety concerns associated with hydrogen transport and distribution. Butanol can be easily reformed for its hydrogen content and can be distributed through existing gas stations in the purity required for either fuel cells or combustion engines in vehicles.
Finally the present invention produces 2-butanol from plant derived carbon sources, avoiding the negative environmental impact associated with standard petrochemical processes for butanol production.
The present invention also provides recombinant microorganisms and methods for producing 2-butanone, an intermediate in the 2-butanol biosynthetic pathways disclosed herein. 2-Butanone, also known as methyl ethyl ketone (MEK), is useful as a solvent in paints and other coatings. It is also used in the synthetic rubber industry and in the production of paraffin wax.
The following definitions and abbreviations are to be used for the interpretation of the claims and the specification.
The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.
The term “2-butanol biosynthetic pathway” refers to the enzyme pathways to produce 2-butanol from pyruvate.
The term “2-butanone biosynthetic pathway” refers to the enzyme pathways to produce 2-butanone from pyruvate.
The term “acetolactate synthase”, also known as “acetohydroxy acid synthase”, refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of two molecules of pyruvic acid to one molecule of alpha-acetolactate. Acetolactate synthase, known as EC 2.2.1.6 [formerly 4.1.3.18] (Enzyme Nomenclature 1992, Academic Press, San Diego) may be dependent on the cofactor thiamin pyrophosphate for its activity. Suitable acetolactate synthase enzymes are available from a number of sources, for example, Bacillus subtilis [GenBank Nos: AAA22222 NCBI (National Center for Biotechnology Information) amino acid sequence (SEQ ID NO:77), L04470 NCBI nucleotide sequence (SEQ ID NO:76)], Klebsiella terrigena [GenBank Nos: AAA25055 (SEQ ID NO:79), L04507 (SEQ ID NO:78)], and Klebsiella pneumoniae [GenBank Nos: AAA25079 (SEQ ID NO:4), M73842 (SEQ ID NO:3)].
The term “acetolactate decarboxylase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Acetolactate decarboxylases are known as EC 4.1.1.5 and are available, for example, from Bacillus subtilis [GenBank Nos: AAA22223 (SEQ ID NO:81), L04470 (SEQ ID NO:80)], Klebsiella terrigena [GenBank Nos: AAA25054 (SEQ ID NO:83), L04507 (SEQ ID NO:82)] and Klebsiella pneumoniae [GenBank Nos: AAU43774 (SEQ ID NO:2), AY722056 (SEQ ID NO:1)].
The term “acetoin aminase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 3-amino-2-butanol. Acetoin aminase may utilize the cofactor pyridoxal 5′-phosphate or NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate). The resulting product may have (R) or (S) stereochemistry at the 3-position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate as the amino donor. The NADH- and NADPH-dependent enzymes may use ammonia as a second substrate. A suitable example of an NADH-dependent acetoin aminase, also known as amino alcohol dehydrogenase, is described by Ito et al. (U.S. Pat. No. 6,432,688). An example of a pyridoxal-dependent acetoin aminase is the amine:pyruvate aminotransferase (also called amine:pyruvate transaminase) described by Shin and Kim (J. Org. Chem. 67:2848-2853 (2002)).
The term “butanol dehydrogenase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the interconversion of 2-butanone and 2-butanol. Butanol dehydrogenases are a subset of a broad family of alcohol dehydrogenases. Butanol dehydrogenase may be NAD- or NADP-dependent. The NAD-dependent enzymes are known as EC 1.1.1.1 and are available, for example, from Rhodococcus ruber [GenBank Nos: CAD36475 (SEQ ID NO:14), AJ491307 (SEQ ID NO:13)]. The NADP-dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus [GenBank Nos: AAC25556 (SEQ ID NO:91), AF013169 (SEQ ID NO:90)]. Additionally, a butanol dehydrogenase is available from Escherichia coli [GenBank Nos: NP—417-484 (SEQ ID NO:75), NC-000913 (SEQ ID NO:74)] and a cyclohexanol dehydrogenase is available from Acinetobacter sp. [GenBank Nos: AAG10026 (SEQ ID NO:72), AF282240 (SEQ ID NO:71)].
The term “acetoin kinase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to phosphoacetoin. Acetoin kinase may utilize ATP (adenosine triphosphate) or phosphoenolpyruvate as the phosphate donor in the reaction. Although there are no reports of enzymes catalyzing this reaction on acetoin, there are enzymes that catalyze the analogous reaction on the similar substrate dihydroxyacetone, for example, enzymes known as EC 2.7.1.29 (Garcia-Alles et al. (2004) Biochemistry 43:13037-13046).
The term “acetoin phosphate aminase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of phosphoacetoin to 3-amino-2-butanol O-phosphate. Acetoin phosphate aminase may use the cofactor pyridoxal 5′-phosphate, NADH or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate. The NADH- and NADPH-dependent enzymes may use ammonia as a second substrate. Although there are no reports of enzymes catalyzing this reaction on phosphoacetoin, there is a pyridoxal phosphate-dependent enzyme that is proposed to carry out the analogous reaction on the similar substrate serinol phosphate (Yasuta et al. (2001) Appl. Environ. Microbiol. 67:4999-5009).
The term “aminobutanol phosphate phospho-lyase”, also called “amino alcohol O-phosphate lyase”, refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 3-amino-2-butanol O-phosphate to 2-butanone. Aminobutanol phosphate phospho-lyase may utilize the cofactor pyridoxal 5′-phosphate. There are no previous reports of enzymes catalyzing this reaction on aminobutanol phosphate, though there are reports of enzymes that catalyze the analogous reaction on the similar substrate 1-amino-2-propanol phosphate (Jones et al. (1973) Biochem J. 134:167-182). The present invention describes a newly identified aminobutanol phosphate phospho-lyase (SEQ ID NO: 126) from the organism Erwinia carotovora, with the activity demonstrated in Example 15 herein.
The term “aminobutanol kinase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 3-amino-2-butanol to 3-amino-2-butanol O-phosphate. Aminobutanol kinase may utilize ATP as the phosphate donor. Although there are no reports of enzymes catalyzing this reaction on 3-amino-2-butanol, there are reports of enzymes that catalyze the analogous reaction on the similar substrates ethanolamine and 1-amino-2-propanol (Jones et al., supra). The present invention describes, in Example 14, an amino alcohol kinase of Erwinia carotovora subsp. atroseptica (SEQ ID NO:124). The term “butanediol dehydrogenase” also known as “acetoin reductase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanediol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of (R)— or (S)-stereochemistry in the alcohol product. (S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (GenBank Nos: BBA13085 (SEQ ID NO:6), D86412 (SEQ ID NO:5)). (R)-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus [GenBank Nos. NP—830481 (SEQ ID NO:85), NC—004722 (SEQ ID NO:84); AAP07682 (SEQ ID NO:87), AE017000 (SEQ ID NO:86)], and Lactococcus lactis [GenBank Nos. AAK04995 (SEQ ID NO:89), AE006323 (SEQ ID NO:88)].
The term “butanediol dehydratase”, also known as “diol dehydratase” or “propanediol dehydratase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone. Butanediol dehydratase may utilize the cofactor adenosyl cobalamin (also known as coenzyme B12, or vitamin B12; although vitamin B12 may refer also to other forms of cobalamin that are not coenzyme B12). Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca [GenBank Nos: BAA08099 (alpha subunit) (SEQ ID NO:8), D45071 (SEQ ID NO:7); BAA08100 (beta subunit) (SEQ ID NO:10), D45071 (SEQ ID NO:9); and BBA08101 (gamma subunit) (SEQ ID NO:12), D45071 (SEQ ID NO:11) (Note all three subunits are required for activity)], and Klebsiella pneumoniae [GenBank Nos: AAC98384 (alpha subunit) (SEQ ID NO:105), AF102064 (SEQ ID NO:104); GenBank Nos: AAC98385 (beta subunit) (SEQ ID NO:107), AF102064 (SEQ ID NO:106), GenBank Nos: AAC98386 (gamma subunit) SEQ ID NO:109), AF102064 (SEQ ID NO:108)]. Other suitable diol dehydratases include, but are not limited to, B12-dependent diol dehydratases available from Salmonella typhimurium [GenBank Nos: AAB84102 (large subunit) (SEQ ID NO:93), AF026270 (SEQ ID NO:92); GenBank Nos: AAB84103 (medium subunit) (SEQ ID NO:95), AF026270 (SEQ ID NO:94); GenBank Nos: AAB84104 (small subunit) (SEQ ID NO:97), AF026270 (SEQ ID NO:96)]; and Lactobacillus collinoides [GenBank Nos: CAC82541 (large subunit) (SEQ ID NO:99), AJ297723 (SEQ ID NO:98); GenBank Nos: CAC82542 (medium subunit) (SEQ ID NO:101); AJ297723 (SEQ ID NO:100); GenBank Nos: CAD01091 (small subunit) (SEQ ID NO:103), AJ297723 (SEQ ID NO:102)]; and enzymes from Lactobacillus brevis (particularly strains CNRZ 734 and CNRZ 735, Speranza et al., supra), and nucleotide sequences that encode the corresponding enzymes. Methods of diol dehydratase gene isolation are well known in the art (e.g., U.S. Pat. No. 5,686,276). Additional diol dehydratases are listed in Table 2.
The term “glycerol dehydratase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde. Adenosyl cobalamin-dependent glycerol dehydratases are known as EC 4.2.1.30. The glycerol dehydratases of EC 4.2.1.30 are similar to the diol dehydratases in sequence and in having three subunits. The glycerol dehydratases can also be used to convert 2,3-butanediol to 2-butanone. Some examples of glycerol dehydratases of EC 4.2.1.30 include those from Klebsiella pneumoniae (alpha subunit, SEQ ID NO:145, coding region and SEQ ID NO:146, protein; beta subunit, SEQ ID NO:147, coding region and SEQ ID NO:148, protein; and gamma subunit SEQ ID NO:149, coding region and SEQ ID NO:150, protein); from Clostridium pasteurianum [GenBank Nos: 3360389 (alpha subunit, SEQ ID NO:135), 3360390 (beta subunit, SEQ ID NO:136), and 3360391 (gamma subunit, SEQ ID NO:137)]; from Escherichia blattae [GenBank Nos: 60099613 (alpha subunit, SEQ ID NO:138), 57340191 (beta subunit, SEQ ID NO:139), and 57340192 (gamma subunit, SEQ ID NO:140)]; and from Citrobacter freundii [GenBank Nos: 1169287 (alpha subunit, SEQ ID NO:141), 1229154 (beta subunit, SEQ ID NO:142), and 1229155 (gamma subunit, SEQ ID NO:143)]. Note that all three subunits are required for activity. Additional glycerol dehydratases are listed in Table 2.
Diol and glycerol dehydratases may undergo suicide inactivation during catalysis. A reactivating factor protein, also referred to herein as “reactivase”, can be used to reactivate the inactive enzymes (Mori et al., J. Biol. Chem. 272:32034 (1997)). Preferably, the reactivating factor is obtained from the same source as the diol or glycerol dehydratase used. For example, suitable diol dehydratase reactivating factors are available from Klebsiella oxytoca [GenBank Nos: MC15871 (large subunit) (SEQ ID NO:111), AF017781 (SEQ ID NO:110); GenBank Nos: AAC15872 (small subunit) (SEQ ID NO:113), AF017781 (SEQ ID NO:112)]; Salmonella typhimurium [GenBank Nos: AAB84105 (large subunit) (SEQ ID NO:115), AF026270 (SEQ ID NO:114), GenBank Nos: AAD39008 (small subunit) (SEQ ID NO:117), AF026270 (SEQ ID NO:116)]; and Lactobacillus collinoides [GenBank Nos: CAD01092 (large subunit) (SEQ ID NO:119), AJ297723 (SEQ ID NO:118); GenBank Nos: CAD01093 (small subunit) (SEQ ID NO:121), AJ297723 (SEQ ID NO:120)]. Both the large and small subunits are required for activity. For example, suitable glycerol dehydratase reactivating factors are available from Klebsiella pneumoniae (large subunit, SEQ ID NO:151, coding region and SEQ ID NO:152, protein; and small subunit, SEQ ID NO:153, coding region and SEQ ID NO:154, protein).
Diol or glycerol dehydratases may also function independently of coenzyme B12. Such dehydratases are often known as B12-independent diol or glycerol dehydratases. These are described further in the Pathway 3 description below. The term “a facultative anaerobe” refers to a microorganism that can grow in both aerobic and anaerobic environments.
The term “carbon substrate” or “fermentable carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.
The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
As used herein, an “isolated nucleic acid fragment” or “isolated nucleic acid molecule” or “genetic construct” will be used interchangeably and will mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
A nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2 ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular fungal proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.
The terms “homology” and “homologous” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.
Moreover, the skilled artisan recognizes that homologous nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under moderately stringent conditions (e.g., 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein.
“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).
Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlignTM v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.
It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 75% to 100% may be useful in describing the present invention, such as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.
As used herein the term “coding sequence” or “CDS” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid” and “vector” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation vector” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell.
As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.
The term “fermentation product medium” refers to a medium in which fermentation has occurred such that product is present in the medium.
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).
The 2-Butanol and 2-Butanone Biosynthetic Pathways
Carbohydrate utilizing microorganisms employ the Embden-Meyerhof-Parnas (EMP) pathway, the Entner-Doudoroff pathway and the pentose phosphate cycle as the central, metabolic routes to provide energy and cellular precursors for growth and maintenance. These pathways have in common the intermediate glyceraldehyde-3-phosphate, and, ultimately, pyruvate is formed directly or in combination with the EMP pathway. The combined reactions of sugar conversion to pyruvate produce energy (e.g. adenosine-5′-triphosphate, ATP) and reducing equivalents (e.g. reduced nicotinamide adenine dinucleotide, NADH, and reduced nicotinamide adenine dinucleotide phosphate, NADPH). NADH and NADPH must be recycled to their oxidized forms (NAD+ and NADP+, respectively). In the presence of inorganic electron acceptors (e.g. O2, NO3− and SO42−), the reducing equivalents may be used to augment the energy pool; alternatively, a reduced carbon by-product may be formed.
The invention enables the production of 2-butanone or 2-butanol from carbohydrate sources with recombinant microorganisms by providing a complete biosynthetic pathway from pyruvate to 2-butanone or 2-butanol. Three additional pathways are described. Although 2-butanol is not known to be the major product of any bacterial fermentation, there are a number of possible pathways for the production of 2-butanol via known biochemical reaction types. These pathways are shown in FIG. 1. The letters and roman numerals cited below correspond to the letters and roman numerals in FIG. 1, which are used to depict the conversion steps and products, respectively. As described below, 2-butanone is an intermediate in all of these 2-butanol biosynthetic pathways.
All of the pathways begin with the initial reaction of two pyruvate molecules to yield alpha-acetolactate (I), shown as the substrate to product conversion (a) in FIG. 1. From alpha-acetolactate, there are 4 possible pathways to 2-butanone (V), referred to herein as 2-butanone biosynthetic pathways:
Pathway

    
    
        1) I→II→III→IV→V (substrate to product conversions b, c, d, e);
        2) I→II→VII→IV→V (substrate to product conversions b, g, h, e)
        3) I→II→VIII→V (substrate to product conversions b, i, j): This is the pathway of the present invention.
        4) I→IX→X→V (substrate to product conversions k, l, m)

The 2-butanol biosynthetic pathways conclude with the conversion of 2-butanone (V) to 2-butanol (VI). A detailed discussion of the substrate to product conversions in each pathway is given below.

Pathway 1:

    
    


(a) Pyruvate to Alpha-Acetolactate
The initial step in pathway 1 is the conversion of two molecules of pyruvate to one molecule of alpha-acetolactate (compound I in FIG. 1) and one molecule of carbon dioxide catalyzed by a thiamin pyrophosphate-dependent enzyme. Enzymes catalyzing this substrate to product conversion (generally called either acetolactate synthase or acetohydroxy acid synthase; EC 2.2.1.6 [switched from 4.1.3.18 in 2002]) are well-known, and they participate in the biosynthetic pathway for the proteinogenic amino acids leucine and valine, as well as in the pathway for fermentative production of 2,3-butanediol and acetoin of a number of organisms.
The skilled person will appreciate that polypeptides having acetolactate synthase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. Some example of suitable acetolactate synthase enzymes are available from a number of sources, for example, Bacillus subtilis [GenBank Nos: AAA22222 NCBI (National Center for Biotechnology Information) amino acid sequence (SEQ ID NO:77), L04470 NCBI nucleotide sequence (SEQ ID NO:76)], Klebsiella terrigena [GenBank Nos: AAA25055 (SEQ ID NO:79), L04507 (SEQ ID NO:78)], and Klebsiella pneumoniae [GenBank Nos: AAA25079 (SEQ ID NO:4), M73842 (SEQ ID NO:3)]. Preferred acetolactate synthase enzymes are those that have at least 80%-85% identity to SEQ ID NO's 4, 77, and 79, where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
(b) Alpha-Acetolactate to Acetoin
Alpha-acetolactate (I) is converted to acetoin (II) by the action of an enzyme such as acetolactate decarboxylase (EC 4.1.1.5). Like acetolactate synthase, this enzyme is thiamin pyrophosphate-dependent and is also involved in the production of 2,3-butanediol and acetoin by a number of organisms. The enzymes from different sources vary quite widely in size (25-50 kilodaltons), oligomerization (dimer-hexamer), localization (intracellular of extracellular), and allosteric regulation (for example, activation by branched-chain amino acids). For the purpose of the present invention, an intracellular location is preferable to extracellular, but other variations are generally acceptable.
The skilled person will appreciate that polypeptides having acetolactate decarboxylase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. Some example of suitable acetolactate decarboxylase enzymes are available from a number of sources, for example, Bacillus subtilis [GenBank Nos: AAA22223 (SEQ ID NO:81), L04470 (SEQ ID NO:80)], Klebsiella terrigena [GenBank Nos: AAA25054 (SEQ ID NO:83), L04507 (SEQ ID NO:82)] and Klebsiella pneumoniae [GenBank Nos: AAU43774 (SEQ ID NO:2), AY722056 (SEQ ID NO:1)].
Preferred acetolactate decarboxylase enzymes are those that have at least 80%-85% identity to SEQ ID NO's 2, 81 and 83, where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
(c) Acetoin to 3-Amino-2-Butanol
There are two known types of biochemical reactions that could effect the substrate to product conversion of acetoin (II) to 3-amino-2-butanol (III), specifically, pyridoxal phosphate-dependent transamination utilizing an accessory amino donor and direct reductive amination with ammonia. In the latter case, the reducing equivalents are supplied in the form of a reduced nicotinamide cofactor (either NADH or NADPH). An example of an NADH-dependent enzyme catalyzing this reaction with acetoin as a substrate is reported by Ito et al. (U.S. Pat. No. 6,432,688). Any stereospecificity of this enzyme has not been assessed. An example of a pyridoxal phosphate-dependent transaminase that catalyzes the conversion of acetoin to 3-amino-2-butanol has been reported by Shin and Kim (supra). This enzyme was shown in Example 13 herein to convert both the (R) isomer of acetoin to the (2R,3S) isomer of 3-amino-2-butanol and the (S) isomer of acetoin to the (2S,3S) isomer of 3-amino-2-butanol. Either type of enzyme (i.e., transaminase or reductive aminase) is considered to be an acetoin aminase and may be utilized in the production of 2-butanol. Other enzymes in this group may have different stereospecificities.
The skilled person will appreciate that polypeptides having acetoin aminase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. One example of this activity has is described herein and is identified as SEQ ID NO:122. Accordingly preferred acetoin aminase enzymes are those that have at least 80%-85% identity to SEQ ID NO:122, where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
(d) 3-Amino-2-Butanol to 3-Amino-2-Butanol O-Phosphate
There are no enzymes known in the art that catalyze the substrate to product conversion of 3-amino-2-butanol (III) to 3-amino-2-butanol phosphate (IV). However, a few Pseudomonas and Erwinia species have been shown to express an ATP-dependent ethanolamine kinase (EC 2.7.1.82) which allows them to utilize ethanolamine or 1-amino-2-propanol as a nitrogen source (Jones et al. (1973) Biochem. J. 134:167-182). It is likely that this enzyme also has activity towards 3-amino-2-butanol or could be engineered to do so, thereby providing an aminobutanol kinase. The present invention describes in Example 14 a gene of Erwinia carotovora subsp. atroseptica (SEQ ID NO:123) that encodes a protein (SEQ ID NO:24) that is identified as an amino alcohol kinase. This enzyme may be used to convert 3-amino-2-butanol to 3-amino-2-butanol O-phosphate.
The skilled person will appreciate that polypeptides having aminobutanol kinase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. One example of this activity has is described herein and is identified as SEQ ID NO:124. Accordingly preferred aminobutanol kinase enzymes are those that have at least 80%-85% identity to SEQ ID NO:124, where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
(e) 3-Amino-2-Butanol Phosphate to 2-Butanone
Although there are no enzymes reported to catalyze the substrate to product conversion of 3-amino-2-butanol phosphate (IV) to 2-butanone (V), the substrate is very similar to those utilized by the pyridoxal phosphate-dependent phosphoethanolamine phospho-lyase enzyme, which has been found in a small number of Pseudomonas and Erwinia species. These enzymes have activity towards phosphoethanolamine and both enantiomers of 2-phospho-1-aminopropane (Jones et al. (1973) Biochem. J. 134:167-182), and may also have activity towards 3-amino-2-butanol O-phosphate. The present invention describes a gene of Erwinia carotovora subsp. atroseptica (SEQ ID NO:125) that encodes a protein (SEQ ID NO:126) with homology to class III aminotransferases. Example 15 demonstrates that this enzyme is active on both aminopropanol phosphate and aminobutanol phosphate substrates. The newly identified and characterized enzyme was able to catalyze the conversion of a mixture of (R)-3-amino-(S)-2-butanol and (S)-3-amino-(R)-2-butanol O-phosphate, and a mixture of (R)-3-amino-(R)-2-butanol and (S)-3-amino-(S)-2-butanol O-phosphate to 2-butanone. The newly identified and characterized enzyme was also able to catalyze the conversion of both (R) and (S)-2-amino-1-propanol phosphate to propanone, with a preference for (S)-2-amino-1-propanol phosphate. The highest activity was observed with the proposed natural substrate DL-1-amino-2-propanol phosphate, which was converted to propionaldehyde.
The skilled person will appreciate that polypeptides having aminobutanol phosphate phospho-lyase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. One example of a suitable aminobutanol phosphate phospho-lyase enzyme is described herein as SEQ ID NO: 126. Accordingly preferred aminobutanol phosphate phospho-lyase enzymes are those that have at least 80%-85% identity to SEQ ID NO's 126, where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
(f) 2-Butanone to 2-Butanol
The final step in all pathways to produce 2-butanol from pyruvic acid is the reduction of 2-butanone (V) to 2-butanol (VI). This substrate to product conversion is catalyzed by some members of the broad class of alcohol dehydrogenases (types utilizing either NADH or NADPH as a source of hydride, depending on the enzyme) that may be called butanol dehydrogenases. Enzymes of each type that catalyze the reduction of 2-butanone are well known, as described above in the definition for butanol dehydrogenase.
The skilled person will appreciate that polypeptides having butanol dehydrogenase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. Some example of suitable butanol dehydrogenase enzymes are available from a number of sources, for example, Rhodococcus ruber [GenBank Nos: CAD36475 (SEQ ID NO:14), AJ491307 (SEQ ID NO:13)]. The NADP-dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus [GenBank Nos: AAC25556 (SEQ ID NO:91), AF013169 (SEQ ID NO:90)]. Additionally, a butanol dehydrogenase is available from Escherichia coli [GenBank Nos: NP—417484 (SEQ ID NO:75), NC—000913 (SEQ ID NO:74)] and a cyclohexanol dehydrogenase is available from Acinetobacter sp. [GenBank Nos: AAG10026 (SEQ ID NO:72), AF282240 (SEQ ID NO:71)]. Preferred butanol dehydrogenase enzymes are those that have at least 80%-85% identity to SEQ ID NO's 14, 91, 75, and 72, where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
Pathway 2:
(a) Pyruvate to Alpha-Acetolactate
This substrate to product conversion is the same as described above for Pathway 1.
(b) Alpha-Acetolactate to Acetoin
This substrate to product conversion is the same as described above for Pathway 1.
(g) Acetoin to Phosphoacetoin
Although enzymes that catalyze the substrate to product conversion of acetoin (II) to phosphoacetoin (VII) have not been described, the structure of the substrate acetoin is very similar to that of dihydroxyacetone, and therefore acetoin may be an acceptable substrate for dihydroxyacetone kinase (EC 2.7.1.29), an enzyme which catalyzes phosphorylation of dihydroxyacetone. Protein engineering techniques for the alteration of substrate specificity of enzymes are well known (Antikainen and Martin (2005) Bioorg. Med. Chem. 13:2701-2716) and may be used to generate an enzyme with the required specificity. In this conversion, the phosphate moiety may be supplied by any high energy biological phosphate donor, with the common substrates being phosphoenolpyruvate (as in the E. coli dihydroxyacetone kinase) and ATP (as in the Citrobacter freundii dihydroxyacetone kinase) (Garcia-Alles et al. (2004) Biochemistry 43:13037-13045).
(h) Phosphoacetoin to 3-Amino-2-Butanol O-Phosphate
Although enzymes that catalyze the substrate to product conversion of phosphoacetoin (VII) to 3-amino-2-butanol O-phosphate (IV) have not been described, the structure of the substrate is very similar to that of dihydroxyacetone phosphate, a substrate for the proposed serinol phosphate aminotransferase encoded by the 5′ portion of the rtxA gene in some species of Bradyrhizobium (Yasuta et al., supra). Thus a serinol phosphate aminotransferase may be functional in this step.
(e) 3-Amino-2-Butanol O-Phosphate to 2-Butanone
This substrate to product conversion is the same as described above for Pathway 1.
(f) 2-Butanone to 2-Butanol
This substrate to product conversion is the same as described above for Pathway 1.
Pathway 3:
(a) Pyruvate to Alpha-Acetolactate
This substrate to product conversion is the same as described above for Pathway 1.
(b) Alpha-Acetolactate to Acetoin
This substrate to product conversion is the same as described above for Pathway 1.
(i) Acetoin to 2,3-Butanediol
The substrate to product conversion of acetoin (II) to 2,3-butanediol (VIII) may be catalyzed by a butanediol dehydrogenase that may either utilize NADH or NADPH as the source of reducing equivalents when carrying out reductions. Enzymes with activity towards acetoin participate in the pathway for production of 2,3-butanediol in organisms that produce that compound. The reported enzymes (e.g., BudC from Klebsiella pneumoniae (Ui et al. (2004) Letters in Applied Microbiology 39:533-537) generally utilize NADH. Either cofactor is acceptable for use in the production of 2-butanol by this pathway.
The skilled person will appreciate that polypeptides having butanediol dehydrogenase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. Some example of suitable butanediol dehydrogenase enzymes are available from a number of sources, for example, Klebsiella pneumoniae (GenBank Nos: BBA13085 (SEQ ID NO:6), D86412 (SEQ ID NO:5)). (R)-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus [GenBank Nos. NP—830481 (SEQ ID NO:85), NC—004722 (SEQ ID NO:84); AAP07682 (SEQ ID NO:87), AE017000 (SEQ ID NO:86)], and Lactococcus lactis [GenBank Nos. AAK04995 (SEQ ID NO:89), AE006323 (SEQ ID NO:88)]. Preferred butanediol dehydrogenases enzymes are those that have at least 80%-85% identity to SEQ ID NO's 6, 85, 87, and 89, where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
(j) 2,3-Butanediol to 2-Butanone
The substrate to product conversion of 2,3-butanediol (VIII) to 2-butanone (V) may be catalyzed by diol dehydratase enzymes (EC 4.2.1.28) and glycerol dehydratase enzymes (EC 4.2.1.30). The best characterized diol dehydratase is the coenzyme B12-dependent Klebsiella oxytoca enzyme, but similar enzymes are found in a number of enteric bacteria. The K. oxytoca enzyme has been shown to accept meso-2,3-butanediol as a substrate (Bachovchin et al. (1977) Biochemistry 16:1082-1092), producing the desired product 2-butanone. Example 17 demonstrates that the Klebsiella pneumoniae glycerol dehydratase was able to convert meso-2,3-butanediol to 2-butanone. The three subunit of the Klebsiella pneumoniae glycerol dehydratase (alpha: SEQ ID NO:145 (coding region) and 146 (protein); beta: SEQ ID NO: 147 (coding region) and 148 (protein); and gamma: SEQ ID NO: 149 (coding region) and 150 (protein)) were expressed in conjunction with the two subunits of the Klebsiella pneumoniae glycerol dehydratase reactivase (large subunit, SEQ ID NO: 151 (coding region) and 152 (protein); and small subunit, SEQ ID NO: 153 (coding region) and 154 (protein)) to provide activity.
There are also reports in the literature of a B12-independent diol dehydratase from Clostridium glycolicum (Hartmanis et al. (1986) Arch. Biochem. Biophys. 245:144-152). This enzyme has activity towards 2,3-butanediol, although this activity is less than 1% of the activity towards ethanediol, but the enzyme may be engineered to improve that activity. A better-characterized B12-independent dehydratase is the glycerol dehydratase from Clostridium butyricum (protein SEQ ID NO:280; coding region SEQ ID NO:281; O'Brien et al. (2004) Biochemistry 43:4635-4645), which has high activity towards 1,2-propanediol as well as glycerol. This enzyme uses S-adenosylmethionine as a source of adenosyl radical. There are no reports of activity towards 2,3-butanediol, but such activity, if not already present, may possibly be engineered. The glycerol dehydratase reactivase from Clostridium butyricum has also been reported (protein SEQ ID NO:282; coding region SEQ ID NO:283).
In addition, a putative B12-independent (coenzyme B12-independent) propanediol dehydratase of Roseburia inulinivorans (encoded by ORF18) has been identified as induced under fucose utilization conditions and by sequence homology to the Clostridium butyricum B12-independent glycerol dehydratase (53% identity; Scott et al., J. of Bacteriology (2006) 188:4340-4349)). A Roseburia inulinivorans putative propanediol dehydratase activator was similarly identified (encoded by ORF 19) with fucose induction and homology to the Clostridium butyricum glycerol dehydratase activase (35% identity; Scott et al. ibid.).
Applicants have demonstrated for the first time that the Roseburia inulinivorans ORF18 and ORF19 encoded proteins, named herein RdhtA and RdhtB, respectively, provide 1,2 propanediol dehydratase activity when expressed in a recombinant microbial host cell (Example 19). Further, applicants have found that expression of RdhtA and RdhtB provides activity for conversion of meso-2,3-butanediol to 2-butanone, indicating that the expressed enzyme activity includes butanediol dehydratase activity. In addition, applicants have demonstrated herein that when RdhtA and RdhtB are expressed in a recombinant microbial host cell having a source of 2,3-butanediol and having butanol dehydrogenase activity, 2-butanol is produced (Example 21). 2-Butanone may be produced if the host cell is lacking butanol dehydrogenase activity.
Thus in one aspect of the invention, preferred B12-independent glycerol/diol dehydratase activity, more specifically butanediol dehydratase activity, is provided by the RdhtA and RdhtB proteins for conversion of 2,3-butanediol to 2-butanone in biosynthetic Pathway 3, for production of 2-butanone or 2-butanol. Using these proteins, 2-butanol may be produced in the absence of coenzyme B12, as demonstrated in Example 21 herein, where the recombinant microbial host cell used for production is incapable of synthesizing coenzyme B12 and no vitamin B12 is added to the medium. The vitamin B12 that is generally used as an additive in the medium is not coenzyme B12 (adenosyl cobalamin), but is another form of cobalamin that is converted by cells to coenzyme B12.
A source of 2,3-butanediol in the recombinant microbial host cells may be provided naturally, exogenously, or by engineering a biosynthetic pathway to produce 2,3-butanediol. Such a pathway is described above in steps that include providing enzymes for conversion of pyruvate to alpha-acetolactate, alpha-acetolactate to acetoin, and acetoin to 2,3-butanediol. RdhtA and RdhtB may be expressed in a cell having enzymes able to carry out these steps for production of 2-butanone. Expression of butanol dehydrogenase activity, for conversion of 2-butanone to 2-butanol, is described above as an additional step.
Any B12-independent diol dehydratase or glycerol dehydratase, in conjunction with a B12-independent diol dehydratase reactivase or glycerol dehydratase reactivase, with butanediol dehydratase activity may be used in the present invention. A BLAST search of publicly available sequences reveals that the closest protein sequences to RdhtA are an unknown protein of Ruminococcus gnavus (Accession # Z7B0E1) that is about 69% identical, and a hypothetical protein of Ruminococcus obeum (Accession # A5ZM80) that is about 66% identical. The closest protein sequences to RdhtB are a hypothetical protein of Ruminococcus gnavus (Accession # ZP—02040254) that is about 67% identical, and a hypothetical protein of Ruminococcus obeum (Accession # ZP—01962383) that is about 66% identical.
Proteins having at least about 75% or greater amino acid identity to either SEQ ID NO:276 or 278 which together provide B12-independent butanediol dehydratase activity, and isolated DNA molecules encoding said proteins, may be used in the present invention. Such proteins may have identity of 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%. Isolated DNA molecules encoding said proteins that have sequence identities to SEQ ID NOs: 277 or 279 that are at least about 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100% may be used. Most suitable are DNA molecules with SEQ ID NOs:277 and 279, encoding the Roseburia inulinivorans B12-independent butanediol dehydratase RdhtA and butanediol dehydratase reactivase RdhtB, respectively. A nucleic acid molecule encoding the Roseburia inulinivorans B12-independent butanediol dehydratase, such as SEQ ID NO:277, may be used to isolate nucleic acid molecules encoding homologous proteins, that have at least 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100% sequence identity to this nucleic acid fragment, from the same or other microbial species. A nucleic acid molecule encoding the Roseburia inulinivorans B12-independent butanediol dehydratase reactivase, such as SEQ ID NO:279, may be used to isolate nucleic acid molecules encoding homologous proteins, that have at least 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100% sequence identity to this nucleic acid fragment, from the same or other microbial species.
Isolation of homologs using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g. polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Natl. Acad. Sci. USA 82, 1074, (1985; or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89: 392, (1992)).
For example, nucleic acid fragments of the instant invention may be isolated directly by using all or a portion of the nucleic acid fragment of SEQ ID NO:277 or 279 as a DNA hybridization probe to screen libraries from any desired bacteria using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon SEQ ID NO: 277 or 279 can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of or the full-length of homologs of SEQ ID NO: 277 or 279. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.
Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art. (Thein and Wallace, “The use of oligonucleotide as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp. 33-50 IRL Press, Herndon, Va.); Rychlik, W. (1993) In White, B. A. (ed.), Methods in Molecular Biology, Vol. 15, pages 31-39, PCR Protocols: Current Methods and Applications. Humania Press, Inc., Totowa, N.J.)
Generally, two short segments of the instant nucleic acid sequence may be used to design primers for use in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous coding regions from DNA or RNA. PCR may be performed using as template any DNA that contains a nucleic acid sequence homologous to SEQ ID NO: 277 or 279, including for example, genomic DNA, cDNA or plasmid DNA as template. When using a library of cloned cDNA, the sequence of one primer is derived from SEQ ID NO: 277 or 279, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts at the 3′ end of the mRNA precursor encoding microbial genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol using mRNA as template (Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant nucleic acid sequence. Using commercially available 3′ RACE or 5′ RACE systems (Life Technologies, Rockville, Md.), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci. USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
Alternatively a nucleic acid molecule of SEQ ID NO: 277 or 279 or its complement may be employed as a hybridization reagent for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes of the present invention are typically single stranded nucleic acid sequences which are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions which will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration the shorter the hybridization incubation time needed. Optionally a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).
Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9), about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons (kD)), polyvinylpyrrolidone (about 250-500 kD), and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA, e.g., calf thymus or salmon sperm DNA, or yeast RNA, and optionally from about 0.5 to 2% wt./vol. glycine. Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents, such as polyethylene glycol, anionic polymers such as polyacrylate or polymethylacrylate, and anionic saccharidic polymers, such as dextran sulfate.
Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
In addition, since sequences of microbial genomes are rapidly becoming available to the public, homologs may be identified using bioinformatics approaches alone.
One skilled in the art can readily assess butanediol dehydratase activity of a protein encoded by a DNA molecule identified by sequence-based methodologies described above. The protein is expressed in a microbial host cell as described below and a butanediol dehydratase activity assay is performed in culture medium, cell extracts, crude enzyme preparations, or purified enzyme preparations. For example, meso-2,3-butanediol is added to the culture medium and after about 24 hours of culturing of the expressing host, 2-butanone is detected in the culture medium by HPLC if the activity is present. Through this or other readily performable assays butanediol dehydratase function is linked to structure of a protein encoded by a DNA molecule identified by a sequence-based methodology.
The skilled person will appreciate that polypeptides having butanediol dehydratase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. As noted above a variety of diol and glycerol dehydratases have been described in the literature and will be suitable for use in the present invention. Accordingly, in one aspect of the invention preferred B12-dependent diol and glycerol dehydratase enzymes are those that have at least 80%-85% identity to enzymes having the large, medium and small subunits, respectively of the sequences listed below:
a) SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12;
b) SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:97;
c) SEQ ID NO:99, SEQ ID NO:101, and SEQ ID NO:103;
d) SEQ ID NO:105, SEQ ID NO:107, and SEQ ID NO:109;
e) SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137;
f) SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140;
g) SEQ ID NO:146, SEQ ID NO:148, and SEQ ID NO:150;
h) SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143; and
i) SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166.
where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
Similarly preferred diol and glycerol dehydratase enzymes are those that have at least 80%-85% identity to enzymes having the large, medium and small subunits, respectively of the sequences listed below: Large subunit: SEQ ID NOs: 8, 99, 105, 135, 138, 141, 146, and 164; Medium subunit: SEQ ID NOs: 10, 101, 107, 136, 139, 142, 148, and 165; Small subunit: SEQ ID NOs:12, 103, 109, 137, 140, 143, 150, and 166; where at least 85%-90% identity is more preferred and where at least 95% identity based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, is most preferred.
Additional diol and glycerol dehydratase enzymes that may be used in the biosynthetic pathway 3 of the present invention were identified through a bioinformatics structure/function analysis that is described below and in Example 18.
(f) 2-Butanone to 2-Butanol
This substrate to product conversion is the same as described above for Pathway 1.
Diol and Glycerol Dehydratases for Biosynthetic Pathway 3
Any enzyme that is a diol or glycerol dehydratase may be used in the present invention for the conversion of 2,3-butanediol to 2-butanone. A structure/function relationship for diol and glycerol dehydratases in the enzyme classes EC 4.2.1.28 and EC 4.2.1.30, respectively, was established herein in Example 18. The function is provided by experimental data and the structure is provided by bioinformatics analysis. Eight diol and glycerol dehydratase enzymes with activities that have been experimentally demonstrated were analyzed. In this group (listed in Table 10), the Klebsiella oxytoca diol dehydratase and the Klebsiella pneumoniae glycerol dehydratase enzymes were both shown to convert 2,3-butanediol to 2-butanone (Bachovchin et al. (1977) Biochemistry 16:1082-1092 and Example 17 herein, respectively), while the activities of the additional six enzymes were demonstrated using their natural substrates (references given in Table 10). This set of eight diol and glycerol dehydratases was analyzed using the hmmsearch algorithm of the HMMER software package (Janelia Farm Research Campus, Ashburn, Va.). The Z parameter of the hmmsearch algorithm was set to 1 billion. The output of the HMMER analysis using a set of protein sequences is a Profile Hidden Markov Model (Profile HMM). The theory behind Profile HMMs is described in Durbin et al., Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531, incorporated herein by reference) that characterizes the set of proteins based on the probability of each amino acid occurring at each position in the alignment of the proteins of the set.
Since the eight diol and glycerol dehydratases (diol/glycerol dehydratases) with experimentally verified function that were used for the analysis each have three subunits (large or alpha, medium or beta, and small or gamma), a separate Profile HMM was prepared for each subunit. The large subunit Profile HMM (Table 12) was built using proteins with SEQ ID NOs: 8, 99, 105, 135, 138, 141, 146, and 164 that are described in Tables 1 and 2. The medium subunit Profile HMM (Table 13) was built using proteins with SEQ ID NOs: 10, 101, 107, 136, 139, 142, 148, and 165 that are described in Tables 1 and 2. The small subunit Profile HMM (Table 14) was built using proteins with SEQ ID NOs:12, 103, 109, 137, 140, 143, 150, and 166 that are described in Tables 1 and 2. References that provide the functional assay data are given in Table 10. The Profile HMM prepared for the large subunit gives a structural characterization for the functional large subunit of diol/glycerol dehydratases. Similarly the Profile HMMs for the medium and small subunits give structural characterizations for the functional medium and small subunits, respectively, of diol/glycerol dehydratases. Therefore any protein that has a significant match to either the large, medium, or small subunit Profile HMM is directly linked to the function of the subunit to which the profile was prepared. To be significant, the matching has an E-value of 0.01 or less, and further use of “match” is understood to be with this E-value criterion. Thus diol/glycerol dehydratase subunits that may be used in the present invention are proteins that match the Profile HMMs, that were prepared using the proteins with SEQ ID NOs listed above, with an E-value of 0.01 or less.
Proteins that are full length and have functional linkage to the large subunit of diol/glycerol dehydratases, through matching the large subunit Profile HMM, include but are not limited to, proteins with SEQ ID NOs; 93, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 130, 243, 254, 255, 256, 257, 258, and 259. Proteins that are full length and have functional linkage to the medium subunit of diol/glycerol dehydratases, through matching the medium subunit Profile HMM, include but are not limited to, proteins with SEQ ID NOs; 95, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 244, 250, 252, 260, 261, 262, 263, 364, 265, 266, and 167. Proteins that are full length and have functional linkage to the small subunit of diol/glycerol dehydratases, through matching the small subunit Profile HMM, include but are not limited to, proteins with SEQ ID NOs; 97, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 234, 236, 238, 240, 242, 245, 248, 249, 251, 253, 268, 270, 271, 272, 273, and 274. In addition, proteins that are fused full length large and medium subunits that have functional linkage to the large and medium subunits of diol/glycerol dehydratases, through matching the large and medium subunit Profiles HMM, include but are not limited to, proteins with SEQ ID NOs; 233, 235, 237, 239, 241, 246, and 247.
Since the Profile HMMs described above provide a structure/function relationship for diol/glycerol dehydratases, newly identified proteins that match these profiles may also be used in the present invention. In addition, diol/glycerol dehydratase subunit protein sequences that may be used in the present invention include proteins with amino acid changes that have minimal effects on subunit function, which are substantially similar to the sequences of the SEQ ID NOs listed above. It is well known in the art that substitution of a chemically equivalent amino acid at a given site which does not effect the functional properties of the encoded protein is common. For the purposes of the present invention substitutions providing substantially similar proteins are defined as exchanges within one of the following five groups:

    1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);
    2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln;
    3. Polar, positively charged residues: His, Arg, Lys;
    4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and
    5. Large aromatic residues: Phe, Tyr, Trp.

Thus, substitutions of one amino acid for another in these groups can be expected to produce a functionally equivalent protein. In many cases, changes which result in alteration of the N-terminal and C-terminal portions of the protein would also not be expected to alter the activity of the protein.



Substantially similar proteins to those of SEQ IDs that match the Profile HMMs may be 90% or 95% identical in amino acid sequence to one of the matching proteins, and these may be used in the present invention.
One skilled in the art can readily identify a set of three subunits that may be used together to provide a functional diol/glycerol dehydratase. Particularly suitable is a combination of a large, medium and small subunit from the same strain of organism, whose coding regions are located near one another in the genome. These subunits would be most likely to form a natural diol or glycerol dehydratase. Many large, medium, and small, subunits are grouped in this manner in Table 2. A combination of subunits from closely related strains or species is suitable for composing a diol dehydratase or a glycerol dehydratase. Any combination of subunits that catalyzes the conversion of 2,3-butanediol to 2-butanone may be used. Effective subunit combinations may readily be determined by one skilled in the art through amino acid sequence comparisons and/or functional assays.
Accordingly the invention provides diol and glycerol dehydratase enzymes having amino acid sequences comprising full length large, medium and small subunits that each give an E-value parameter of 0.01 or less when queried using a Profile Hidden Markov Model prepared using the large subunits of SEQ ID NOs: 8, 99, 105, 135, 138, 141, 146, and 164; the medium subunits of SEQ ID NOs: 10, 101, 107, 136, 139, 142, 148, and 165; and the small subunits of SEQ ID NOs:12, 103, 109, 137, 140, 143, 150, and 166; each query being carried out using the hmmsearch algorithm wherein the Z parameter is set to 1 billion.

    
    
        Alternatively the invention provides diol and glycerol dehydratase enzymes having amino acid sequences identified by a process comprising a) generating a Profile Hidden Markov Model from the alignment of the amino acid sequences corresponding to the large, medium and small subunits of diol and glycerol dehydratase enzymes wherein;
        i) the large subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 99, 105, 135, 138, 141, 146, and 164;
        ii) the medium subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 101, 107, 136, 139, 142, 148, and 165; and
        iii) the small subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:12, 103, 109, 137, 140, 143, 150, and 166;
        b) querying at least one public database of protein sequences containing sequences of diol and glycerol dehydratases with the Profile Hidden Markov Model of (a) using the hmmsearch algorithm wherein the Z parameter is set to 1 billion and the E-value parameter is set to 0.01, to identify a first data set of diol and glycerol dehydratase amino acid sequences; and
        c) removing any partial sequences from the first data set of (b) to generate a second data set of diol and glycerol dehydratase amino acid sequences, wherein diol dehydratase and the glycerol dehydratase enzymes are identified.
    
    


With respect to large subunits of the diol and glycerol dehydratases of the invention the enzymes may comprise a large subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 93, 99, 105, 135, 138, 141, 146, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 130, 243, 254, 255, 256, 257, 258 and 259, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
With respect to the medium subunits of the diol and glycerol dehydratases of the invention the enzymes may comprise a medium subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 95, 101, 107, 136, 139, 142, 148, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 244, 250, 252, 260, 261, 262, 263, 364, 265, 266, and 167 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
With respect to the small subunits of the diol and glycerol dehydratases of the invention the enzymes may comprise a medium subunit comprising a small subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 97, 103, 109, 137, 140, 143, 150, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 234, 236, 238, 240, 242, 245, 248, 249, 251, 253, 268, 270, 271, 272, 273, and 274, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
Alternatively the diol dehydratase or glycerol dehydratase may comprise fused large, medium and small subunits comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 233, 235, 237, 239, 241, 246, and 247, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
Alternatively the diol dehydratase or glycerol dehydratase enzymes may comprise a fused large, medium and small subunits and has at least 95% identity to an amino acid sequence comprising all three of the amino acid sequences encoding large, medium and small subunits, selected from the group consisting of:
a) SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12;
b) SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:97;
c) SEQ ID NO:99, SEQ ID NO:101, and SEQ ID NO:103;
d) SEQ ID NO:105, SEQ ID NO:107, and SEQ ID NO:109;
e) SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137;
f) SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140;
g) SEQ ID NO:146, SEQ ID NO:148, and SEQ ID NO:150;
h) SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143; and
i) SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166;
based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
Pathway 4:
(a) Pyruvate to Alpha-Acetolactate
This substrate to product conversion is the same as described above for Pathway 1.
(k) Alpha-Acetolactate to 2,3-Dihydroxy-2-Methylbutanoic Acid
The substrate to product conversion of acetolactate (I) to 2,3-dihydroxy-2-methylbutanoic acid (IX) is not known in the art. However, the product of this conversion has been reported as a component of fermentation broths (Ziadi et al. (1973) Comptes Rendus des Seances de l'Academie des Sciences, Serie D: Sciences Naturelles 276:965-8), but the mechanism of formation is unknown. The likely mechanism of formation is reduction of acetolactate with NADH or NADPH as the electron donor. To utilize this pathway for production of 2-butanol, an enzyme catalyzing this reaction needs to be identified or engineered. However, the precedent for enzymatic reduction of ketones to alcohols is well established.
(l) 2,3-Dihydroxy-2-Methylbutanoic Acid to 2-Hydroxy-2-Methyl-3-Phosphobutanoic Acid
There are no enzymes known that catalyze the substrate to product conversion of 2,3-dihydroxy-2-methylbutanoic acid (IX) to 2-hydroxy-2-methyl-3-phosphobutanoic acid (X). However, there are a large number of kinases in Nature that possess varying specificity. It is therefore likely that an enzyme could be isolated or engineered with this activity.
(m) 2-Hydroxy-2-Methyl-3-Phosphobutanoic Acid to 2-Butanone
There are no known enzymes that catalyze the substrate to product conversion of 2-hydroxy-2-methyl-3-phosphobutanoic acid (X) to 2-butanone (V). The combination of this reaction with the previous one is very similar to the multi-step reaction catalyzed by mevalonate-5-pyrophosphate (M5PP) decarboxylase, which consists of initial phosphorylation of M5PP to 3-phosphomevalonate-5-PP, followed by decarboxylation-dependent elimination of phosphate (Alvear et al. (1982) Biochemistry 21:4646-4650).
(f) 2-Butanone to 2-Butanol
This substrate to product conversion is the same as described above for Pathway 1.
Thus, in providing multiple recombinant pathways from pyruvate to 2-butanol, there exists a number of choices to fulfill the individual conversion steps, and the person of skill in the art will be able to utilize publicly available sequences and sequences disclosed herein to construct the relevant pathways. A listing of a representative number of genes known in the art and useful in the construction of 2-butanol biosynthetic pathways is given above in Tables 1 and 2.
Microbial Hosts for 2-Butanol and 2-Butanone Production
Microbial hosts for 2-butanol or 2-butanone production may be selected from bacteria, cyanobacteria, filamentous fungi and yeasts. The microbial host used for 2-butanol or 2-butanone production should be tolerant to the product produced, so that the yield is not limited by toxicity of the product to the host. The selection of a microbial host for 2-butanol production is described in detail below. The same criteria apply to the selection of a host for 2-butanone production.
Microbes that are metabolically active at high titer levels of 2-butanol are not well known in the art. Although butanol-tolerant mutants have been isolated from solventogenic Clostridia, little information is available concerning the butanol tolerance of other potentially useful bacterial strains. Most of the studies on the comparison of alcohol tolerance in bacteria suggest that butanol is more toxic than ethanol (de Cavalho et al., Microsc. Res. Tech. 64:215-22 (2004) and Kabelitz et al., FEMS Microbiol. Lett. 220:223-227 (2003)). Tomas et al. (J. Bacteriol. 186:2006-2018 (2004)) report that the yield of 1-butanol during fermentation in Clostridium acetobutylicum may be limited by butanol toxicity. The primary effect of 1-butanol on Clostridium acetobutylicum is disruption of membrane functions (Hermann et al., Appl. Environ. Microbiol. 50:1238-1243 (1985)).
The microbial hosts selected for the production of 2-butanol should be tolerant to 2-butanol and should be able to convert carbohydrates to 2-butanol using the introduced biosynthetic pathway. The criteria for selection of suitable microbial hosts include the following: intrinsic tolerance to 2-butanol, high rate of carbohydrate utilization, availability of genetic tools for gene manipulation, and the ability to generate stable chromosomal alterations.
Suitable host strains with a tolerance for 2-butanol may be identified by screening based on the intrinsic tolerance of the strain. The intrinsic tolerance of microbes to 2-butanol may be measured by determining the concentration of 2-butanol that is responsible for 50% inhibition of the growth rate (IC50) when grown in a minimal medium. The IC50 values may be determined using methods known in the art. For example, the microbes of interest may be grown in the presence of various amounts of 2-butanol and the growth rate monitored by measuring the optical density at 600 nanometers. The doubling time may be calculated from the logarithmic part of the growth curve and used as a measure of the growth rate. The concentration of 2-butanol that produces 50% inhibition of growth may be determined from a graph of the percent inhibition of growth versus the 2-butanol concentration. Preferably, the host strain should have an IC50 for 2-butanol of greater than about 0.5%. More suitable is a host strain with an IC50 for 2-butanol that is greater than about 1.5%. Particularly suitable is a host strain with an IC50 for 2-butanol that is greater than about 2.5%.
The microbial host for 2-butanol production should also utilize glucose and/or other carbohydrates at a high rate. Most microbes are capable of utilizing carbohydrates. However, certain environmental microbes cannot efficiently use carbohydrates, and therefore would not be suitable hosts.
The ability to genetically modify the host is essential for the production of any recombinant microorganism. Modes of gene transfer technology that may be used include by electroporation, conjugation, transduction or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors used with an organism are tailored to the host organism based on the nature of antibiotic resistance markers that can function in that host.
The microbial host also may be manipulated in order to inactivate competing pathways for carbon flow by inactivating various genes. This requires the availability of either transposons or chromosomal integration vectors to direct inactivation. Additionally, production hosts that are amenable to chemical mutagenesis may undergo improvements in intrinsic 2-butanol tolerance through chemical mutagenesis and mutant screening.
Based on the criteria described above, suitable microbial hosts for the production of 2-butanol and 2-butanone include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Kluyveromyces and Saccharomyces. Preferred hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Pediococcus pentosaceus, Pediococcus acidilactici, Bacillus subtilis, Kluyveromyces lactis and Saccharomyces cerevisiae. 
Construction of Production Host
Recombinant organisms containing the necessary genes that encode the enzymatic pathway for the conversion of a fermentable carbon substrate to 2-butanol or 2-butanone may be constructed using techniques well known in the art. In the present invention, genes encoding the enzymes of the 2-butanol biosynthetic Pathway 3: acetolactate synthase, acetolactate decarboxylase, butanediol dehydrogenase, butanediol dehydratase, and butanol dehydrogenase; or 2-butanone biosynthetic Pathway 3 omitting the butanol dehydrogenase, may be isolated from various sources, as described above.
Methods of obtaining desired genes from a bacterial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, primers may be designed and the desired sequence amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for cloning into expression vectors. If a gene that is heterologous to a known sequence is to be isolated, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes having complementary sequence to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for cloning into expression vectors, which are then transformed into appropriate host cells.
In addition, given the amino acid sequence of a protein with desired enzymatic activity, the coding sequence may be ascertained by reverse translating the protein sequence. A DNA fragment containing the coding sequence may be prepared synthetically and cloned into an expression vector, then transformed into the desired host cell.
In preparing a synthetic DNA fragment containing a coding sequence, this sequence may be optimized for expression in the target host cell. Tools for codon optimization for expression in a heterologous host are readily available. Some tools for codon optimization are available based on the GC content of the host organism. The GC contents of some exemplary microbial hosts are given Table 3.







TABLE 3







GC Contents of Microbial Hosts










Strain
% GC






B. licheniformis
46



B. subtilis
42



C. acetobutylicum
37



E. coli
50



P. putida
61



A. eutrophus
61



Paenibacillus macerans
51



Rhodococcus erythropolis
62



Brevibacillus
50



Paenibacillus polymyxa
50









Once the relevant pathway genes are identified and isolated they may be transformed into suitable expression hosts by means well known in the art. Vectors useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.), Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly, Mass.). Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors comprise a promoter region which harbors transcriptional initiation controls and a transcriptional termination control region, between which a coding region DNA fragment may be inserted, to provide expression of the inserted coding region. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific species chosen as a production host.
Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention including, but not limited to, promoters derived from the following genes: CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1, FBA, GPD, and GPM (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); as well as the lac, ara, tet, trp, IPL, IPR, T7, tac, and trc promoters (useful for expression in Escherichia coli, Alcaligenes, and Pseudomonas); the amy, apr, and npr promoters, and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans; nisA (useful for expression Gram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152:1011-1019 (2006)).
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.
Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors: pRK437, pRK442, and pRK442(H), are available. These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid 50(1):74-79 (2003)). Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 are also available with promoters that can function in a range of Gram-negative bacteria. Plasmid pAYC36 and pAYC37, have active promoters along with multiple cloning sites to allow for heterologous gene expression in Gram-negative bacteria.
Chromosomal gene replacement tools are also widely available. For example, a thermosensitive variant of the broad-host-range replicon pWV101 has been modified to construct a plasmid pVE6002 which can be used to effect gene replacement in a range of Gram-positive bacteria (Maguin et al., J. Bacteriol. 174(17):5633-5638 (1992)). Additionally, in vitro transposomes are available from commercial sources such as EPICENTRE® to create random mutations in a variety of genomes.
The expression of a 2-butanol biosynthetic pathway in various preferred microbial hosts is described in more detail below. For the expression of a 2-butanone biosynthetic pathway, the same description applies, but the final substrate to product conversion of 2-butanone to 2-butanol is omitted.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in E. coli 
Vectors useful for the transformation of E. coli are common and commercially available from the companies listed above. For example, the genes of a 2-butanol biosynthetic pathway may be isolated from various sources, as described above, cloned onto a modified pUC19 vector and transformed into E. coli NM522, as described in Examples 6 and 7. Alternatively, the genes encoding a 2-butanol biosynthetic pathway may be divided into multiple operons, cloned onto expression vectors, and transformed into various E. coli strains, as described in Examples 9, 10, and 11. The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in Rhodococcus erythropolis 
A series of E. coli-Rhodococcus shuttle vectors are available for expression in R. erythropolis, including, but not limited to pRhBR17 and pDA71 (Kostichka et al., Appl. Microbiol. Biotechnol. 62:61-68 (2003)). Additionally, a series of promoters are available for heterologous gene expression in R. erythropolis (see for example Nakashima et al., Appl. Environ. Microbiol. 70:5557-5568 (2004), and Tao et al., Appl. Microbiol. Biotechnol. 2005, DOI 10.1007/s00253-005-0064). Targeted gene disruptions in chromosomal genes of R. erythropolis may be created using the methods described by Tao et al., supra, and Brans et al. (Appl. Envion. Microbiol. 66: 2029-2036 (2000)).
The heterologous genes required for the production of 2-butanol, as described above, may be cloned initially in pDA71 or pRhBR71 and transformed into E. coli. The vectors may then be transformed into R. erythropolis by electroporation, as described by Kostichka et al., supra. The recombinants may be grown in synthetic medium containing glucose and the production of 2-butanol can be followed using fermentation methods known in the art. The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in B. Subtilis 
Methods for gene expression and creation of mutations in B. subtilis are also well known in the art. For example, the genes of a 2-butanol biosynthetic pathway may be isolated from various sources, as described above, cloned into a modified E. coli-Bacillus shuttle vector and transformed into Bacillus subtilis BE1010, as described in Example 8, The desired genes may be cloned into a Bacillus expression vector and transformed into a strain to make a production host. Alternatively, the genes may be integrated into the Bacillus chromosome using conditional replicons or suicide vectors that are known to one skilled in the art. For example, the Bacillus Genetic Stock Center carries numerous integration vectors. The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in B. licheniformis 
Most of the plasmids and shuttle vectors that replicate in B. subtilis may be used to transform B. licheniformis by either protoplast transformation or electroporation. The genes required for the production of 2-butanol may be cloned in plasmids pBE20 or pBE60 derivatives (Nagarajan et al., Gene 114:121-126 (1992)). Methods to transform B. licheniformis are known in the art (for example see Fleming et al. Appl. Environ. Microbiol., 61(11):3775-3780 (1995)). The plasmids constructed for expression in B. subtilis may be transformed into B. licheniformis to produce a recombinant microbial host that produces 2-butanol. The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in Paenibacillus macerans 
Plasmids may be constructed as described above for expression in B. subtilis and used to transform Paenibacillus macerans by protoplast transformation to produce a recombinant microbial host that produces 2-butanol. The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in Alcaligenes (Ralstonia) eutrophus 
Methods for gene expression and creation of mutations in Alcaligenes eutrophus are known in the art (see for example Taghavi et al., Appl. Environ. Microbiol., 60(10):3585-3591 (1994)). The genes for a 2-butanol biosynthetic pathway may be cloned in any of the broad host range vectors described above, and electroporated into Alcaligenes eutrophus to generate recombinants that produce 2-butanol. The poly(hydroxybutyrate) pathway in Alcaligenes has been described in detail, a variety of genetic techniques to modify the Alcaligenes eutrophus genome are known, and those tools can be applied for engineering a 2-butanol biosynthetic pathway. The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in Pseudomonas putida 
Methods for gene expression in Pseudomonas putida are known in the art (see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which is incorporated herein by reference). The genes of a 2-butanol biosynthetic pathway may be inserted into pPCU18, and this ligated DNA may be electroporated into electrocompetent Pseudomonas putida DOT-T1 C5aAR1 cells to generate recombinants that produce 2-butanol. The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in Lactobacillus plantarum 
The Lactobacillus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus may be used for Lactobacillus. Non-limiting examples of suitable vectors include pAMβ1 and derivatives thereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl. Environ. Microbiol. 62:1481-1486 (1996)); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903 (1994)). Several plasmids from Lactobacillus plantarum have also been reported (van Kranenburg et al., Appl. Environ. Microbiol. 71(3):1223-1230 (2005)).
The various genes for a 2-butanol biosynthetic pathway may be assembled into any suitable vector, such as those described above. The codons can be optimized for expression based on the codon index deduced from the genome sequences of Lactobacillus plantarum or Lactobacillus arizonensis. The plasmids may be introduced into the host cell using methods known in the art, such as electroporation (Cruz-Rodz et al. Molecular Genetics and Genomics 224:1252-154 (1990), Bringel, et al. Appl. Microbiol. Biotechnol. 33: 664-670 (1990), Alegre et al., FEMS Microbiology letters 241:73-77 (2004)), and conjugation (Shrago et al., Appl. Environ. Microbiol. 52:574-576 (1986)). The 2-butanol biosynthetic pathway genes can also be integrated into the chromosome of Lactobacillus using integration vectors (Hols et al., Appl. Environ. Microbiol. 60:1401-1403 (1990), Jang et al., Micro. Lett. 24:191-195 (2003)). The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in Enterococcus faecium, Enterococcus qallinarium, and Enterococcus faecalis 
The Enterococcus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Lactobacillus, Bacillus subtilis, and Streptococcus, described above, may be used for Enterococcus. Expression vectors for E. faecalis using the nisA gene from Lactococcus may also be used (Eichenbaum et al., Appl. Environ. Microbiol. 64:2763-2769 (1998). Additionally, vectors for gene replacement in the E. faecium chromosome may be used (Nallaapareddy et al., Appl. Environ. Microbiol. 72:334-345 (2006)).
The various genes for a 2-butanol biosynthetic pathway may be assembled into any suitable vector, such as those described above. The codons can be optimized for expression based on the codon index deduced from the genome sequences of Enterococcus faecalis or Enterococcus faecium. The plasmids may be introduced into the host cell using methods known in the art, such as electroporation, as described by Cruz-Rodz et al. (Molecular Genetics and Genomics 224:1252-154 (1990)) or conjugation, as described by Tanimoto et al. (J. Bacteriol. 184:5800-5804 (2002)) and Grohamann et al. (Microbiol. Mol. Biol. Rev. 67:277-301 (2003)). The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Expression of a 2-Butanol or 2-Butanone Biosynthetic Pathway in Pediococcus pentosaceus and Pediococcus acidilactici. 
The Pediococcus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus, described above, may be used for Pediococcus. A non-limiting example of a suitable vector is pHPS9 (Bukhtiyarova et al. Appl. Environ. Microbiol. 60:3405-3408 (1994)). Several plasmids from Pediococcus have also been reported (Alegre et al., FEMS Microbiol. Lett. 250:151-156 (2005); Shareck et al. Crit. Rev Biotechnol. 24:155-208 (2004)).
The genes for a 2-butanol biosynthetic pathway may be assembled into any suitable vector, such as those described above. The codons can be optimized for expression based on the codon index deduced from the genome sequence of Pediococcus pentosaceus. The plasmids may be introduced into the host cell using methods known in the art, such as electroporation (see for example, Osmanagaoglu et al., J. Basic Microbiol. 40:233-241 (2000); Alegre et al., FEMS Microbiol. Lett. 250:151-156 (2005)) and conjugation (Gonzalez and Kunka, Appl. Environ. Microbiol. 46:81-89 (1983)). The 2-butanol biosynthetic pathway genes can also be integrated into the chromosome of Pediococcus using integration vectors (Davidson et al. Antonie van Leeuwenhoek 70:161-183 (1996)). The 2-butanone biosynthesis pathway may be similarly expressed, omitting the butanol dehydrogenase.
Fermentation Media
Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1-Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, as well as mixtures of any of these sugars. Sucrose may be obtained from feedstocks such as sugar cane, sugar beets, cassava, and sweet sorghum. Glucose and dextrose may be obtained through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, and oats.
In addition, fermentable sugars may be obtained from cellulosic and lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in co-owned and co-pending US patent application US20070031918A1, which is herein incorporated by reference. Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of an enzymatic pathway necessary for 2-butanol or 2-butanone production.
Culture Conditions
Typically cells are grown at a temperature in the range of about 25° C. to about 40° C. in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, may also be incorporated into the fermentation medium.
Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition.
Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.
Industrial Batch and Continuous Fermentations
The present process employs a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.
A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and fed-batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.
Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by the turbidity of the culture medium, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
It is contemplated that the present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for 2-butanol or 2-butanone production.
Methods for 2-Butanol and 2-Butanone Isolation from the Fermentation Medium
The bioproduced 2-butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the 2-butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation. These same methods may be adapted to isolate bioproduced 2-butanone from the fermentation medium.
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating a preferred embodiment of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
General Methods
Standard recombinant DNA and molecular cloning techniques described in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).
Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following Examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials described for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified. Bacterial strains are obtained from the American Type Culture Collection (ATCC, Manassas, Va.) unless otherwise noted.
Oligonucleotide primers described in the following Examples are given in Table 4. All oligonucleotide primers were synthesized by Sigma-Genosys (Woodlands, Tex.).







TABLE 4







Cloning and Screening Primers












Primer

SEQ ID



Gene
Name
Sequence
NO:
Description














budB
B1
CACCATGGACAAACAGTA
15
budB




TCCGGTACGCC

forward


 


budB
B2
CGAAGGGCGATAGCTTTA
16
budB




CCAATCC

reverse


 


budA
B3
CACCATGAATCATTCTGC
17
budA forward




TGAATGCACCTGCG




 


budA
B4
GATACTGTTTGTCCATGT
18
budA reverse




GACC




 


budC
B5
CACCATGAAAAAAGTCGC
19
budC




ACTTGTTACC

forward


 


budC
B6
TTAGTTAAATACCAT
20
budC reverse


 


pddA
B7
CACCATGAGATCGA
21
pddABC




AAAGATTTG

forward


 


pddC
B8
CTTAGAGAAGTTAATCGT
22
pddABC




CGCC

reverse


 


sadh
B9
CACCATGAAAGCCCTCCA
23
sadh




GTACACC

forward


 


sadh
B10
CGTCGTGTCATGCCCGG
24
sadh




G

reverse


 


budA
B11
GATCGAATTCGTTTAAACT
25
budABC




TAGTTTTCTACCGCACG

forward


 


budC
B12
GATCGCATGCAAGCTTTC
26
budABC




ATATAGTCGGAATTCC

reverse


 


pddA
B13
GATCGAATTCGTTTAAACA
27
pddABC




AAGGAGGTCTGATTCATG

forward




AGATCG




 


pddC
B14
GATCGGATTCTTAATCGT
28
pddABC




CGCC

reverse


 


sadh
B15
GATCGGATCCAAAGGAGG
29
sadh




TCGGGCGCATGAAAGCC

forward




C




 


sadh
B16
GATCTCTAGAAAGCTTTC
30
sadh




AGCCCGGGACGACC

reverse


 


—
BenF
ACTTTCTTTCGCCTGTTTC
31
—




AC




 


—
BenBPR
CATGAAGCTTGTTTAAACT
32
—




CGGTGACCTTGAAAATAA






TGAAAACTTATATTGTTTT






GAAAATAATGAAAACTTAT






ATTG




 


budAB
BABC F
GAGCTCGAATTCAAAGGA
33
budAB




GGAAGTGTATATGAATCA

forward




TTC




 


budAB
BAB R
GGATCCTCTAGAATTAGT
34
budAB




TAAATACCATCCCGCCG

reverse


 


budC
BC Spe F
ACTAGTAAAGGAGGAAAG
40
budC forward




AGTATGAAGAAGGTCGCA






CT




 


budC
BC Xba R
TCTAGAAAGCAGGGGCAA
41
budC reverse




GCCATGTC




 


pddAB
DDo For
AAGCTTAAAGGAGGCTGA
44
pddABC-ddrAB


C-

TTCATGAGATCGAAAAGA

forward


ddrAB

TT




 


pddAB
DDo Rev
TCTAGATTATTCATCCTGC
45
pddABC-ddrAB


C-

TGTTCTCC

reverse


ddrAB






 


chnA
ChnA F
CATCAATTGACTACGTAG
54
chnA forward




TCGTACGTGTAAGGAGGT






TTGAAATGGAAAAAATTAT






G




 


chnA
ChnA R
CATGCTAGCCCCGGGTAT
55
chnA reverse




CTTCTACTCATTTTTTATTT






CG




 


—
Top ter
CTAGAAGTCAAAAGCCTC
58
forward



F1
CGACCGGAGGCTTTTGA




 


—
Top ter
CTGCTCGAGTTGCTAGC
59
forward



F2
AAGTTTAAACAAAAAAAA






GCCCGCTCATTAGGCGG






GCTGAGCT




 


—
Bot ter
CAGCCCGCCTAATGAGC
60
reverse



R1
GGGCTTTTTTTTGTTTAA






AC




 


—
Bot ter
TTGCTAGCAACTCGAGCA
61
reverse



R2
GTCAAAAGCCTCCGGTC






GGAGGCTTTTGACTT




 


KA-AT
OT872
CTCCGGAATTCATGTCTG
127
Aminoalcohol




ACGGACGACTCACCGCA

kinase/lyase






operon






forward


KA-AT
OT873
TTCCAATGCATTGGCTGC
128
Aminoalcohol




AGTTATCTCTGTGCACGA

kinase/lyase




GTGCCGATGA

operon






reverse


 


KA
OT879
AACAGCCAAGCTTGGCT
129
Aminoalcohol




GCAGTCATCGCGCATTCT

kinase




CCGGG

reverse


 


AT
OT880
TCTCCGGAATTCATGACG
130
Aminoalcohol




TCTGAAATGACAGCGACA

lyase




GAAG

forward


 


pBAD.
OT909
GCTAACAGGAGGAAGAA
131
Adds EcoRI


HisB

TTCATGGGGGGTTCTC

site to replace






NcoI site


 


pBAD.
OT910
GAGAACCCCCCATGAATT
132
Adds EcoRI


HisB

CTTCCTCCTGTTAGC

site to replace






NcoI site


 


BudAB
N84seqR3
GGACCTGCTTCGCTTTAT
159
reverse




CG




 


APT
APTfor
GCGCGCCCGGGAAGAAG
162
APT forward




GAGCTCTTCACCATGAAC






AAACCACAGTCTTGG




 


APT
APTrev
GCGCGCCCGGGTTCATG
163
APT reverse




CCACCTCTGCG
















TABLE 5







Sequencing Primers













SEQ




Gene-
ID


Name
Sequence
specific
NO:













M13 Forward
GTAAAACGACGGCCAGT
—
35


 


M13 Reverse
AACAGCTATGACCATG
—
36


 


N83 SeqF2
GCTGGATTACCAGCTCGACC
—
37


 


N83 SeqF3
CGGACGCATTACCGGCAAAG
—
38


 


N84 SeqR2
GCATCGAGATTATCGGGATG
—
65


 


N84 SeqR4
CGAAGCGAGAGAAGTTATCC
—
39


 


Trc F
TTGACAATTAATCATCCGGC
all
42


 


Trc R
CTTCTCTCATCCGCCAAAAC
all
43


 


DDko seq F2
GCATGGCGCGGATTTGACGAAC
pddABC-
46




ddrAB



 


DDko seq F5
CATTAAAGAGACCAAGTACGTG
pddABC-
47




ddrAB



 


DDko seq F7
ATATCCTGGTGGTGTCGTCGGCGT
pddABC-
48




ddrAB



 


DDko seq F9
TCTTTGTCACCAACGCCCTGCG
pddABC-
49




ddrAB



 


DDko seq R1
GCCCACCGCGCTCGCCGCCGCG
pddABC-
50




ddrAB



 


DDko seq R3
CCCCCAGGATGGCGGCTTCGGC
pddABC-
51




ddrAB



 


DDko seq R7
GGGCCGACGGCGATAATCACTT
pddABC-
52




ddrAB



 


DDko seq R10
TTCTTCGATCCACTCCTTAACG
pddABC-
53




ddrAB



 


chnSeq F1
CTCAACAGGGTGTAAGTGTAGT
chnA
56


 


chnSeq R1
CGTTTTGATATAGCCAGGATGT
chnA
57


 


pCL1925 vec F
CGGTATCATCAACAGGCTTACC
all
62


 


pCL1925 vec R1
AGGGTTTTCCCAGTCACGACGT
all
63


 


pCL1925 vec R2
CGCAATAGTTGGCGAAGTAATC
all
64


 


APTseqRev
GCTAGAGATGATAGC
APT
160


 


APTseqFor
GGAAGAGACTATCCAGCG
APT
161
















TABLE 15







Sequencing and PCR primers used in Examples 19-22









Name
SEQ ID NO
function





Trc99a_For
285
checking construction of pTrc99a::rdhtAB


Trc99a_Rev
286
checking construction of pTrc99a::rdhtAB


N712
287
checking construction of pCL 1925::RdhtAB


N713
288
checking construction of pCL 1925::RdhtAB


N473
291
amplification of sadB


N469
292
amplification of sadB


N695
293
amplification of RdhtA


N696
294
amplification of RdhtA


N697
295
amplification of RdhtB


N698
296
amplification of RdhtB


N742A
302
amplification of FBA-RdhtA + GPM-RdhtB


N743A
303
amplification of FBA-RdhtA + GPM-RdhtB


N657
304
amplification of the MET 15 marker


N653A
305
amplification of the MET 15 marker


N579
306
amplification of budA


N480
307
amplification of budA


N98SeqF1
311
Screening for alsS


N99SeqR2
312
Screening for alsS


N160SegF1
313
Screening for budA


N84SeqR2
314
Screening for budA


N581
315
amplification of budC


N582
316
amplification of budC


N583
319
amplification of sadB


N584
320
amplification of sadB


N142
322
checking construction of




pRS425::GPM-sadB


N459
323
checking construction of




pRS425::GPM-sadB









Methods for Determining 2-Butanol and 2-Butanone Concentration in Culture Media

The concentration of 2-butanol and 2-butanone in the culture media can be determined by a number of methods known in the art. For example, a specific high performance liquid chromatography (HPLC) method utilized a Shodex SH-1011 column with a Shodex SH-G guard column, both purchased from Waters Corporation (Milford, Mass.), with refractive index (RI) detection. Chromatographic separation was achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Under the conditions used, 2-butanone and 2-butanol had retention times of 39.5 and 44.3 min, respectively. Alternatively, gas chromatography (GC) methods are available. For example, a specific GC method utilized an HP-INNOWax column (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies, Wilmington, Del.), with a flame ionization detector (FID). The carrier gas was helium at a flow rate of 4.5 mL/min, measured at 150° C. with constant head pressure; injector split was 1:25 at 200° C.; oven temperature was 45° C. for 1 min, 45 to 220° C. at 10° C./min, and 220° C. for 5 min; and FID detection was employed at 240° C. with 26 mL/min helium makeup gas. The retention times of 2-butanone and 2-butanol were 3.61 and 5.03 min, respectively.
2-Butanone can also be detected by derivatization with 3-methyl-2-benzothiazolinone hydrazone (MBTH). An aqueous solution containing 2-butanone is mixed with an equal volume of an aqueous solution of 6 mg/mL MBTH in 375 mM glycine-HCl (pH 2.7) and incubated at 100° C. for 3 min. The resulting MBTH-derivatized samples are analyzed on a 25 cm×4.6 mm (id) Supelosil LC-18-D5 5 μm column (Supelco) using a mobile phase of 55% acetonitrile in water at a flow rate of 1 mL/min. The 2-butanone derivative appears as two peaks (cis and trans isomers) with retention times of approximately 12.3 and 13.3 min and absorbance maxima of 230 and 307 nm.
The meaning of abbreviations is as follows: “s” means second(s), “min” means minute(s), “h” means hour(s), “psi” means pounds per square inch, “nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” means nanometers, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmol” means micromole(s)”, “g” means gram(s), “μg” means microgram(s) and “ng” means nanogram(s), “PCR” means polymerase chain reaction, “OD” means optical density, “OD600” means the optical density measured at a wavelength of 600 nm, “kDa” means kilodaltons, “g” means the gravitation constant, “bp” means base pair(s), “kbp” means kilobase pair(s), “% w/v” means weight/volume percent, % v/v” means volume/volume percent, “wt %” means percent by weight, “HPLC” means high performance liquid chromatography, and “GC” means gas chromatography. The term “molar selectivity” is the number of moles of product produced per mole of sugar substrate consumed and is reported as a percent.
Example 1
Cloning and Expression of Acetolactate Synthase
The purpose of this Example was to clone and express in E. coli the budB gene that encodes the enzyme acetolactate synthase. The budB gene was amplified from Klebsiella pneumoniae strain ATCC 25955 genomic DNA using PCR.
The budB sequence which encodes acetolactate synthase was amplified from Klebsiella pneumoniae (ATCC 25955) genomic DNA by PCR using the primer pair B1 (SEQ ID NO:15) and B2 (SEQ ID NO:16). Other PCR amplification reagents (e.g. Kod HiFi DNA Polymerase (Novagen Inc., Madison, Wis.; catalog no. 71805-3)) were supplied in manufacturers' kits and used according to the manufacturer's protocol. Klebsiella pneumoniae genomic DNA was prepared using the Gentra Puregene Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.; catalog number D-5000A). Amplification was carried out in a DNA Thermocycler GeneAmp 9700 (PE Applied Biosystems, Foster city, CA). The nucleotide sequence of the open reading frame (ORF) and the predicted amino acid sequence of the enzyme are given as SEQ ID NO:3 and SEQ ID NO:4, respectively.
For expression studies the Gateway cloning technology (Invitrogen Corp., Carlsbad, Calif.) was used. The entry vector pENTR/SD/D-TOPO allows directional cloning and provided a Shine-Dalgarno sequence for the gene of interest. The destination vector pDEST14 used a T7 promoter for expression of the gene with no tag. The forward primer incorporated four bases (CACC) immediately adjacent to the translational start codon to allow directional cloning of the budB acetolactate synthase coding region PCR product into pENTR/SD/D-TOPO (Invitrogen), generating the plasmid pENTRSDD-TOPObudB. The pENTR construct was transformed into E. Coli Top10 (Invitrogen) cells and plated according to the manufacturer's recommendations. Transformants were grown overnight and plasmid DNA was prepared using the QIAprep Spin Miniprep kit (Qiagen, Valencia, Calif.; catalog no. 27106) according to the manufacturer's recommendations. To create an expression clone, the budB coding region from PENTRSDD-TOPObudB was transferred to the pDEST 14 vector by in vitro recombination using the LR Clonase mix (Invitrogen, Corp., Carlsbad, Calif.). The resulting vector, pDEST14budB, was transformed into BL-21-AI cells (Invitrogen Corp.). BL-21-AI cells carry a chromosomal copy of the T7 RNA polymerase under control of the arabinose-inducible araBAD promoter.
Transformants are inoculated into LB medium supplemented with 50 μg/mL of ampicillin and grown overnight. An aliquot of the overnight culture is used to inoculate 50 mL of LB medium supplemented with 50 μg/mL of ampicillin. The culture is incubated at 37° C. with shaking until the OD600 reaches 0.6-0.8. The culture is split into two 25-mL portions and arabinose is added to one of the flasks to a final concentration of 0.2% w/v. The negative control flask is not induced with arabinose. The flasks are incubated for 4 h at 37° C. with shaking. Cells are harvested by centrifugation and the cell pellets are resuspended in 50 mM MOPS, pH 7.0 buffer. The cells are disrupted either by sonication or by passage through a French Pressure Cell. Each cell lysate is centrifuged yielding the supernatant and the pellet or the insoluble fraction. An aliquot of each fraction (whole cell lysate, from induced and control cells, is resuspended in SDS (MES) loading buffer (Invitrogen), heated to 85° C. for 10 min and subjected to SDS-PAGE analysis (NuPAGE 4-12% Bis-Tris Gel, catalog no. NP0322Box, Invitrogen). A protein of the expected molecular weight, as deduced from the nucleic acid sequence, is present in the induced culture but not in the uninduced control.
Acetolactate synthase activity in the cell free extracts is measured using the method described by Bauerle et al. (Bauerle et al. (1964) Biochim. Biophys. Acta 92:142-149). Protein concentration is measured by either the Bradford method or by the Bicinchoninic Kit (Sigma, catalog no. BCA-1; St. Louis, Mo.) using Bovine serum albumin (BSA) (Bio-Rad, Hercules, Calif.) as the standard.
Example 2
Cloning and Expression of Acetolactate Decarboxylase
The purpose of this Example was to clone and express in E. coli the budA gene that encodes the enzyme acetolactate decarboxylase. The budA gene was amplified from Klebsiella pneumoniae strain ATCC 25955 genomic DNA using PCR.
The budA sequence which encodes acetolactate decarboxylase, was cloned in the same manner as described for budB in Example 1, except that the primers used for PCR amplification were B3 (SEQ ID NO:17) and B4 (SEQ ID NO:18). The nucleotide sequence of the open reading frame (ORF) and the predicted amino acid sequence of the enzyme are given as SEQ ID NO:1 and SEQ ID NO:2, respectively. The resulting plasmid was named pENTRSDD-TOPObudA.
Acetolactate decarboxylase activity in the cell free extracts is measured using the method described by Bauerle et al., supra.
Example 3
Prophetic
Cloning and Expression of Butanediol Dehydrogenase
The purpose of this prophetic Example is to describe how to clone and express in E. coli the budC gene that encodes the enzyme butanediol dehydrogenase. The budC gene is amplified from Klebsiella pneumoniae strain IAM1063 genomic DNA using PCR.
The budC sequence encoding butanediol dehydrogenase is cloned and expressed in the same manner as described for budA in Example 1, except that the primers used for PCR amplification are B5 (SEQ ID NO:19) and B6 (SEQ ID NO:20) and the genomic template DNA is from Klebsiella. pneumoniae IAM1063 (which is obtained from the Institute of Applied Microbiology Culture Collection, Tokyo, Japan). Klebsiella pneumoniae IAM1063 genomic DNA is prepared using the Gentra Puregene Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.; catalog number D-5000A). The nucleotide sequence of the open reading frame (ORF) and the predicted amino acid sequence of the enzyme are given as SEQ ID NO:5 and SEQ ID NO:6, respectively.
Butanediol dehydrogenase activity in the cell free extracts is measured spectrophotometrically by following NADH consumption at an absorbance of 340 nm.
Example 4
Prophetic
Cloning and Expression of Butanediol Dehydratase
The purpose of this prophetic Example is to describe how to clone and express in E. coli the pddA, pddB and pddC genes that encode butanediol dehydratase. The pddA, pddB and pddC genes are amplified from Klebsiella oxytoca ATCC 8724 genomic DNA using PCR.
The pddA, pddB and pddC sequences which encode butanediol dehydratase are cloned and expressed in the same manner as described for budA in Example 1, except that the genomic template DNA is from Klebsiella oxytoca ATCC 8724, and the primers are B7 (SEQ ID NO:21) and B8 (SEQ ID NO:22). Klebsiella oxytoca genomic DNA is prepared using the Gentra Puregene Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.; catalog number D-5000A). A single PCR product including all three open reading frames (ORFs) is cloned, so that all three coding regions are expressed as an operon from a single promoter on the expression plasmid. The nucleotide sequences of the open reading frames for the three subunits are given as SEQ ID NOs:7, 9, and 11, respectively, and the predicted amino acid sequences of the three enzyme subunits are given as SEQ ID NOs:8, 10, and 12, respectively.
Butanediol dehydratase activity in the cell free extracts is measured by derivatizing the ketone product with 2,4-dinitrophenylhydrazine (DNPH). Briefly, 100 μL of reaction mixture, cell extract containing approximately 0.0005 units of enzyme, 40 mM potassium phosphate buffer (pH 8.0), 2 μg of adenosylcobalamin, 5 μg of 2,3,-butanediol, and 1 μg of bovine serum albumin, is quenched by addition of an equal volume of 0.05 wt % DNPH in 1.0 N HCl. After 15 min at room temperature, the color is developed by addition of 100 μL of 4 N NaOH. The amount of product is determined from the absorbance of the final solution at 550 nm compared to a standard curve prepared with 2-butanone. All reactions are carried out at 37° C. under dim red light.
Example 5
Prophetic
Cloning and Expression of Butanol Dehydrogenase
The purpose of this prophetic Example is to describe how to clone and express in E. coli the sadh gene that encodes butanol dehydrogenase. The sadh gene is amplified from Rhodococcus ruber strain 219 genomic DNA using PCR.
The sadh sequence encoding butanol dehydrogenase is cloned and expressed in the same manner as described for budA in Example 1, except that the genomic template DNA is from Rhodococcus ruber strain 219 (Meens, Institut fuer Mikrobiologie, Universitaet Hannover, Hannover, Germany) and the primers are B9 (SEQ ID NO:23) and B10 (SEQ ID NO:24). Rhodococcus ruber genomic DNA is prepared using the Ultra CleanTM Microbial DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, Calif.), according to the manufacturer's protocol. The nucleotide sequence of the open reading frame (ORF) and the predicted amino acid sequence of the enzyme are given as SEQ ID NO:13 and SEQ ID NO:14, respectively.
Butanol dehydrogenase activity in cell free extracts is measured by following the increase in absorbance at 340 nm resulting from the conversion of NAD to NADH when the enzyme is incubated with NAD and 2-butanol.
Example 6
Prophetic
Construction of a Transformation Vector for the Genes in a 2-Butanol Biosynthetic Pathway
The purpose of this prophetic Example is to describe the preparation of a transformation vector for the genes in a 2-butanol biosynthetic pathway (i.e., Pathway 3 as described above). Like most organisms, E. coli converts glucose initially to pyruvic acid. The enzymes required to convert pyruvic acid to 2-butanol following Pathway 3, i.e., acetolactate synthase, acetolactate decarboxylase, butanediol dehydrogenase, butanediol dehydratase, and butanol dehydrogenase, are encoded by the budA, budB, budC, pddA, pddB, pddC and sadh genes. To simplify building the 2-butanol biosynthetic pathway in a recombinant organism, the genes encoding the 5 steps in the pathway are divided into two operons. The upper pathway comprises the first three steps catalyzed by acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase. The lower pathway comprises the last two steps catalyzed by butanediol dehydratase and butanol dehydrogenase.
The coding sequences are amplified by PCR with primers that incorporate restriction sites for later cloning, and the forward primers contain an optimized E. coli ribosome binding site (AAAGGAGG). PCR products are TOPO cloned into the pCR4Blunt-TOPO vector and transformed into Top10 cells (Invitrogen). Plasmid DNA is prepared from the TOPO clones, and the sequence of the cloned PCR fragment is verified. Restriction enzymes and T4 DNA ligase (New England Biolabs, Beverly, Mass.) are used according to manufacturer's recommendations. For cloning experiments, restriction fragments are gel-purified using QIAquick Gel Extraction kit (Qiagen).
After confirmation of the sequence, the coding regions are subcloned into a modified pUC19 vector as a cloning platform. The pUC19 vector is modified by a HindIII/SapI digest, followed by treatment with Klenow DNA polymerase to fill in the ends. The 2.4 kB vector fragment is gel-purified and religated creating pUC19dHS. Alternatively the pUC19 vector is modified by a SphI/SapI digest, followed by treatment with Klenow DNA polymerase to blunt the ends. The 2.4 kB vector fragment is gel-purified and religated creating pUC19dSS. The digests remove the lac promoter adjacent to the MCS (multiple cloning sites), preventing transcription of the operons from the vector.
Upper Pathway:
The budABC coding regions are amplified from Klebsiella pneumoniae genomic DNA by PCR using primer pair B11 and B12 (Table 4), given as SEQ ID NOs:25 and 26, respectively. The forward primer incorporates an EcoRI restriction site and a ribosome binding site (RBS). The reverse primer incorporates an SphI restriction site. The PCR product is cloned into pCR4 Blunt-TOPO creating pCR4 Blunt-TOPO-budABC.
To construct the upper pathway operon pCR4 Blunt-TOPO-budABC is digested with EcoRI and SphI releasing a 3.2 kbp budABC fragment. The pUC19dSS vector is also digested with EcoRI and SphI, releasing a 2.0 kbp vector fragment. The budABC fragment and the vector fragment are ligated together using T4 DNA ligase (New England Biolabs) to form pUC19dSS-budABC.
Lower Pathway:
The pddABC coding regions are amplified from Klebsiella oxytoca ATCC 8724 genomic DNA by PCR using primers B13 and B14 (Table 4), given as SEQ ID NOs:27 and 28, respectively, creating a 2.9 kbp product. The forward primer incorporates EcoRI and PmeI restriction sites and a RBS. The reverse primer incorporates the BamHI restriction site. The PCR product is cloned into pCRBluntII-TOPO creating pCRBluntII-pdd.
The sadh gene is amplified from Rhodococcus ruber strain 219 genomic DNA by PCR using primers B15 and B16 (Table 4), given as SEQ ID NOs:29 and 30, respectively, creating a 1.0 kbp product. The forward primer incorporates a BamHI restriction site and a RBS. The reverse primer incorporates an XbaI restriction site. The PCR product is cloned into pCRBlunt II-TOPO creating pCRBluntII-sadh.
To construct the lower pathway operon, a 2.9 kbp EcoRI and BamHI fragment from pCRBluntII-pdd, a 1.0 kbp BamHI and XbaI fragment from pCRBluntII-sadh, and the large fragment from an EcoRI and XbaI digest of pUC19dHS are ligated together. The three-way ligation creates pUC19dHS-pdd-sadh.
The pUC19dSS-budABC vector is digested with PmeI and HindIII, releasing a 3.2 kbp fragment that is cloned into pBenBP, an E. coli-B. subtilis shuttle vector. Plasmid pBenBP is created by modification of the pBE93 vector, which is described by Nagarajan (WO 93/2463, Example 4). To generate pBenBP, the Bacillus amyloliquefaciens neutral protease promoter (NPR) signal sequence and the phoA gene are removed from pBE93 with an NcoI/HindIII digest. The NPR promoter is PCR amplified from pBE93 by primers BenF and BenBPR, given by SEQ ID NOs:31 and 32, respectively. Primer BenBPR incorporates BstEII, PmeI and HindIII sites downstream of the promoter. The PCR product is digested with NcoI and HindIII, and the fragment is cloned into the corresponding sites in the vector pBE93 to create pBenBP. The upper operon fragment is subcloned into the PmeI and HindIII sites in pBenBP creating pBen-budABC.
The pUC19dHS-pdd-sadh vector is digested with PmeI and HindIII releasing a 3.9 kbp fragment that is cloned into the PmeI and HindIII sites of pBenBP, creating pBen-pdd-sadh.
Example 7
Prophetic
Expression of a 2-Butanol Biosynthetic Pathway in E. coli 
The purpose of this prophetic Example is to describe how to express a 2-butanol biosynthetic pathway in E. coli. 
The plasmids pBen-budABC and pBen-pdd-sadh, prepared as described in Example 6, are separately transformed into E. coli NM522 (ATCC No. 47000), and expression of the genes in each operon is monitored by SDS-PAGE analysis and enzyme assay. After confirmation of expression of all genes, pBen-budABC is digested with EcoRI and HindIII to release the NPR promoter-budABC fragment. The fragment is blunt ended using the Klenow fragment of DNA polymerase (New England Biolabs, catalog no. M0210S). The plasmid pBen-pdd-sadh is digested with EcoRI and similarly blunted to create a linearized, blunt-ended vector fragment. The vector and NPR-budABC fragments are ligated, creating p2BOH. This plasmid is transformed into E. coli NM522 to give E. coli NM522/p2BOH, and expression of the genes is monitored as previously described.
E. coli NM522/p2BOH is inoculated into a 250 mL shake flask containing 50 mL of medium and shaken at 250 rpm and 35° C. The medium is composed of: dextrose, 5 g/L; MOPS, 0.05 M; ammonium sulfate, 0.01 M; potassium phosphate, monobasic, 0.005 M; S10 metal mix, 1% (v/v); yeast extract, 0.1% (w/v); casamino acids, 0.1% (w/v); thiamine, 0.1 mg/L; proline, 0.05 mg/L; and biotin 0.002 mg/L, and is titrated to pH 7.0 with KOH. S10 metal mix contains: MgCl2, 200 mM; CaCl2, 70 mM; MnCl2, 5 mM; FeCl3, 0.1 mM; ZnCl2, 0.1 mM; thiamine hydrochloride, 0.2 mM; CuSO4, 172 μM; COCl2, 253 μM; and Na2MoO4, 242 μM. After 18 h, 2-butanol is detected by HPLC or GC analysis using methods that are well known in the art, for example, as described in the General Methods section above.
Example 8
Prophetic
Expression of a 2-Butanol Biosynthetic Pathway in Bacillus subtilis 
The purpose of this prophetic Example is to describe how to express a 2-butanol biosynthetic pathway in Bacillus subtilis. 
The plasmids pBen-budABC and pBen-pdd-sadh, prepared as described in Example 6, are separately transformed into Bacillus subtilis BE1010 (J. Bacteriol. 173:2278-2282 (1991)) and expression of the genes in each operon is monitored as described in Example 7. The plasmid pBen-budABC is digested with EcoRI and HindIII to release the NPR promoter-budABC fragment. The fragment is blunt ended using the Klenow fragment of DNA polymerase (New England Biolabs, catalog no. M0210S). The plasmid pBen-pdd-sadh is digested with EcoRI and similarly blunted to create a linearized, blunt-ended vector fragment. The vector and NPR-budABC fragments are ligated, creating p2BOH. This plasmid is transformed into Bacillus subtilis BE1010 to give Bacillus subtilis BE1010/p2BOH, and expression of the genes is monitored as previously described.
Bacillus subtilis BE1010/p2BOH is inoculated into a 250 mL shake flask containing 50 mL of medium and shaken at 250 rpm and 35° C. for 18 h. The medium is composed of: dextrose, 5 g/L; MOPS, 0.05 M; glutamic acid, 0.02 M; ammonium sulfate, 0.01 M; potassium phosphate, monobasic buffer, 0.005 M; S10 metal mix (as described in Example 7), 1% (v/v); yeast extract, 0.1% (w/v); casamino acids, 0.1% (w/v); tryptophan, 50 mg/L; methionine, 50 mg/L; and lysine, 50 mg/L, and is titrated to pH 7.0 with KOH. After 18 h, 2-butanol is detected by HPLC or GC analysis using methods that are well known in the art, for example, as described in the General Methods section above.
Example 9
Construction of a Transformation Vector for the Genes in a 2-Butanol Biosynthetic Pathway
The purpose of this Example was to prepare a recombinant E. coli host carrying the genes in a 2-butanol biosynthetic pathway (i.e., Pathway 3 as described above). Like most organisms, E. coli converts glucose initially to pyruvic acid. The enzymes required to convert pyruvic acid to 2-butanone in Pathway 3, i.e., acetolactate synthase, acetolactate decarboxylase, butanediol dehydrogenase, and butanediol dehydratase are encoded by the budA, budB, budC, pddA, pddB, and pddC genes. In the last step of the pathway, a butanol dehydrogenase converts 2-butanone to 2-butanol. Dehydrogenases that carry out this last step are promiscuous and may be found in many organisms. To simplify building the 2-butanol biosynthetic pathway in a recombinant organism, the genes encoding the 5 steps in the pathway were divided into multiple operons. The upper pathway operon comprised the first three steps catalyzed by acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase and were cloned onto an expression vector. The lower pathway comprised the last two steps catalyzed by butanediol dehydratase including the reactivating factor (Mori et al., J. Biol. Chem. 272:32034 (1997)) and a butanol dehydrogenase. The diol dehydratase can undergo suicide inactivation during catalysis. The reactivating factor protein encoded by ddrA and ddrB (GenBank AF017781, SEQ ID NO:70) reactivates the inactive enzyme. The ddrA and ddrB genes flank the diol dehydratase operon. The operons for the dehydratase/reactivating factor and the butanol dehydrogenase were either cloned onto another expression vector or the dehydratase/reactivating factor operon was cloned singly onto another expression vector and the last step was provided by an endogenous activity in the demonstration host.
Construction of Vector pTrc99a-budABC
The budAB coding regions were amplified from K. pneumoniae ATCC 25955 genomic DNA by PCR using primer pair BABC F and BAB R, given as SEQ ID NOs:33 and 34, respectively (see Table 4), creating a 2.5 kbp product. The forward primer incorporated SacI and EcoRI restriction sites and a ribosome binding site (RBS). The reverse primer incorporated a SpeI restriction site. The PCR product was cloned into pCR4 Blunt-TOPO creating pCR4 Blunt-TOPO-budAB. Plasmid DNA was prepared from the TOPO clones and the sequence of the genes was verified with primers M13 Forward (SEQ ID NO:35), M13 Reverse (SEQ ID NO:36), N83 SeqF2 (SEQ ID NO:37), N83 SeqF3 (SEQ ID NO:38) and N84 SeqR4 (SEQ ID NO:39) (see Table 5).
The budC coding region was amplified from K. pneumoniae ATCC 25955 genomic DNA by PCR using primer pair BC Spe F and BC Xba R given as SEQ ID NOs:40 and 41, respectively, creating a 0.8 kbp product. The forward primer incorporated a SpeI restriction site, a RBS and modified the CDS by changing the second and third codons from AAA to AAG. The reverse primer incorporated an XbaI restriction site. The PCR product was cloned into pCR4 Blunt-TOPO creating pCR4 Blunt-TOPO-budC. Plasmid DNA was prepared from the TOPO clones and the sequence of the genes was verified with primers M13 Forward (SEQ ID NO:35) and M13 Reverse (SEQ ID NO:36).
To construct the budABC operon, pCR4 Blunt-TOPO-budC was digested with SnaBI and XbaI releasing a 1.0 kbp budC fragment. The vector pTrc99a (Amann et al., Gene 69(2):301-315 (1988)) was digested with SmaI and XbaI creating a 4.2 kbp linearized vector fragment. The vector and the budC fragment were ligated to create pTrc99a-budC and transformed into E. coli Top 10 cells (Invitrogen). Transformants were analyzed by PCR amplification with primers Trc F (SEQ ID NO:42) and Trc R (SEQ ID NO:43) for a 1.2 kbp product to confirm the presence of the budC insert. The budAB genes were subcloned from pCR4 Blunt-TOPO-budAB as a 2.5 kbp EcoRI/SpeI fragment. Vector pTrc99a-budC was digested with EcoRI and SpeI and the resulting 5.0 kbp vector fragment was gel-purified. The purified vector and budAB insert were ligated and transformed into E. coli Top 10 cells. Transformants were screened by PCR amplification with primers Trc F (SEQ ID NO:42) and N84 Seq R2 (SEQ ID NO:65) to confirm creation of pTrc99a-budABC. In this plasmid, the bud A, B, and C coding regions are adjacent to each other, in this order, and between the Trc promoter and the rrnB termination sequence.
Results:
Three independent isolates of E. coli Top 10/pTrc99a-budABC were examined for the production of butanediol, using E. coli Top 10/pCL1925-Kodd-ddr (described below) as a negative control. The strains were grown in LB medium containing 100 μg/mL carbenicillin. The resulting cells were used to inoculate shake flasks (approximately 175 mL total volume) containing 125 mL of TM3a/glucose medium with 100 μg/mL carbenicillin. In addition, the flasks inoculated with strains carrying pTrc99a-budABC contained 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). TM3a/glucose medium contains (per liter): 10 g glucose, 13.6 g KH2PO4, 2.0 g citric acid monohydrate, 3.0 g (NH4)2SO4, 2.0 g MgSO4.7H2O, 0.2 g CaCl2.2H2O, 0.33 g ferric ammonium citrate, 1.0 mg thiamine HCl, 0.50 g yeast extract, and 10 mL trace elements solution, adjusted to pH 6.8 with NH4OH. The solution of trace elements contained: citric acid H2O (4.0 g/L), MnSO4.H2O (3.0 g/L), NaCl (1.0 g/L), FeSO4.7H2O (0.10 g/L), COCl2.6H2O (0.10 g/L), ZnSO4.7H2O (0.10 g/L), CuSO4.5H2O (0.010 g/L), H3BO3 (0.010 g/L), and Na2MoO4.2H2O (0.010 g/L). The flasks, capped with vented caps, were inoculated at a starting OD600 of approximately 0.03 units and incubated at 34° C. with shaking at 300 rpm.
Approximately 23 h after induction, an aliquot of the broth was analyzed by HPLC (Shodex Sugar SH1011 column) and GC (HP-INNOWax), using the same methods described in the General Methods section for 2-butanol and 2-butanone. The results of the analysis are given in Table 6. The three E. coli clones converted glucose to acetoin and meso-2,3-butanediol, the desired intermediates of the pathway, with a molar selectivity of 14%. This selectivity was approximately 35-fold higher than that observed with the E. coli control strain lacking budABC.







TABLE 6







Production of Acetoin and meso-2,3-butanediol


by E. coli Top 10/pTrc99a-budABC














Meso-2,3-






Butanediol,
Molar


Strain
OD600
Acetoin, mM
mM
Selectivitya, %














Negative
1.4
0.07
0.03
0.4


control






Isolate #1
1.5
0.64
1.3
14


Isolate #2
1.4
0.70
1.2
14


Isolate #3
1.4
0.74
1.3
15





aMolar selectivity is (acetoin + meso-2,3-butanendiol)/(glucose consumed).






Construction of vector pCL 1925-KoDD-ddr
The diol dehydratase (GenBank D45071, SEQ ID NO:69) and reactivating factor (GenBank AF017781, SEQ ID NO:70) operons were PCR amplified from Klebsiella oxytoca ATCC 8724 as a single unit with primers DDo For (SEQ ID NO: 44) and DDo Rev (SEQ ID NO:45). The forward primer incorporated an optimized E. coli RBS and a HindIII restriction site. The reverse primer included an XbaI restriction site. The 5318 bp PCR product was cloned into pCR4Blunt-TOPO and clones of the resulting pCR4Blunt-TOPO-Kodd-ddr were sequenced with primers M13 Forward (SEQ ID NO:35), M13 Reverse (SEQ ID NO:36), DDko seq F2 (SEQ ID NO:46), DDko seq F5 (SEQ ID NO:47), DDko seq F7 (SEQ ID NO:48), DDko seq F9 (SEQ ID NO:49), DDko seq R1 (SEQ ID NO:50), DDko seq R3 (SEQ ID NO:51), DDko seq R7 (SEQ ID NO:52), and DDko seq R10 (SEQ ID NO:53). A clone having the insert with the expected sequence was identified.
For expression, the diol dehydratase/reactivating factor genes were subcloned into pCL1925 (U.S. Pat. No. 7,074,608), a low copy plasmid carrying the glucose isomerase promoter from Streptomyces. pCR4Blunt-TOPO-Kodd-ddr was digested with HindIII and XbaI and the resulting 5.3 kbp Kodd-ddr fragment was gel-purified. Vector pCL 1925 was digested with HindIII and XbaI and the resulting 4539 bp vector fragment was gel purified. The vector and Kodd-ddr fragment were ligated and transformed into E. coli Top10. Transformants were screened by PCR with primers DDko Seq F7 (SEQ ID NO:48) and DDko Seq R7 (SEQ ID NO: 52). Amplification of the plasmid (pCL1925-Kodd-ddr) carrying the insert resulted in a product of approximately 797 bp.
Activity of diol dehydratase towards meso-2,3-butanediol was measured by incubating cell extract (total protein  ̃0.8 mg/mL) with 10 mM butanediol and 12 mM coenzyme B12 in 80 mM HEPES (pH 8.2) for 17 h at room temperature. Formation of the expected product, 2-butanone, was determined by HPLC as described in the General Methods.
Construction of vector pCL1925-KoDD-ddr::T5 chnA ter
To provide a heterologous alcohol dehydrogenase activity, the chnA gene encoding cyclohexanol dehydrogenase from Acinetobacter sp. (Cheng et al., J. Bacteriol. 182:4744-4751 (2000)) was cloned into the pCL1925 vector with the diol dehydratase operon, pCL1925-Kodd-ddr. The chnA gene, given as SEQ ID NO:71 (Genbank No: AF282240, SEQ ID NO:73) was amplified from pDCQ2, a cosmid carrying the cyclohexanol gene cluster from Acinetobacter, with primers ChnA F (SEQ ID NO:54) and ChnA R (SEQ ID NO:55). The resulting 828 bp PCR product was cloned into pCR4Blunt-TOPO to create pCR4Blunt-TOPO-chnA and transformants were screened by colony PCR with primers M13 Forward (SEQ ID NO:35) and M13 Reverse (SEQ ID NO:36). Correct clones produced a PCR product of about 1 kbp and were sequenced with primers M13 Forward (SEQ ID NO:35) and M13 Reverse (SEQ ID NO:36).
After sequencing pCR4Blunt-TOPO-chnA to confirm the correct sequence, the chnA gene was subcloned from the plasmid as an 813 bp MfeI/SmaI fragment. The expression vector pQE30 (Qiagen) was digested with MfeI and SmaI and the resulting 3350 bp vector fragment was gel-purified. The chnA fragment and the purified vector were ligated and transformed into E. coli Top10 cells. Transformants were colony PCR screened with primers chnseq F1 (SEQ ID NO:56) and chnseq R1 (SEQ ID NO:57) for a 494 bp PCR product. This cloning placed the chnA gene under the control of the T5 promoter in the plasmid, pQE30-chnA.
To prepare the pCL1925 vector to carry two operons, terminators were added to the vector. A tonB terminator-mcs-trpA terminator fragment was prepared by oligonucleotide annealing with primers Top ter F1 (SEQ ID NO:58), Top ter F2 (SEQ ID NO:59), Bot ter R1 (SEQ ID NO:60) and Bot ter R2 (SEQ ID NO:61). The annealed DNA was gel-purified on a 6% PAGE gel (Embi-tec, San Diego, Calif.). Vector pCL1925 was digested with SacI and XbaI and gel-purified. The annealed DNA and vector fragment were ligated to create pCL1925-ter. Transformants were screened by colony PCR amplification with primers pCL1925 vec F (SEQ ID NO:62) and pCL 1925 vec R1 (SEQ ID NO:63) for the presence of a PCR product of approximately 400 bp. Positive clones from the PCR screen were sequenced with the same primers.
Vector pCL 1925-ter was digested with XhoI and PmeI and the resulting 4622 bp fragment was gel-purified. pQE30-chnA was digested with NcoI and the DNA was treated with Klenow DNA polymerase to blunt the ends. pQE30-chnA was then digested with XhoI and the resulting 1.2 kbp T5 promoter-chnA fragment was gel-purified. The pCL1925-ter vector and the chnA operon fragment were ligated together to give pCL1925-ter-T5chnA and transformed into E. coli Top10. Transformants were screened by colony PCR amplification with primers pCL1925 vec F (SEQ ID NO:64) and chnseq R1 (SEQ ID NO:59) for a product of approximately 1 kbp.
To finish building the pathway vector, the pCL1925-KoDD-ddr plasmid was digested with XbaI and SacI and the resulting 9504 bp vector fragment was gel-purified. The chnA operon flanked by terminators, with the trpA terminator (Koichi et al. (1997) Volume 272, Number 51, pp. 32034-32041) 3′ to the chnA coding sequence, from pCL1925-ter-T5chnA was gel-purified as a 1271 bp XbaI/SacI fragment. After ligation of the fragments and transformation into E. coli Top10, transformants were screened by colony PCR. Primers chnseq F1 (SEQ ID NO:58) and pCL1925 vec R2 (SEQ ID NO:64) amplified the expected 1107 bp PCR product in the resulting plasmid, pCL1925-KoDD-ddr::ter-T5chnA.
Example 10
Expression of a 2-Butanol Biosynthetic Pathway in E. coli with Overexpressed Endogenous Alcohol Dehydrogenase
The purpose of this Example was to express a 2-butanol biosynthetic pathway in several E. coli strains.
Construction of E. coli Strains Constitutively Expressing yqhD:
E. coli contains a native gene (yqhD) that was identified as a 1,3-propanediol dehydrogenase (U.S. Pat. No. 6,514,733). The yqhD gene, given as SEQ ID NO:74, has 40% identity to the gene adhB in Clostridium, a probable NADH-dependent butanol dehydrogenase. The yqhD gene was placed under the constitutive expression of a variant of the glucose isomerase promoter 1.6GI (SEQ ID NO:67) in E. coli strain MG1655 1.6yqhD::Cm (WO 2004/033646) using λ Red technology (Datsenko and Wanner, Proc. Natl. Acad. Sci. U.S.A. 97:6640 (2000)). Similarly, the native promoter was replaced by the 1.5GI promoter (WO 2003/089621) (SEQ ID NO:68), creating strain MG1655 1.5yqhD::Cm, thus, replacing the 1.6GI promoter of MG1655 1.6yqhD::Cm with the 1.5GI promoter. The 1.5GI and 1.6GI promoters differ by 1 bp in the −35 region, thereby altering the strength of the promoters (WO 2004/033646). While replacing the native yqhD promoter with either the 1.5GI or 1.6GI promoter, the yqhC gene encoding the putative transcriptional regulator for the yqh operon was deleted. Butanol dehydrogenase activity was confirmed by enzyme assay using methods that are well known in the art.
Transformation of E. coli Strains:
Pathway plasmids pCL1925-Kodd-ddr and pTrc99a-budABC, described in Example 9, were co-transformed into E. coli strains MG1655, MG1655 1.6yqhD, and MG1655 1.5yqhD. The two latter strains overexpress the 1,3-propanediol dehydrogenase, YqhD, which also has butanol dehydrogenase activity. Strains were examined for the production of 2-butanone and 2-butanol essentially as described above. Cells were inoculated into shake flasks (approximately 175 mL total volume) containing either 50 or 150 mL of TM3a/glucose medium (with 0.1 mg/L vitamin B12, appropriate antibiotics and IPTG) to represent medium and low oxygen conditions, respectively. Spectinomycin (50 μg/mL) and carbenicillin (100 μg/mL) were used for plasmids pCL1925-Kodd-ddr and pTrc99a-budABC, respectively. The flasks were inoculated at a starting OD600 of ≦0.04 units and incubated at 34° C. with shaking at 300 rpm. The flasks containing 50 mL of medium were capped with vented caps; the flasks containing 150 mL, were capped with non-vented caps to minimize air exchange. IPTG was present at time zero at a concentration of zero or 0.04 mM. Analytical results for 2-butanone and 2-butanol production are presented in Table 7. All the E. coli strains comprising a 2-butanol biosynthetic pathway produced 2-butanone under low and medium oxygen conditions and produced 2-butanol under low oxygen conditions.







TABLE 7







Production of 2-Butanone and 2-Butanol by E. coli MG1655 strains


harboring pathway plasmids pCL1925-Kodd-ddr and pTrc99a-budABC













Volume of
2-Butanone,
2-Butanol,


Straina,b
IPTG, mM
Medium, mL
mM
mM














MG1655 #1
0
50
0.08
Not detected


MG1655 #2
0
50
0.11
Not detected


MG1655 #1
0.04
50
0.12
Not detected


MG1655 #2
0.04
50
0.11
Not detected


MG1655 #1
0
150
0.15
0.047


MG1655 #2
0
150
0.19
0.041


MG1655 #1
0.04
150
0.10
0.015


MG1655 #2
0.04
150
0.11
0.015


MG1655
0
50
0.10
Not detected


1.5yqhD #1






MG1655
0
50
0.07
Not detected


1.5yqhD #2






MG1655
0.04
50
0.12
Not detected


1.5yqhD #1






MG1655
0.04
50
0.18
Not detected


1.5yqhD #2






MG1655
0
150
0.16
0.030


1.5yqhD #1






MG1655
0
150
0.18
0.038


1.5yqhD #2






MG1655
0.04
150
0.10
0.021


1.5yqhD #1






MG1655
0.04
150
0.09
0.017


1.5yqhD #2






MG1655
0
50
0.08
Not detected


1.6yqhD #1






MG1655
0
50
0.07
Not detected


1.6yqhD #2






MG1655
0.04
50
0.12
Not detected


1.6yqhD #1






MG1655
0.04
50
0.15
Not detected


1.6yqhD #2






MG1655
0
150
0.17
0.019


1.6yqhD #1






MG1655
0
150
0.18
0.041


1.6yqhD #2






MG1655
0.04
150
0.11
0.026


1.6yqhD #1






MG1655
0.04
150
0.11
0.038


1.6yqhD #2






Control


Not detected
Not detected


(uninoculated






medium)





a#1 and #2 represent independent isolates.


bMG1655 is MG1655/pCL1925-Kodd-ddr/pTrc99a-budABC MG1655 1.6yqhD is MG1655 1.6yqhD/pCL1925-Kodd-ddr/pTrc99a-budABC MG1655 1.6yqhD is MG1655 1.5yqhD/pCL1925-Kodd-ddr/pTrc99a-budABC.






Example 11
Expression of a 2-Butanol Biosynthetic Pathway in E. coli with Heterologous Alcohol Dehydrogenase
Plasmids pCL1925-KoDD-ddr::ter-T5chnA and pTrc99a-budABC, described in Example 9, were transformed into E. coli strains MG1655 and MG1655 ΔyqhCD for a demonstration of the production of 2-butanol.
MG1655 ΔyqhCD carries a yqhCD inactivation that was made using the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. U.S.A. 97(12):6640-6645 (2000)). After replacement of the region with the FRT-CmR-FRT cassette of pKD3, the chloramphenicol resistance marker was removed using the FLP recombinase. The sequence of the deleted region is given as SEQ ID NO:66.
Strains MG1655/pTrc99a-budABC/pCL1925KoDD-ddr::ter-T5 chnA and MG1655 ΔyqhCD/pTrc99a-budABC/pCL1925KoDD-ddr::ter-T5 chnA were examined for the production of 2-butanone and 2-butanol essentially as described above. Strain MG1655 ΔyqhCD/pCL1925 was used as a negative control. Cells were inoculated into shake flasks (approximately 175 mL total volume) containing 50 or 150 mL of TM3a/glucose medium (with 0.1 mg/L vitamin B12 and appropriate antibiotics) to represent medium and low oxygen conditions, respectively. Spectinomycin (50 μg/mL) and ampicillin (100 μg/mL) were used for selection of pCL1925 based plasmids and pTrc99a-budABC, respectively. Enzyme activity derived from pTrc99a-budABC was detected by enzyme assay in the absence of IPTG inducer, thus, IPTG was not added to the medium. The flasks were inoculated at a starting OD600 of ≦0.01 units and incubated at 34° C. with shaking at 300 rpm for 24 h. The flasks containing 50 mL of medium were capped with vented caps; the flasks containing 150 mL, were capped with non-vented caps to minimize air exchange. Analytical results for 2-butanone and 2-butanol production are presented in Table 8. Both E. coli strains comprising a 2-butanol biosynthetic pathway produced 2-butanone under low and medium oxygen conditions and produced 2-butanol under low oxygen conditions, while the negative control strain did not produce detectable levels of either 2-butanone or 2-butanol.







TABLE 8







Production of 2-butanone and 2-butanol by E. coli strains












2-




Volume,
Butanone,
2-Butanol,


Straina
mL
mM
mM













Negative control, MG1655
50
Not
Not detected


ΔyqhCD/pCL1925

detected



MG1655/pTrc99a-
50
0.33
Not detected


budABC/pCL1925KoDD-ddr::T5





chnA ter





MG1655 ΔyqhCD/pTrc99a-
50
0.23
Not detected


budABC/pCL1925KoDD-ddr::T5





chnA ter#1





MG1655 ΔyqhCD/pTrc99a-
50
0.19
Not detected


budABC/pCL1925KoDD-ddr::T5





chnA #2





Negative control, MG1655
150
Not
Not detected


ΔyqhCD/pCL1925

detected



MG1655/pTrc99a-
150
0.41
0.12


budABC/pCL1925KoDD-ddr::T5





chnA ter





MG1655 ΔyqhCD/pTrc99a-
150
0.15
0.46


budABC/pCL1925KoDD-ddr::T5





chnA #1





MG1655 ΔyqhCD/pTrc99a-
150
0.44
0.14


budABC/pCL1925KoDD-ddr::T5





chnA #2





Medium

Not
Not detected




detected





a#1 and #2 represent independent isolates.






Example 12
Cloning of Amino:Pyruvate Transaminase (APT)
An amino:pyruvate transaminase (APT) from Vibrio Fluvialis JS17 was identified by Shin et al. (Appl. Microbiol. Biotechnol. (2003) 61:463-471). The amino acid sequence (SEQ ID NO:122) was found to have significant homology with ω-amino acid:pyruvate transaminases (Shin and Kim (J. Org. Chem. 67:2848-2853 (2002)). It was shown that the Vibrio Fluvialis APT has transaminase activity towards acetoin.
For expression of the APT enzyme in E. coli, a codon optimized APT coding region (SEQ ID NO:144) was designed using the preferred E. coli codons with additional considerations such as codon balance and mRNA stability, and synthesized (by DNA2.0; Redwood City, Calif.). The coding region DNA fragment was subcloned into the pBAD.HisB vector (Invitrogen) between the NcoI and HindIII sites and the resulting plasmid, hereafter referred to as pBAD.APT1, was transformed into TOP10 cells.
Example 13
Characterization of Vibrio Fluvialis APT Alanine:Acetoin Aminotransferase Activity
A 5 mL volume of LB broth+100 μg/mL ampicillin was inoculated with a fresh colony of TOP10/pBAD:APT1 cells. The culture was incubated at 37° C. for approximately 16 h with shaking (225 rpm). A 300 μL aliquot of this culture was used to inoculate 300 mL of the same medium, which was incubated at 37° C. with shaking (225 rpm). When the culture reached an OD600 of 0.8, L-arabinose was added to a final concentration of 0.2% (w/v). The culture was incubated for an additional 16 h, then harvested. The cells were washed once with 100 mM potassium phosphate buffer (pH 7.8) and then frozen and stored at −80° C.
To isolate the enzyme, the cell pellet was thawed and resuspended in 8 mL of 100 mM potassium phosphate buffer (pH 7) containing 0.2 mM ethylenediaminetetraacetate, 1 mM dithiothreitol and 1 tablet of protease inhibitor cocktail (Roche; Indianapolis, Ind.). The cells were lysed by two passes through a French pressure cell at 900 psi, and the resulting lysate was clarified by centrifugation for 30 min at 17000×g. Ammonium sulfate was added to 35% saturation, and the solution was stirred for 30 min at room temperature, at which point precipitated solids were removed by centrifugation (30 min, 17000×g). Additional ammonium sulfate was added to the supernatant to give 55% saturation, and the solution was again stirred for 30 min at room temperature. The precipitated solids were removed by centrifugation (30 min, 17000×g) and then resuspended in 5 mL of 100 mM potassium phosphate buffer (pH 7) containing 10 μM pyridoxal 5′-phosphate and 1 mM dithiothreitol. This solution was desalted by passage through a PD10 column equilibrated with Buffer A (50 mM bis-tris propane buffer (pH 6) containing 10 μM pyridoxal 5′-phosphate and 1 mM dithiothreitol). The desalted extract was then loaded onto a 20 mL Q-Fast Flow column pre-equilibrated with Buffer A. APT was eluted with a linear gradient of 0-0.1 M NaCl in Buffer A. The enzyme was detected in eluted fractions by the presence of a protein band of size  ̃50 kD when analyzed by SDS-polyacrylamide gel electrophoresis and by the characteristic absorbance at 418 nm. Fractions containing the enzyme eluted at  ̃0.3 M NaCl. These fractions were pooled to yield a total of 6 mL of a 5.45 mg/mL solution of enzyme, which was >90% pure, as judged by SDS-polyacrylamide gel electrophoresis.
The alanine:acetoin aminotransferase activity of APT was assayed using a lactic dehydrogenase coupled assay. Reaction mixtures contained 100 mM bis-tris propane (pH 9.0), 10 μM pyridoxal 5′-phosphate, 0-50 mM acetoin, 0-5 mM L-alanine, 0.14 or 0.28 mg/mL purified enzyme, 200 μM NADH and 20 U/mL lactic dehydrogenase (Sigma; St. Louis, Mo.). The reaction was followed by measuring the change in absorbance at 340 nm, indicative of the oxidation of NADH. Under these conditions, the kcat/Km for acetoin was 10 M−1 s−1 and that for L-alanine was 400 M−1 s−1.
The identity of the expected product 3-amino-2-butanol was confirmed by comparison to a synthetic standard. A mixture of (R,R)- and (S,S)-3-amino-2-butanol was synthesized by the method of Dickey et al. [J Amer Chem Soc 74:944 (1952)]: 5 g of trans-2,3-epoxybutane were slowly stirred into 150 mL of cold (4° C.) NH4OH. The reaction was slowly warmed to room temperature, sealed and stirred at room temperature for an additional 10 days. At this time, excess ammonia and water and residual epoxybutane were removed by rotary evaporation under vacuum at 40° C. The resulting clear oil (2.9 g) was resuspended in water to a concentration of 10% (w/v). Production of the desired product was confirmed by NMR analysis and comparison of the spectrum to that reported by Levy et al. [Org. Magnetic Resonance 14:214 (1980)]. A mixture of the corresponding (2R,3S)- and (2S,3R)-isomers was produced using the identical method with the exception that the starting material was the cis-isomer of 2,3-epoxybutane.
An analytical method for detection of 3-amino-2-butanol was developed based on the o-phthaldialdehyde derivatization method for amino acid determination reported by Roth [Anal. Chem. 43:880 (1971)]. A 200 μL aliquot of 1 mM 3-amino-2-butanol (mixture of isomers) was mixed with 200 μL of a 50 mM solution of borate (pH 9.5), to which was added 10 μL of 5 μL/mL 2-mercaptoethanol in ethanol and 10 μL of 10 mg/mL o-phthaldialdehdye in ethanol. The solution was incubated at room temperature for 10 min, at which time the derivative was extracted into 200 μL hexane. The hexane was separated from the aqueous solution by decanting, and 10 μL were injected onto a Chiracel OD HPLC column (Daicel Chemical Industries; Fort Lee, N.J.). The column was run isocratically with a mobile phase of 90:10 hexane:isopropanol at a rate of 1 mL/min. The derivatized isomers of 3-amino-2-butanol were detected by absorbance at 340 nm with retention times of approximately 15.7 and 16.8 min [(2S,3S) and (2R,3R)], and 18.4 and 21.9 min [(2R,3S) and (2S,3R)]. To differentiate the enantiomers in the first mixture, the pure (2R,3R) isomer (Bridge Organics; Vicksburg, Mich.) was also run under the identical conditions and found to be the 16.8 min peak. To differentiate the enantiomers in the second mixture, the mixture was first kinetically resolved using the alanine:acetoin aminotransferase: 0.28 mg of purified enzyme was incubated with 10 mM pyruvate and 10 mM 3-amino-2-butanol [1:1 mixture of (2R,3S) and (2S,3R) isomers] in 1 mL of 100 mM bis-tris propane (pH 9.0). After 24 h at room temperature, an aliquot was removed and analyzed as described above. Analysis revealed that the 18.4 min peak was 95% depleted, while the 21.9 min peak was >90% retained. A 100 μL aliquot of the remaining reaction mixture was mixed with 50 μL of 20 mM NADH and 10 μL of extract from the TOP10/pTrc99a-BudC strain described in Example 9. The BudC enzyme is known to reduce (R)-acetoin to meso-2,3-butanediol and (S)-acetoin to (S,S)-2,3-butanediol [Ui et al. (2004) Letters in Applied Microbiology 39:533-537]. After 3 h, samples were taken from the reaction and analyzed as described above for acetoin and butanediol. The analysis indicated that the primary product of the reduction was meso-2,3-butanediol, indicating that the product of the aminotransferase reaction was (R)-acetoin, and therefore the consumed 3-amino-2-butanol isomer was the (2R,3S) isomer. Thus the retention time of 18.4 min can be assigned to this isomer and 21.9 to the (2S,3R) isomer.
To confirm that the product of the APT-catalyzed alanine:acetoin aminotransferase reaction was 3-amino-2-butanol, 0.28 mg of purified enzyme was incubated with 10 mM acetoin, 10 mM L-alanine, 50 U lactic dehydrogenase and 200 μM NADH in 1 mL of 100 mM bis-tris propane (pH 9.0). The reaction mixture was incubated at room temperature for 20 h, after which a 200 μL aliquot was removed and derivatized as described above. The retention times of the derivatized products were 15.8 min (major product) and 18.5 min (minor product), matching that of the (2S,3S)- and (2R,3S)-3-amino-2-butanol standards.
Example 14
Identification and Cloning of Erwinia carotovora subsp. atroseptica Amino Alcohol Kinase and Amino Alcohol O-Phosphate Lyase
The purpose of this example is to describe the identification and cloning of sequences encoding an amino alcohol kinase and amino alcohol O-phosphate lyase from the bacterium Erwinia carotovora. These two enzymes are part of Pathway 1 for the conversion of 3-amino-2-butanol to 2-butanone via the intermediate 3-amino-2-butanol phosphate as shown in FIG. 1.
Prediction of the Erwinia Amino Alcohol Kinase and the Amino Alcohol O-Phosphate Lyase
ATP-dependent amino alcohol kinase and amino alcohol O-phosphate lyase activities have been detected in several Pseudomonas and Erwinia species, including Pseudomonas sp. P6 (NCIB10431), Pseudomonas putida NCIB 10558 (Jones et al. (1973) Biochem. J. 134:167-182), Erwinia carotovora, Erwinia amanas, Erwina milletiae, and Erwinia atroseptica (Jones et al. (1973) Biochem. J. 134:959-968). In these studies, the extracts of the above species were shown to have activity for the enzymatic conversion of aminopropanol through aminopropanol O-phosphate to propionaldehyde, and the conversion of ethanolamine through ethanolamine O-phosphate to acetaldehyde.
The genomic sequence of the Erwinia atroseptica strain in which these activities were reported to exist (now designated as Erwinia carotovora subsp. atroseptica strain SCR11043 (ATCC BAA-672)) has been determined at the Sanger Institute (Bell et al. Proc. Natl. Acad. Sci. USA 101 (30): 11105-11110). Analysis of the putative kinases in the Erwinia carotovora subsp. atroseptica genome revealed an operon sequence (SEQ ID NO: 275) encoding a putative protein (ECA2059; SEQ ID NO:124) that is 39% identical to a Rhizobium loti homoserine kinase and a putative class-III pyridoxal phosphate (PLP)-dependent aminotransferase (ECA2060; SEQ ID NO:126) that is 58% identical to a putative aminotransferase from Rhizobium meliloti. It was expected that ECA2059 was an amino alcohol kinase and ECA2060 was an amino alcohol O-phosphate lyase which uses PLP as cofactor.
Cloning of the Putative Amino Alcohol Kinase and Putative Amino Alcohol O-Phosphase Lyase from Erwinia carotovora subsp. atroseptica 
Genomic DNA of Erwinia carotovora subsp. atroseptica (ATCC #: BAA-672D) was obtained from American Type Culture Collection (ATCC). The operon encoding the putative amino alcohol kinase (KA) and amino alcohol O-phosphate lyase (AT) was named KA-AT (SEQ ID NO: 275. This operon was amplified from the Erwinia genomic DNA by Phusion DNA polymerase (Finnzymes; via New England Biolabs; Ipswich, Mass.) using primers OT872 (SEQ. ID. No. 127) and OT873 (SEQ. ID. No128). A DNA fragment of 2.4 kb was obtained by the PCR reaction, which corresponds to the size of the KA-AT operon. The PCR product was digested with EcoRI and PstI restriction enzymes, and cloned into vector pKK223-3 (Amersham Biosciences; Piscataway, N.J.) which was digested with the same restriction enzymes. This produced plasmid pKK223.KA-AT, which contained the putative Erwinia amino alcohol kinase-lyase operon under control of the tac promoter. Similarly, plasmids pKK223.KA and pKK223.AT were made which placed the putative Erwinia kinase and the putative Erwinia lyase coding regions in separate vectors, each under the control of the tac promoter. For the PCR cloning of the KA coding region (SEQ ID NO:123), primers OT872 (SEQ. ID. No. 127) and OT879 (SEQ. ID. No. 129) were used; and for the PCR cloning of AT coding region (SEQ ID NO:125), primers OT873 (SEQ. ID. No. 128) and OT880 (SEQ. ID. No. 130) were used in the PCR amplifications, which generated PCR products of 1.1 kb and 1.3 kb respectively. The PCR products were each digested with EcoRI and PstI, and ligated into vector pKK223-3 to generate pKK223.KA and pKK223.AT.
In Vivo Activity of the Putative Amino Alcohol Kinase and Putative Amino Alcohol O-Phosphase Lyase from Erwinia carotovora subsp. atroseptica 
Plasmids pKK223.KA-AT, pKK223.KA, pKK223.AT and pKK223-3 were transformed into the E. coli MG1655 strain. The transformants were restreaked onto a MOPS minimal media plate containing 1% glucose, 0.5% aminopropanol as a sole nitrogen source, 1 mM IPTG and 100 μg/mL ampicillin. Expression of KA-AT, KA and AT genes were induced by the IPTG. A control plate had no IPTG included. The plates were incubated at 37° C. for 7 days. On the plate with IPTG, only the strain MG1655/pKK223.KA-AT grew, while all the other three strains did not grow. On the plate without added IPTG, the strain MG1655/pKK223.KA-AT grew, but the colonies were significantly smaller than those on the IPTG-containing plate, which corresponds to the lower expression levels of KA and AT in the uninduced cells. None of the other three strains grew on this plate. This indicates that the co-expression of the putative Erwinia KA and AT genes provided sufficient enzyme activities that allowed the E. Coli strain MG1655/pKK223.KA-AT to utilize aminopropanol as a sole nitrogen source. Expression of each individual enzyme of either KA or AT was not sufficient to provide such enzyme activity in vivo.
Example 15
In vitro Activity of Erwinia putative Amino Alcohol Kinase and Amino Alcohol O-Phosphate Lyase
Subcloning of the Erwinia KA-AT Operon into the pBAD.HisB Vector and Induction of Protein Expression
The protein expression levels of Erwinia putative KA and AT enzymes expressed in MG1655 cells from the pKK223.KA-AT vector were analyzed by SDS-PAGE analysis. The expression level of the Erwinia AT enzyme was relatively low, with a new protein band detected at the correct molecular weight of 46 kD in the soluble fraction of a cell extract, while no new protein band was detected at the size predicted for the KA enzyme.
In an effort to improve the expression of the Erwinia putative KA and AT genes, the KA-AT operon was subcloned into the EcoRI and HindIII sites of vector pBAD.HisB-EcoRI. pBAD.HisB-EcoRI was derived from the pBAD.HisB vector (Invitrogen), by replacing the NcoI site in pBAD.HisB with an EcoRI site via QuickChange site-directed mutagenesis (Stratagene, La Jolla, Calif.) using primers OT909 (SEQ ID.#131) & OT910 (SEQ ID.#132). In the constructed plasmid pBAD.KA-AT, the KA-AT operon was placed directly under control of the araB promoter (without His-tag).
The PBAD.KA-AT plasmid was transformed into the E. coli TOP10 strain. A 50 mL culture of TOP10/pBAD.KA-AT strain was grown to mid log phase (OD600=0.6) in LB, 100 μg/mL ampicillin media at 37° C. with shaking at 250 rpm. The culture was induced by addition of L-arabinose to a final concentration of 0.1% (w/v), and it was further incubated at 37° C. for 5 h before harvesting by centrifugation. The cell pellet was resuspended in ice cold 50 mM Tris-HCl, pH 8.0, and disrupted by sonication on ice with a Fischer Sonic Model 300 Dismembrator (Fischer, Pittsburgh, Pa.) at 50% power, repeating four cycles of 30 seconds sonication with 60 seconds rest in-between each cycle. Each sonicated sample was centrifuged (15,000×g, 4 min, 4° C.). Clarified cell free extracts were analyzed for protein expression level and amino alcohol O-phosphate lyase activity.
Chemical Synthesis of Aminobutanol O-Phosphate and Aminopropanol O-Phosphate
The substrate (R,R)-3-amino-2-butanol O-phosphate was synthesized by a method based on that reported by Ferrari and Ferrari (U.S. Pat. No. 2,730,542 [1956]) for phosphoethanolamine: 10 mmol of H3PO4 in a 50% (w/v) aqueous solution was mixed with a 50% (w/v) solution of (R,R)-3-amino-2-butanol (Bridge Organics; Vicksburg, Mich.) while stirring on ice. After mixing, the solution was slowly warmed to room temperature and then stirred under vacuum and heated to 70° C. After 1 h at 70° C., the temperature was slowly increased to 185° C. and maintained there for an additional 2 h. At that time, the reaction was cooled to room temperature and the vacuum released. The remaining material was dissolved in water, and analysis by NMR indicated that 80% of the starting material was converted to product with 20% remaining unreacted. No additional products were observed.
The additional substrates (2R,3S)-3-amino-2-butanol O-phosphate and (2S,3R)-3-amino-2-butanol O-phosphate were synthesized by the same procedure using a 1:1 mixture of (2R,3S)-3-amino-2-butanol and (2S,3R)-3-amino-2-butanol (synthesized as described in Example 13) as the starting material. DL-1-amino-2-propanol O-phosphate, (S)-2-amino-1-propanol O-phosphate, and (R)-2-amino-1-propanol O-phosphate were synthesized by the same procedure using DL-1-amino-2-propanol, (R)-2-amino-1-propanol, or (S)-2-amino-1-propanol as the starting material.
Analysis of the Aminopropanol O-Phosphate Lyase Activity Encoded by the Putative Erwinia KA-AT Operon
The aminopropanol O-phosphate lyase assay was performed as described by Jones et al. (1973, Biochem. J. 134:167-182) and G. Gori et al. (1995, Chromatographia 40:336) The formation of propionaldehyde from aminopropanol O-phosphate was assayed calorimetrically with MBTH, which allows the detection of aldehyde formation. The reaction was performed as follows. In a 1 mL reaction, 100 μg cell free extract of E. coli TOP10/pBAD.KA-AT was added to 10 mM DL-1-amino-2-propanol O-phosphate in 100 mM Tris-HCl, pH 7.8, with 0.1 mM PLP. The reaction was incubated at 37° C. for 10 min and 30 min, with an aliquot of 100 μL reaction mixture removed at each time point and mixed with 100 μL of 6 mg/mL MBTH in 375 mM glycine-HCl, pH 2.7. This mixture was incubated at 100° C. for 3 min, cooled on ice for 15-30 s, and 1 mL of 3.3 mg/mL FeCl3.6H2O (in 10 mM HCl) was added, followed by incubation for 30 min at room temperature. The absorbance of the reaction mixture which contains the aldehyde-MBTH adduct, was measured at 670 nm. The results of the assay are listed in Table 9. In the presence of the aminopropanol phosphate substrate, PLP and cell free extract, formation of aldehyde was detected, as indicated by an Abs670 that was higher than the control background of up to 0.3. In the absence of either the substrate or the cell free extract, no aldehyde formation was detected. In the absence of added PLP, somewhat less amount aldehyde was detected, presumably due to the presence of PLP in the cell free extract. Cell free extract of the uninduced TOP10/pBAD.KA-AT-culture did not produce any detectable aldehyde in the reaction. These results indicated that the putative Erwinia amino alcohol O-phosphate lyase does catalyze the conversion of aminopropanol O-phosphate to propionaldehyde.







TABLE 9







Aminopropanol O-phosphate lyase assay. Sample 1 was the cell free


extract of a non-induced control of E. coli TOP10/pBAD.KA-AT.


Samples 2-5 contained the cell free extract of the induced


culture E. coli TOP10/pBAD.KA-AT.

















Enzyme





Induction by
Aminopro-

extract


Sample
0.1%
panol O-

(100 μg/
OD670,
OD670,


Number
arabinose
phosphate
PLP
mL)
10 min
30 min





1
uninduced
(+)
(+)
(+)
0.262
0.255


2
induced
(+)
(+)
(+)
1.229
2.264


3
induced
(−)
(+)
(+)
0.303
0.223


4
induced
(+)
(−)
(+)
0.855
1.454


5
induced
(+)
(+)
(−)
0.156
0.065









Analysis of the Activity of the Erwinia Amino Alcohol O-Phosphate Lyase Towards Aminobutanol O-Phosphate Substrate

The activity of the amino alcohol O-phosphate lyase towards the aminobutanol O-phosphate substrates was studied under the same conditions as described above. The reaction was carried out at 37° C. overnight in a 1 mL reaction that contained 100 μg of cell free extract of E. coli TOP10/pBAD.KA-AT, 10 mM aminobutanol O-phosphate (either the mixture of (R,R)+(S,S) or the mixture of (R,S)+(S,R) isomers described in Example 15) in 100 mM Tris-HCl, pH 7.8, with 0.1 mM PLP. An aliquot of 100 μL reaction mixture was removed and the 2-butanone product was detected using the MBTH derivatization method described in the General Methods. The two peaks representing the derivatized 2-butanone isomers were observed. Therefore the Erwinia amino alcohol O-phosphate lyase is an aminobutanol phosphate phospho-lyase in addition to an aminopropanol phosphate phospho-lyase.
Analysis of the Activity of the Erwinia Amino Alcohol O-Phosphate Lyase Towards Stereoisomers of Aminopropanol O-Phosphate and Aminobutanol O-Phosphate
The activity of the Erwinia amino alcohol O-phosphate lyase towards various stereoisomers of aminopropanol O-phosphate and aminobutanol O-phosphate was studied under the same conditions as described above. In the presence of the Erwinia amino alcohol O-phosphate lyase, both (R) and (S)-2-amino-1-propanol O-phosphate were converted to propanone by the enzyme, but the product yield was much higher with the (S) isomer. The enzyme also produced butanone from both mixtures of 3-amino)-2-butanol O-phosphate isomers, with a higher product yield found in the reaction containing the (R,S) and (S,R) substrate isomers. Both propanone and butanone products were derivatized by MBTH, and detected by HPLC as described in General Methods.
Optimization of the Gene Expression Level for the Erwinia Amino Alcohol Kinase and Amino Alcohol O-Phosphate Lyase
In order to improve the expression levels for the Erwinia amino alcohol kinase and the amino alcohol O-phosphate lyase in E. coli, codon optimized coding regions for both enzymes (named EKA: SEQ ID NO:155 and EAT: SEQ ID NO:156 respectively) were synthesized by DNA2.0 (Redwood City, Calif.). Each coding region was synthesized with 5′ and 3′ tails including restriction sites for cloning: EKA has 5′ BbsI and 3′ EcoRI, HindIII sites; EAT has 5′ EcoRI and 3′ HindIII sites. The EKA and EAT coding regions were provided from DNA2.0 as plasmids pEKA and pEAT, which were in the pJ51 vector of DNA2.0. The EKA optimized coding region was subcloned by ligating a BbsI and HindIII digested fragment of pEKA into the pBAD.HisB vector between the NcoII and HindIII sites, to generate plasmid PBAD.EKA. In the resulting plasmid the coding region is 5′ to the His tag, so a coding region for an N-terminus His6 tag fused to the Erwinia amino alcohol kinase was constructed by performing a QuickChange site-directed mutagenesis reaction using primers SEQ ID NO:157 and SEQ ID NO:158 to generate vector pBAD.His-EKA.
pBAD.His-EKA was transformed into E. coli strain BL21AI (F− ompT hsdSB (rB− mB−) gal dcm araB::T7RNAP-tetA; Invitrogen) to produce strain BL21AI/pBAD.HisA-EKA. A 50 mL culture of BL21AI/pBAD.HisA-EKA was grown to mid-log stage (OD600=0.6), induced with 0.1% arabinose, and further incubated at 30° C. overnight. Cell free extracts were prepared by sonication. The His6-tagged fusion protein of Erwinia amino alcohol kinase was purified using the ProBondTM Purification System (Invitrogen) under non-denaturing purification conditions following the manufacture's instructions.
Prophetic Result
The kinase activity of the His6-tagged Erwinia amino alcohol kinase is analyzed by the ADP Quest Assay (DiscoveRx, Fremont, Calif.) following the manufacture's instructions. This is a biochemical assay that measures the accumulation of ADP, a product of the amino alcohol kinase reaction using either aminopropanol or aminobutanol as substrate. 10 mM substrate is mixed with His6-tagged Erwinia amino alcohol kinase, in 100 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 2 mM KCl, 0.1 mM ATP, and incubated at 37° C. for 1 h in a 0.2 mL reaction. ADP reagent A (100 μL) and ADP reagent B (200 μL) are added and the mixture is incubated at room temperature for 30 min. The fluorescence signal indicating activity is measured with excitation wavelength of 530 nm and emission wavelength of 590 nm.
Example 16
Expression of Entire Pathway 3
Construction of Vector pCLBudAB-ter-T5chnA
The vector pTrc99a::BudABC (described in Example 9) is digested with EcoRI, and the DNA is treated with Klenow DNA polymerase to blunt the ends. The blunted vector is subsequently digested with SpeI to yield a 2.5 kb fragment containing the budA and budB genes. The vector pCL1925-ter-T5chnA (described in Example 9) is digested with HindIII, and the DNA was treated with Klenow DNA polymerase to blunt the ends. The blunted vector is subsequently digested with XbaI to yield a 4.6 kb fragment which is then ligated to the budAB fragment from pTrc99a::BudABC. The resulting plasmid, designated pCLBudAB-ter-T5chnA, is used to transform E. coli Top10 cells, and single colonies are screened for proper plasmid structure by PCR using primers pCL1925vecF (SEQ ID NO:62) and N84seqR3 (SEQ ID NO:159). Plasmid is prepared from a single colony which yields a PCR product of the expected size of 1.4 kb.
Construction of Vector pKK223.KA-AT-APT
The APT gene is amplified from the vector PBAD.APT (described in Example 12) by PCR using primers APTfor (SEQ ID NO:162; 5′ includes RBS and SmaI site) and APTrev (SEQ ID NO:163; 3′ adds SmaI site). The product of expected size of 1.7 kbp is gel purified and digested with SmaI to yield blunt ends. The vector pKK223.KA-AT (described in Example 14) is digested with PstI, and the DNA is treated with Klenow DNA polymerase to blunt the ends. The resulting DNA fragment is ligated with the SmaI-digested PCR product, and the ligation product is used to transform E. coli Top10 cells. Individual ampicillin resistant colonies are screened by PCR using primers OT872 (SEQ ID NO:127) and APTrev (SEQ ID NO:163). The presence of a PCR product of the expected size of 4.1 kbp indicates that the gene encoding APT is present and oriented in the same direction as the genes encoding KA and AT. The sequence of the insert is verified using the primers APTseqRev (SEQ ID NO:160) and APTseqFor (SEQ ID NO:161). This plasmid is named pKK223.KA-AT-APT. Proper expression of all three genes is verified by growing a 5 mL culture of Top10/pKK223.KA-AT-APT in LB+100 μg/mL ampicillin at 37° C. with shaking. When the OD600 reaches  ̃0.8, expression of the genes on the plasmid is induced by addition of IPTG to 0.4 mM. The expression is evaluated by SDS PAGE and activity assays as described above.
Construction of 2-Butanol Production Strain and Production of 2-Butanone and 2-Butanol
E. coli strain MG1655 is transformed with both pKK223.KA-AT-APT and pCLBudAB-ter-T5chnA, and transformants selected for ampicillin and spectinomycin resistance, indicative of the presence of the plasmids. The cells are inoculated into shake flasks (approximately 175 mL total volume) containing 50 or 150 mL of TM3a/glucose medium (with appropriate antibiotics) to represent medium and low oxygen conditions, respectively. IPTG is added to 0.4 mM to induce expression of genes from pKK223.KA-AT-APT. As a negative control, MG1655 cells are grown in the same medium lacking antibiotics. The flasks are inoculated at a starting OD600 of ≦0.01 and incubated at 34° C. with shaking at 300 rpm for 24 h. The flasks containing 50 mL of medium are capped with vented caps; the flasks containing 150 mL are capped with non-vented caps to minimize air exchange. The MG1655/pKK223.KA-AT-APT/pCLBudAB-ter-T5chnA strain comprising a 2-butanol biosynthetic pathway produces both 2-butanone and 2-butanol under low and medium oxygen conditions while the negative control strain does not produce detectable levels of either 2-butanone or 2-butanol.
Example 17
Characterization of Glycerol Dehydratase Butanediol Dehydratase Activity
Glycerol dehydratase (E.C. 4.2.1.30) and diol dehydratase (E.C. 4.2.1.28), while structurally related, are often distinguished in the art based on various differences that include substrate specificity. This example demonstrates that glycerol dehydratase converts meso-2,3-butanediol to 2-butanone. The recombinant E. coli strain KLP23/pSYCO12, comprising Klebsiella pneumoniae genes encoding the multiple subunits of glycerol dehydratase (alpha: SEQ ID NO:145 (coding region) and 146 (protein); beta: SEQ ID NO: 147 (coding region) and 148 (protein); and gamma: SEQ ID NO: 149 (coding region) and 150 (protein)) and Klebsiella pneumoniae genes encoding the multiple subunits of glycerol dehydratase reactivase (large subunit, SEQ ID NO: 151 (coding region) and 152 (protein); and small subunit, SEQ ID NO: 153 (coding region) and 154 (protein)), is described in Emptage et al. U.S. Pat. No. 6,514,733 and in WO 2003089621, which are herein incorporated by reference. A crude, cell free extract of KLP23/pSYCO12 was prepared by methods known to one skilled in the art. Enzyme assay was performed in the absence of light in 80 mM HEPES buffer, pH 8.2 at 37° C. with 12 μM coenzyme B12 and 10 mM meso-2,3-butanediol. The formation of 2-butanone was monitored by HPLC (Shodex SH-1011 column and SH-G guard column with refractive index detection; 0.01 M H2SO4 as the mobile phase at a flow rate of 0.5 mL/min and a column temperature of 50° C.; 2-butanone retention time=40.2 min). The rate of 2-butanone formation by the glycerol dehydratase preparation was determined to be 0.4 nmol/min/mg of crude protein.
Example 18
Structural Analysis of Diol/Glycerol Dehydratases through Generation and Validation of a Profile HMM for Experimentally Proven Diol/Glycerol Dehydratases
The diol dehydratase and glycerol dehydratase enzymes belong to the enzyme classes 4.2.1.28 and 4.2.1.30, respectively. The enzymes in both classes are each a complex of three subunits: large (also called alpha), medium (also called beta) and small (also called gamma). In some glycerol dehydratases the large and medium subunits were found to be fused.
Identifying Family Members by Sequence
The Klebsiella oxytoca butanediol dehydratase enzyme was used as a prototype enzyme for identifying a family of diol and glycerol dehydratase enzymes. The amino acid sequences of the alpha (GenBank No: BAA08099; SEQ ID NO: 8), beta (GenBank No: BAA08100; SEQ ID NO: 10) and gamma (GenBank No: BAA08101; SEQ ID NO: 12) subunits were each run as the query sequence in a BLASTP search against the GenBank non-redundant protein database using default parameters. Sequences with relevant matches were extracted. Relevance was judged by the E-value score, protein definition, details included in the GenBank report for the matched proteins, and literature review of the topic. For the large subunit, the BLAST output showed an abrupt decrease in the E-value from e-20 to an E-value of 1.5. All sequence matches with an E-value of 1.5 or larger had definitions inconsistent with them being dehydratases. Many of these sequences were labeled as DNA-directed RNA polymerase beta subunits. There were matches with E-values around e-20, which were partial sequences. Sequences with no annotation were included if the E-value was less than 1.5.
Using the Klebsiella oxytoca butanediol dehydratase alpha subunit as a query, 50 homologs were identified as members of this family of proteins. This group included some sequences that were not full length proteins. The full length sequences identified for the alpha subunit family of diol/glycerol dehydratases are the prototype SEQ ID No: 8 and SEQ ID NOs: 93, 99, 105, 135, 138, 141, 146, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 130, 243, 254, 255, 256, 257, 258, and 259. SEQ ID NOs: 233, 235, 237, 239, 241, 246, 247 include both the alpha and beta subunits, which are fused in these cases.
Using the Klebsiella oxytoca butanediol dehydratase beta subunit as a query, 51 homologs were identified as members of this family of proteins. This group included some sequences that were not full length proteins. The full length sequences identified for the beta subunit family of diol/glycerol dehydratases are the prototype SEQ ID No: 10 and SEQ ID NOs: 95, 101, 107, 136, 139, 142, 148, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 244, 250, 252, 260, 261, 262, 263, 364, 265, 266, and 167.
Using the Klebsiella oxytoca butanediol dehydratase gamma subunit as a query, 48 homologs were identified as members of this family of proteins. This group included some sequences that were not full length proteins. The full length sequences identified for the gamma subunit family of diol/glycerol dehydratases are the prototype SEQ ID No: 12 and SEQ ID NOs: 97, 103, 109, 137, 140, 143, 150, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 234, 236, 238, 240, 242, 245, 248, 249, 251, 253, 268, 270, 271, 272, 273, and 274.
Identifying Family Members with Experimentally Assessed Function
For each sequence identified through the analysis described above, a search for experimental evidence of its biochemical function was conducted in the BRENDA, UniProt and NCBI Entrez databases. BRENDA is a human-curated database that contains detailed information about enzyme kinetic, physical, and biochemical properties extracted from the experimental literature and with links to the relevant databases (Cologne University BioInformatics Center). The UniProt Knowledgebase is composed of a human-curated part the Swiss-Prot database and of a machine annotated supplement the TrEMBL database. The curated Swiss-Prot database (Swiss Institute of Bioinformatics) provides a high level of protein annotation that includes domain architecture, post-translational modifications and sequence variants. NCBI Entrez is the integrated, text-based search and retrieval system used at NCBI (National Center for Biotechnology Information, Bethesda, Md.) for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, and Taxonomy.
Through the analysis of information and references identified from these databases, eight diol/glycerol dehydratases with experimentally verified function as a diol or glycerol dehydratase were identified. These are given in Table 10.







TABLE 10







Diol/glycerol dehydratases with experimentally verified function















SEQ




organism
subunit
Genbank #
ID No
type
reference















Klebsiella
large
gi|6980836
8
diol
Shibata et al.,


oxytoca




Structure 1999;


K. oxytoca
medium
gi|6980837
10
diol
7: 997-1008


K. oxytoca
small
gi|6980838
12
diol


Klebsialla
large
gi|4063702
105
diol
Tobimatsu et al.,


pneumoniae




1998; Biosci.


K. pneumoniae
medium
gi|94470233
107
diol
Biotechnol. Biochem.


K. pneumoniae
small
gi|4063704
109
diol
62: 1774-1777


Clostridium
large
gi|3360389
135
glycerol
Macis et al., FEMS


pasteurianum




Microbiol Lett. 1998;


C. pasteurianum
medium
gi|3360390
136
glycerol
164(1): 21-8


C. pasteurianum
small
gi|3360391
137
glyceral


Escherichia
large
gi|60099613
138
glycerol
Sönke et al.


blattae




J. of Mol. Micro. and


E. blattae
medium
gi|57340191
139
glycerol
Biotech. 2004; 8: 150-168


E. blattae
small
gi|57340192
140
glycerol
168


Klebsialla
large
gi|24158719
146
glycerol
Willard, Thesis


pneumoniae




(1994), U of


K. pneumoniae
medium
gi|24158720
148
glycerol
Wisconsin-Madison


K. pneumoniae
small
gi|24158721
150
glycerol


Citrobacter
large
gi|1169287
141
glycerol
Seyfried, Gottschalk;


freundii




J. Bacteriol.


C. freundii
medium
gi|1229154
142
glycerol
178: 5793-5796


C. freundii
small
gi|1229155
143
glycerol
(1996)


Lactobacillus
large
gi|116334196
164
diol
Schuetz and Radler


brevis




1984; Arch. Microbiol.


L. brevis
medium
gi|116334195
165
diol
139, 366-370


L. brevis
small
gi|116334194
166
diol


Lactobacillus
large
gi|18857678
99
diol
Sauvageot et al.,


collinoides




2002; Eur J Biochem.


L. collinoides
medium
gi|18857679
101
diol
269(22): 5731-7.


L. collinoides
small
gi|18857680
103
diol









The set of 8 amino acid sequences of each subunit from diol/glycerol dehydratases with experimentally determined function, listed in Table 10, were compared by making a multiple sequence alignment using ClustalW with default parameters. The % identity for the large subunit sequences ranged from 97.6% to 58.4%. The % identity for the medium subunit sequences ranged from 89.5% to 41.7%. The % identity for the small subunit sequences ranged from 83.3% to 36.4%. Thus the amount of sequence identity between some subunit sequences was very low (such as 36.4%, 41.7%) even though these subunits were known to be components of enzymes known through experimental data to perform the same function. The low level of % sequence identity made it impractical to use this criterion for structure/function correlations
Sequence Relationship of Experimentally Verified Diol/Glyderol Dehydratases to Other Diol/Glycerol Dehydratases
To perform this analysis, highly redundant sequences that are >95% identical were removed from the sequence set for large, medium or small subunits, except that all experimentally verified function sequences were retained. Truncated or partial protein sequences were also removed. A multiple sequence alignment was performed on the remaining sequences using ClustalW with default parameters. The % identity for the large subunits ranged from 97.6% (highest % is from multiple experimentally verified sequences) to 42.8%. The % identity for the medium subunits ranged from 91.9% to 26.4%. The % identity for the small subunits ranged from 85.2% to 20.5%. These % identities are similar ranges to the % identities for the experimentally verified sequences.
Based on the multiple sequence alignments, phylogenetic trees were built using the neighbor-joining algorithm (as implemented in the MEGA software version 3.1 package; Kumar et al., 2004 Briefings in Bioinformatics 5:150-163.) The phylogenetic trees are shown in FIGS. 2 (large subunit), 3 (medium subunit), and 4 (small subunit), with the identities of the mapped sequences listed in a key for each figure. As seen from the positions marked for the experimentally verified function sequences (in dark and light grey circles for diol dehydratase and glycerol dehydratase, respectively), these sequences are spread over most of the tree. However, each tree does include a branch with no experimentally verified members, but which appears to belong to the diol/glycerol dehydratase family.
Building a Profile Hidden Markov Model (HMM) of the Diol/Glycerol-Dehydratase Family Based on the Sets of Eight Subunit Sequences
An alternative structure/function characterization of the sets of subunits of the diol/glycerol dehydratase family of enzymes was performed using the HMMER software package (The theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531), following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, Va.).
Each set of 8 amino acid sequences for the large, medium, and small subunits of functionally characterized diol/glycerol dehydratases (in Table 10) was separately analyzed using the HMMER software program. The output of the HMMER software program is a Profile Hidden Markov Model (HMM) that characterizes the input sequences. As stated in the user guide, Profile HMMs are statistical models of multiple sequence alignments. They capture position-specific information about how conserved each column of the alignment is, and which amino acid residues are most likely to occur at each position. Thus HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus, Ashburn, Va.).
Each Profile HMM was built as follows:
Step 1. Build a Sequence Alignment
The eight sequences for the large subunit of the functionally verified diol/glycerol dehydratases (SEQ ID NOs:8, 99, 105, 135, 138, 141, 146, and 164) were aligned using Clustal W with default parameters. The same was done for the set of medium subunit sequences (SEQ ID NOs:10, 101, 107, 136, 139, 142, 148, and 165) and the set of small subunit sequences (SEQ ID NOs:12, 103, 109, 137, 140, 143, 150 and 166).
Step 2. Build a Profile HMM
The hmmbuild program was run on each set of the aligned sequences using default parameters. hmmbuild reads the multiple sequence alignment file, builds a new Profile HMM, and saves the Profile HMM to file. Using this program an un-calibrated profile was generated from the multiple alignment for each set of subunit sequences described above.
The following information based on the HMMER software user guide gives some description of the way that the hmmbuild program prepares a Profile HMM. A Profile HMM is capable of modeling gapped alignments, e.g. including insertions and deletions, which lets the software describe a complete conserved domain (rather than just a small ungapped motif). Insertions and deletions are modeled using insertion (I) states and deletion (D) states. All columns that contain more than a certain fraction x of gap characters will be assigned as an insert column. By default, x is set to 0.5. Each match state has an I and a D state associated with it. HMMER calls a group of three states (M/D/I) at the same consensus position in the alignment a “node”. These states are interconnected with arrows called state transition probabilities. M and I states are emitters, while D states are silent. The transitions are arranged so that at each node, either the M state is used (and a residue is aligned and scored) or the D state is used (and no residue is aligned, resulting in a deletion-gap character, ‘-’). Insertions occur between nodes, and I states have a self-transition, allowing one or more inserted residues to occur between consensus columns.
The scores of residues in a match state (i.e. match state emission scores), or in an insert state (i.e. insert state emission scores) are proportional to Log—2 (p_x)/(null_x). Where p_x is the probability of an amino acid residue, at a particular position in the alignment, according to the Profile HMM and null_x is the probability according to the Null model. The Null model is a simple one state probabilistic model with pre-calculated set of emission probabilities for each of the 20 amino acids derived from the distribution of amino acids in the SWISSPROT release 24.
State transition scores are also calculated as log odds parameters and are proportional to Log—2 (t_x). Where t_x is the probability of transiting to an emitter or non-emitter state.
Step 3. Calibrate the Profile HMM
Each Profile HMM was read using hmmcalibrate which scores a large number of synthesized random sequences with the Profile (the default number of synthetic sequences used is 5,000), fits an extreme value distribution (EVD) to the histogram of those scores, and re-saves the HMM file now including the EVD parameters. These EVD parameters (μ and λ) are used to calculate the E-values of bit scores when the profile is searched against a protein sequence database. hmmcalibrate writes two parameters into the HMM file on a line labeled “EVD”: these parameters are the μ (location) and λ (scale) parameters of an extreme value distribution (EVD) that best fits a histogram of scores calculated on randomly generated sequences of about the same length and residue composition as SWISS-PROT. This calibration was done once for each Profile HMM.
The calibrated Profiles HMM for the large subunit, medium subunit, and small subunit sets of sequences are provided in the appendix as alpha Profile HMM, beta Profile HMM, and gamma Profile HMM Excel charts. Each Profile HMM is provided in a chart that gives the probability of each amino acid occurring at each position in the amino acid sequence. The highest probability is highlighted for each position. Table 11 shows a few lines of the Profile HMM prepared for the large subunits of diol/glycerol dehydratases with function experimentally verified.







TABLE 11





A portion of the large subunit Profile HMM.


































The amino acids are represented by the one letter code.

The first line for each position reports the match emission scores: probability for each amino acid to be in that state (highest score is highlighted). The second line reports the insert emission scores, and the third line reports on state transition scores: M→M, M→I, M→D; I→M, I→I; D→M, D→D; B→M; M→E.

Table 11 shows that for the large subunits, methionine has a 4141 probability of being in the first position, the highest probability which is highlighted. In the second position lysine has the highest probability, which is 1954. In the third position arginine has the highest probability, which is 3077.
Step 4. Test the Specificity and Sensitivity of the Built Profile HMMs
Each Profile HMM was evaluated using hmmsearch, which reads a Profile HMM from hmmfile and searches a sequence file for significantly similar sequence matches. The sequence file searched was the GenBank non-redundant protein database. The size of the database (Z parameter) was set to 1 billion. This size setting ensures that significant E-values against the current database will remain significant in the foreseeable future. The E-value cutoff was set at 10.
The Profile HMMs for the large, medium and small subunits of diol/glycerol dehydratases with experimentally verified function were specific in that only diol/glycerol dehydratase subunits were recovered, as indicated by the annotation of the matched sequences, and sensitive in that even partial sequences of diol/glycerol dehydratase subunits were recovered. Each of the recovered sequences had an E-value of 0.01 or less.
All of the sequences on the phylogenetic trees in FIGS. 2, 3, and 4 were recovered in the Profile HMM matching. All sequences in the branches of the trees which contain no sequence with experimentally verified function were matched. Thus all of the diol and glycerol dehydratases were linked to the 8 diol and glycerol dehydratases with experimentally verified function through matching with the Profile HMMs for the large, medium, or small subunits of the enzymes. The full length diol and glycerol dehydratase subunits that match the Profile HMMs have the following SEQ ID NOs:
Large (alpha) subunits: 8, 93, 99, 105, 135, 138, 141, 146, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 130, 243, 254, 255, 256, 257, 258, and 259.
Large+medium subunits fused (large subunit and medium subunit portion matches the large Profile and medium Profile, respectively): 233, 235, 237, 239, 241, 246, and 247.
Medium (beta) subunits: 10, 95, 101, 107, 136, 139, 142, 148, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 244, 250, 252, 260, 261, 262, 263, 364, 265, 266, and 167.
Small (gamma) subunits: 12, 97, 103, 109, 137, 140, 143, 150, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 234, 236, 238, 240, 242, 245, 248, 249, 251, 253, 268, 270, 271, 272, 273, and 274.
This analysis shows that the Profile HMM for each subunit, that was prepared using sequences with experimentally verified function, provides a structure that is linked to function of diol/glycerol dehydratase enzymes. Matching of all of the above sequences to the Profiles HMM in turn provides a structure/function link for these sequences.
Example 19
Recombinant Expression of a B12-Independent Diol Dehydratase for Conversion of 2,3-Butanediol to 2-Butanone
The sequences encoding a putative B12-independent (S-adenosylmethionine (SAM)-dependent) propanediol dehydratase (SEQ ID NO: 277) and its putative associated reactivase (SEQ ID NO:279) in the bacterium Roseburia inulinivorans (Scott et al. (2006) J. Bacteriol. 188:4340-9), hereafter referred to as rdhtA and rdhtB, respectively, were synthesized as one DNA fragment (SEQ ID NO:284) by standard methods and cloned into an E. coli vector (by DNA2.0, Inc., Menlo Park, Calif.). The resulting clone was named pJ206::rdhtAB. The synthetic DNA fragment also contained a consensus ribosome binding site 5′ of the rdhtA coding region and terminal restriction sites recognized by BamHI (5′ end) and SalI (3′ end). The DNA fragment was transferred to pTrc99a (Amann et al. (1988) Gene 69:301-315) between the Trc promoter and rrnB termination sequence for expression in E. coli as an rdhtAB operon. The synthetic construct (about 150 ng) and the pTrc99a vector (about 50 ng) were digested with BamHI and SalI and ligated to form pTrc99a::rdhtAB, which was used to transform E. coli TOP10 cells. The structure of the plasmid was verified by PCR screening using primers Trc99a_For and Trc99a_Rev (provided as SEQ ID NOs:285 and 286), and the plasmid was then used to transform E. coli strain MG1655 (type strain, ATCC47076). The resulting strain (MG1655/pTrc99a::rdhtAB) was tested for its ability to metabolize 1,2-propanediol (5 g/L) during growth in M9 minimal medium (Davis et al., 1980, A Manual for Genetic Engineering, Advanced Bacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) supplemented with 2 g/L yeast extract and 50 μg/ml ampicillin. Cultures (40 mL in 50 ml serum vials sealed with narrow-seated rubber stoppers held by aluminum crimps) were incubated at 37° C. Propionaldehyde, the expected product of 1,2-propanediol dehydration, and its related metabolites propanol and propionate, were identified in the culture supernatants by the HPLC method described in General Methods giving the following retention times: propionaldehyde 32.6 min; propanol 38.4 min; and propionate 26.6 min; confirming that expression of the rdhtA and rdhtB genes provides 1,2-propanediol dehydratase activity.
After confirming function of the RdhtAB diol dehydratase with the proposed physiological substrate, in vivo activity was tested against two isomers of 2,3-butanediol: meso-BDO and (2S,3S)-(+)-BDO under the same culture conditions as described above, with either of the BDO isomers supplied at 5 g/L. At 24 hours supernatant samples were collected and analyzed by HPLC, and 2-butanone was identified in the culture containing meso-2,3-BDO, but not in the culture containing (+)-2,3-BDO or in control cultures of strains that did not contain the rdhtAB operon. These data demonstrated that this B12-independent enzyme is a butanediol dehydratase.
Example 20
Construction of a 2-Butanol Producing E. coli Strain
The vector pCL1925-ter (described in Example 9 above) was linearized by restriction digestion with HindIII, treated with the Klenow fragment of DNA polymerase (New England Biolabs, Beverly Mass., Cat. No. M0210), and digested with XhoI. The resulting 4.6 kb fragment was then treated with alkaline phosphatase (New England Biolabs, Cat. No. M0290). An rdhtAB gene fragment was prepared by first digesting pJ206::rdhtAB, described in Example 19, with BamHI and then treating with Klenow fragment. The product was digested with SalI, and the 3.4 kb rdhtAB fragment was isolated by gel purification. The 3.4 kb and 4.6 kb fragments were ligated to generate pCL1925::rdhtAB, and this plasmid was used to transform E. coli TOP10 chemically competent cells (Invitrogen, Cat. No. C404003) according to the manufacturer's instructions. In this construct, the rdhtAB operon is expressed from the glucose isomerase promoter. Transformed colonies (obtained by spectinomycin selection) were screened by PCR using primers N712 and N713 (SEQ ID NOs:287 and 288). The verified plasmid (pCL1925::RdhtAB) was prepared and transferred to E. coli MG1655 by electroporation and the resulting strain was named MG1655/pCL1925:rdhtAB. In vivo activity of the diol dehydratase was confirmed in this strain as described in Example 19.
A second plasmid was constructed to encode the remaining genes necessary for production of 2-butanol from glucose. First, a DNA fragment encoding a butanol dehydrogenase (sadB; protein of SEQ ID NO:289) from Achromobacter xylosoxidans (disclosed in co-owned and co-pending US patent application CL3926) was cloned into pTrc99a. The coding region (SEQ ID NO:290) was amplified using standard conditions from A. xylosoxidans genomic DNA, prepared using a Gentra Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.; catalog number D-5500A) following the recommended protocol for gram negative organisms using forward and reverse primers N473 and N469 (SEQ ID NOs:291 and 292), respectively. The PCR product was TOPO-Blunt cloned into pCR4 BLUNT (Invitrogen) to produce pCR4Blunt::sadB, which was transformed into E. coli Mach-1 cells. Plasmid was subsequently isolated from four clones, and the sequence verified. The plasmid was then digested with EcoRI, releasing the sadB fragment, which was ligated with EcoRI-digested pTrc99a (see Example 19) to generate pTrc99a::sadB. The sadB gene was added to pTrc99a::budABC (described in Example 9) by digesting pTrc99a:budABC and pTrc99a:sadB with EcoRI and then ligating the sadB fragment with linearized pTrc99a:budABC, yielding pTrc99a:sadBbudABC.
Example 21
Production of 2-Butanol by Recombinant E. coli Strain Expressing B12: Independent Diol Dehydratase
The plasmid pTrc99a:sadBbudABC (described in Example 20) was used to transform E. coli MG1655/pCL1925:rdhtAB (described in Example 20) to generate MG1655/pCL1925:rdhtAB/pTrc99a:sadBbudABC, a strain which contains all of the genes required for production of 2-butanol from glucose. The strain was grown in 25 mL M9 medium+2 g/L yeast extract+100 μg/mL ampicillin+50 μg/mL spectinomycin in sealed 50 mL serum vials, in four separate cultures. IPTG was added to 0.4 mM after 6 h, and samples were taken for HPLC analysis at 24, 48 and 96 h. A control strain containing pCL1925:budAB and pTrc99a was grown under the same conditions. 2-Butanol was detected in all 4 cultures of the production strain and in neither of the two cultures of the control strain, indicating that the pathway was fully functional and produced 2-butanol in the absence of coenzyme B12.
Example 22
Prophetic
Production of 2-Butanol by Recombinant S. cerevisiae Strain Expressing B12-Independent Diol Dehydratase
The clone described in Example 19 with a synthetic DNA fragment containing the sequences encoding RdhtA and RdhtB was used as a PCR template to prepare separate RdhtA and RdhtB coding region fragments. The RdhtA coding region for the diol dehydratase was amplified by PCR using primers N695 and N696 (SEQ ID NOs:293 and 294). The RdhtB coding region for the diol dehydratase activase, was amplified by PCR using primers N697 and N698 (SEQ ID NOs:295 and 296). The two DNA fragments were combined with a dual terminator DNA fragment (SEQ ID NO:297) that has an ADH terminator (SEQ ID NO:298) and a CYC1 terminator (SEQ ID NO:299) adjacent to each other in opposing orientation using SOE PCR (Horton et al. (1989) Gene 77:61-68). The dual terminator fragment was isolated as a 0.6 kb fragment following PacI digestion of pRS426::FBA-ILV5+GPM-kivD (described in co-owned and co-pending US Patent Publication #20070092957 A1, Example 17). The resulting 4 kb DNA fragment had the RdhtA and RdhtB coding regions in opposing orientation on either side of the dual terminator, with the 3′ end of each coding region adjacent to the dual terminator sequence. This DNA fragment was then cloned by gap repair methodology (Ma et al. (1987) Genetics 58:201-216) into the yeast shuttle vector pRS426::FBA-ILV5+GPM-kivD that was prepared by digestion with BbvCI to remove the ILV5 and kivD coding regions and dual terminator sequence between their 3′ ends. The resulting plasmid, pRS426::RdhtAB, contained the RdhtA gene under the control of the FBA promoter (SEQ ID NO:300) and the RdhtB gene under control of the GPM promoter (SEQ ID NO:301). The activity of the diol dehydratase in several of the yeast clones was confirmed by growing the yeast cells anaerobically in the presence of 1,2-propanediol and analyzing culture supernatants for the presence of propanol by GC or HPLC (as described in General Methods).
The FBA-RdhtA+GPM-RdhtB portion of pRS426::RdhtAB is then integrated into the yeast genome by homologous recombination, as follows. The region is amplified from the plasmid construct using primers N742A and N743A (SEQ ID NOs:302 and 303). Similarly, the MET15 marker is amplified from pRS421 (ATCC No. 87475) using primers N657 and N653A (SEQ ID NOs:304 and 305). These two DNA fragments are then combined using SOE PCR (Horton et al. (1989) Gene 77:61-68). The linear product is transformed into strain BY4741. Transformants are obtained on medium lacking methionine. Integration at the met15 locus is confirmed by PCR. Clones are tested for diol dehydratase activity as described above.
The resulting strain, BY4741 Δmet15::RdhtAB::MET15, is then transformed with butanediol pathway plasmids pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadB that were constructed as follows.
Construction of pRS423::CUP1-alsS+FBA-budA
The budA gene, encoding acetolactate decarboxylase, was amplified from genomic DNA prepared from Klebsiella pneumonia (ATCC #25955) using PhusionTM Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Inc.). The primers used (N579 and N580: SEQ ID NOs:306 and 307) added sequence upstream of the start codon that was homologous to the yeast FBA promoter and sequence downstream of the stop codon that was homologous to the yeast ADH terminator. Plasmid pRS423::CUP1-alsS+FBA-ILV3, which has a chimeric gene containing the CUP1 promoter (SEQ ID NO:308), alsS coding region from Bacillus subtilis (SEQ ID NO:309), and CYC1 terminator (SEQ ID NO:299) as well as a chimeric gene containing the FBA promoter (SEQ ID NO:300), ILV3 coding region from S. cerevisiae (SEQ ID NO:310), and ADH1 terminator (SEQ ID NO:298) (described in co-owned and co-pending US Patent Publication # US20070092957 A1, Example 17) was restriction digested with NcoI and PmlI to remove the ILV3 coding region. The 11.1 kb vector band was gel purified. Approximately 1 μg of cut vector DNA was combined with 1 μg of the budA PCR product and transformed into S. cerevisiae strain BY4741. The insert and vector were combined by homologous recombination in vivo to form a circular vector (also known as “gap repair cloning”; described in Ma et al. (1987) Genetics 58:201-216) that allows retention of the selectable marker (in this case, HIS3). Transformants were selected on synthetic complete medium lacking histidine. Colonies were patched to a new plate and cells from these patches were used to prepare plasmid DNA (ZymoprepTM Yeast Plasmid Miniprep Kit, Zymo Research). PCR was used to screen plasmids for the presence of alsS (primers N98SeqF1 and N99SeqR2: SEQ ID NOs:311 and 312) and for proper insertion of budA (N160SeqF1 and N84SeqR2, SEQ ID NOs:313 and 314).
Construction of pRS426::FBA-budC
The budC gene, encoding butanediol dehydrogenase, was amplified from genomic DNA prepared from Klebsiella pneumonia (ATCC #25955) using PhusionTM Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Inc.). The primers used (N581 and N582; SEQ ID NOs:315 and 316) added sequence upstream of the start codon that was homologous to the yeast FBA promoter and sequence downstream of the stop codon that was homologous to the yeast CYC1 terminator.
Expression plasmid pRS426::FBA::alsS was constructed via the following steps. The 1.7 kb alsS coding region fragment of pRS426::GPD::alsS::CYC was isolated by gel purification following BbvCI and PacI digestion. This plasmid has a chimeric gene containing the GPD promoter (SEQ ID NO:317), the alsS coding region from Bacillus subtilis (SEQ ID NO:309]), and the CYC1 terminator (SEQ ID NO:299) and was described in co-owned and co-pending US Patent Publication # US20070092957 A1, Example 17 which is herein incorporated by reference. The ILV5 fragment from plasmid pRS426::FBA::ILV5::CYC, also described in US20070092957 A1, Example 17, was removed by restriction digestion with BbvCI and PacI and the remaining 6.6 kb vector fragment was gel purified. This plasmid has a chimeric gene containing the FBA promoter (SEQ ID NO:300) and CYC1 terminator (SEQ ID NO:299) bounding the coding region of the ILV5 gene of S. cerevisiae (SEQ ID NO:318). These two purified fragments were ligated overnight at 16° C. and transformed into E. coli TOP10 chemically competent cells (Invitrogen). Transformants were obtained by plating cells on LB Amp100 medium. Insertion of alsS into the vector was confirmed by restriction digestion pattern and PCR using primers N98SeqF1 and N99SeqR2 (SEQ ID NOs:311 and 312).
The plasmid pRS426::FBA::alsS was digested with BbvCI and PacI to release the alsS fragment. The remaining linear vector was gel purified. Approximately 1 μg of vector was combined with 1 μg of budC PCR product and transformed into BY4741 to obtained gap repair clones (see above). Transformants were selected on synthetic complete medium lacking uracil. Plasmids were prepared from patches of 5 transformant colonies. The presence of FBA-budC was screened using PCR using primers N160SeqF1 and N582 (SEQ ID NOs:313 and 314).
Construction of pRS425::GPM-sadB
The sadB gene, encoding secondary alcohol dehydrogenase, was PCR amplified from pCR4Blunt::sadB, described in Example 20. The PCR primers contained additional 5′ sequences that overlap with the yeast GPM1 promoter and the ADH terminator (N583 and N584: SEQ ID NOs:322 and 323). The PCR product was then cloned using “gap repair” methodology in Saccharomyces cerevisiae (Ma et al. ibid) as follows. The yeast-E. coli shuttle vector pRS425::GPM::kivD::ADH which contains the GPM promoter (SEQ ID NO:301), kivD coding region from Lactococcus lactis (SEQ D NO:321), and ADH1 terminator (SEQ ID NO:298) (described in co-owned and co-pending US Patent Publication # US20070092957 A1, Example 17 which is herein incorporated by reference) was digested with BbvCI and PacI restriction enzymes to release the kivD coding region. Approximately 1 μg of the remaining vector fragment was transformed into S. cerevisiae strain BY4741 along with 1 μg of sadB PCR product. Transformants were selected on synthetic complete medium lacking leucine. The proper recombination event, generating pRS425::GPM-sadB, was confirmed by PCR using primers N142 and N459 (SEQ ID NOs:322 and 323).
Construction of pRS426::FBA-budC+GPM-sadB
The GPM-sadB-ADH promoter-gene-terminator cassette was transferred to pRS426 (ATCC No. 77107), a yeast-E. coli shuttle vector carrying the URA3 selection marker, by gap repair cloning. The cassette was isolated by digestion with SalI and SacII, and the vector was linearized with BamHI prior to ligation. The resulting vector, pRS426::GPM-sadB was confirmed by PCR using primers N142 and N459 (SEQ ID NOs:322 and 323. In order to add the budC gene encoding acetoin reductase from Klebsiella pneumonia to this vector, a fragment containing budC was excised from pRS423::FBA-budC+FBA-budA using SphI and SapI.
For construction of pRS423::FBA-budC+FBA-budA, the pRS423::CUP1-alsS+FBA-budA vector described above was digested with SacI and MluI to remove CUP1-alsS. SacII/MluI digestion was also used to isolate FBA-budC from pRS426::FBA-budC (described above). The appropriate fragments (7.6 kb vector fragment and 1.6 kb FBA-budC fragment) were gel purified, ligated and transformed into E. coli TOP10 competent cells (Invitrogen). Transformant colonies were screened by PCR to confirm incorporation of the budC fragment using primers N581 and N582 (SEQ ID NOs:315 and 316).
The SphI-SapI budC fragment from pRS423::FBA-budC+FBA-budA carries portions of the vector upstream of the FBA promoter as well as part of the ADH terminator to allow for cloning by gap repair cloning into the pRS426::GPM-sadB vector that was linearized with SacII. Transformants were plated on medium lacking uracil to select for recombination of the two linear sequences. The resulting vector, pRS426::FBA-budC+GPM-sadB was confirmed by PCR using primers N581 and N582 (SEQ ID NOs:315 and 316).
Strains of BY4741 Δmet15::RdhtAB::MET15 that are transformed with butanediol pathway plasmids pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadB are grown and the culture media assayed as described in General Methods. BDO and/or 2-butanol production is confirmed. It is expected that cells grown with vigorous aeration on glucose will produce BDO and ethanol, and that cells grown under more anaerobic conditions will convert some BDO to 2-butanol.








TABLE 12







HMMER2.0 [2.3.2]
Program name and version


NAME alpha_exp_seqs
Name of the input sequence alignment file


LENG 557
Length of the alignment: include indels


ALPH Amino
Type of residues


MAP yes
Map of the match states to the columns of the alignment


COM hmmbuild alpha.hmm alpha_exp_seqs.aln
Commands used to generate the file: this one means that hmmbuild (default patrameters)



was applied to the alignment file


COM hmmcalibrate alpha.hmm
Commands used to generate the file: this one means that hmmcalibrate (default parametrs)



was applied to the hmm profile


NSEQ 8
Number of sequences in the alignment file


DATE Fri Mar 30 19:02:15 2007
When was the file generated


XT-8455-4-1000-1000-8455-4-8455-4



NULT-4-8455
The transition probability distribution for the null model (single G state).


EVD -264.989197 0.112643
The extreme value distribution parameters μ and lambda respectively; both floating



point values. Lambda is positive and nonzero. These values are set when the model is



calibrated with hmmca


NULE
The symbol emission probability distribution for the null model (G state); consists of



K (e.g. 4 or 20) integers. The null probability used to convert these back to model



probabilities is nonzero 


 













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE 13













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE 14







HMMER2.0 [2.3.2]


NAME gamma_exp_seqs


LENG 175


ALPH Amino


RF no


CS no


MAP yes


COM hmmbuild gamma_hmm3 gamma_exp_s


COM hmmcalibrate gamma_hmm3


NSEQ 8


DATE Fri Mar 30 18:50:16 2007


CKSUM 2849

## Claims

1. A recombinant microbial host cell comprising at least one heterologous DNA molecule encoding a polypeptide that catalyzes the substrate to product conversions:
i) pyruvate to alpha-acetolactate;
ii) alpha-acetolactate to acetoin;
iii) acetoin to 2,3-butanediol; and
iv) 2,3-butanediol to 2-butanone;
wherein the at least one DNA molecule that catalyzes the substrate to product conversion of 2,3-butanediol to 2-butanone encodes a coenzyme B12-independent butanediol dehydratase, and further wherein the B12-independent butanediol dehydratase has an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:276 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix;

and wherein the microbial host cell produces 2 butanol.

2. The recombinant microbial host cell according to claim 1 wherein the B12-independent butanediol dehydratase has the amino acid sequence of SEQ ID NO:276.

3. A recombinant microbial host cell according to claim 1 further comprising a DNA molecule encoding a B12-independent butanediol dehydratase reactivase.

4. A recombinant microbial host cell according to claim 3 wherein the reactivase has an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:278 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

5. A recombinant microbial host cell according to claim 4 wherein the reactivase has the amino acid sequence of SEQ ID NO:278.

6. A method for the production of 2-butanone comprising:
a) providing the recombinant microbial host cell of claim 1 and a source of 2,3-butanediol; and
b) growing the microbial host cell of (a) under conditions whereby the 2,3-butanediol is converted to 2-butanone; and
c) optionally recovering the 2-butanone.

7. A method for the production of 2-butanol comprising:
a) providing the recombinant microbial host cell of claim 1 and a source of 2,3-butanediol; wherein the at least one DNA molecule is heterologous to said microbial host cell; and
b) growing the microbial host cell of (a) under conditions whereby the isolated nucleic acid molecule is expressed and the 2,3-butanediol is converted to 2-butanone, and the 2-butanone is converted to 2-butanol; and
c) optionally recovering the 2-butanol.

8. A recombinant microbial host cell according to claim 3 wherein the reactivase has an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:282 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

9. A recombinant microbial host cell according to claim 8 wherein the reactivase has the amino acid sequence of SEQ ID NO:282.

10. The recombinant microbial host cell of claim 1 further comprising at least one heterologous DNA molecule encoding a polypeptide that catalyzes the substrate to product conversion of 2-butanone to 2-butanol is heterologous to the microbial host cell.