Team:TorontoMaRSDiscovery/Bioinformatics

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<p style="font-size:18pt;">The diversity of bacterial microcompartments in nature and their potential for biotechnological applications</p>
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=Background information=
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==Summary==
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==Microcompartments:==
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Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2].  It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored.  We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology.  With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?
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===Overview:===
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==Background==
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Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2].  It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported (as reviewed below) the diversity of this phenomenon as well as the variety of enzymes involved have not been fully explored.  We used publicly available, sequence-based resources to extend what is known about the diversity of microcompartments and their associated enzymes.  An understanding of the natural biology of microcompartments will allow us to explore a variety of alternative approaches for our engineered system as well as help us to identify potential downstream applications.
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Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.
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===Classifications:===
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So far, most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.
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 +
[[image:TMDT_Nature.jpg|right|thumb|Figure 1:  The carbon concentrating mechanism in carboxysome.]]
#Carboxysomes
#Carboxysomes
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#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2].
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#*First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.
#''Pdu'' microcompartment
#''Pdu'' microcompartment
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#*In 1994, homologues of carboxysome shell proteins were reported in S. enterica. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment.  The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].
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#*In 1994, homologues of carboxysome shell proteins were reported in ''S. enterica''. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment.  The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].
#''Eut'' microcompartment
#''Eut'' microcompartment
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#*Later, similar structures were also found in E.coli and S. enterica when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].
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#*Later, similar structures were also found in ''E.coli'' and ''S. enterica'' when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].''   
#*''Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].''   
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#Other putative microcompartment structures
 
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#*In addition to the three major systems mentioned above, several other putative microcompartments have been inferred from gene clustering analyses:
 
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##A microcompartment was suggested to be involved in the oxidation of ethanol by Clostridium kluyveri. This was explored as an alternative platform to our current construction of encapsulin nanocomparment [9].
 
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##A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].
 
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##A putative microcompartment in Rhodopirellula baltica is proposed to be associated with a lactate dehydrogenase homologue [1].
 
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##A putative microcompartment in Carboxydothermus hydrogenoformans is associated with an isochorismatase-family protein [1].
 
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##A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].
 
 +
These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment.  However, several other putative microcompartments have been inferred by sequence similarity.  These include
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*A microcompartment was suggested to be involved in the oxidation of ethanol by ''Clostridium kluyveri''. [9].
 +
*A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].
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*A putative microcompartment in ''Rhodopirellula baltica'' is proposed to be associated with a lactate dehydrogenase homologue [1].
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*A putative microcompartment in ''Carboxydothermus hydrogenoformans'' is associated with an isochorismatase-family protein [1].
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*A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].
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===Identification of conserved protein domain===
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The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936  Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]
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Identification of new microcompartment is often achieved through bioinformatics analyses. (ie The pdu operon was first discovered as homologous to carboxysome genes.) The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain, Pfam00936, is known as the Bacterial Microcompartment (BMC) domain. It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]
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Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency.  Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in ''pdu'' and ''eut'' microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.
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==Methods/Results==
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===a) Search for enzyme pairs===
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[[image:TMDT_Pathway.jpg|right|thumb|Figure 2:  Complete pathway map obtained from KEGG database with the first enzyme colored in red:.]]
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In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:
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#Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion.
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#*Predictions based on free energy values: [[Media:Scored.xls|Scored.xls]]
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#Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.
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#*Enzyme pairs that possess a common metabolite that is known to be toxic: [[Media:ToxicPairs.xls|ToxicPairs.xls]]
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#Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.
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#*Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: [[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]
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#*Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: [[Media:SwissProtPairs.xls|SwissProtPairs.xls]]
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These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2).  In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated.  The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:
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===Applications in nature===
 
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[[image:TMDT_Nature.jpg|right|thumb|Figure 1:  The carbon concentrating mechanism in carboxysome.]]
 
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#Microcompartments are used to substantially catalyze a particular metabolic pathway by sequestering or co-localizing multiple metabolically related enzymes. This is true for all microcompartment structures discussed above because the product of one enzymes reaction will be in extreme vicinity to the next enzyme and will be delivered at high concentration as a substrate for the next enzyme. This prevents any potential diffusion loss of substrate and greatly enhances the enzymatic efficiency.
 
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#Microcompartments are used to occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in pdu and eut microcompartments. The degradation pathway of 1,2 propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria are using microcompartment to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.
 
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=Future Directions=
 
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[[image:TMDT_Comparison.jpg|right|thumb|Figure 2:  Table containing cross-sections between thermodynamic, toxic intermediate, SwissProt, and RossetteStone categories.]]
 
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Our ultimate goal is to exploit an understanding of bacterial microcompartments to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With those two objectives in mind, we attempted to address the following two questions using various bioinformatics tools: (1) What enzymes will benefit the most from enzymatic channeling? and (2) Is there any other alternative microcompartment chassis that could be explored for enzymatic channeling engineering?
 
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==1. What enzymes will benefit the most from enzymatic channeling?'''==
 
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We’d like to identify several enzyme pairs that display observable benefit from enzymatic channeling. Our approach to address this question is proposed based on the following assumptions:
 
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#Enzymes pair with specific thermodynamic energetic properties will likely to benefit from enzymatic channeling. For two sequential reactions, the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion. 
 
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#Enzymes involved in reactions with toxic intermediates will likely to benefit from enzymatic channeling.
 
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#Enzymes that were identified as gene fusion products in other organisms will likely to benefit from enzymatic channeling.
 
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===Based on these three premises, the following enzyme tables were constructed:===
 
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*Thermodynamic properties:
 
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**Scored.csv: This is the list of all predictions based on free energy values.
 
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*Toxic intermediates:
 
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**[[Media:ToxicPairs.xls|ToxicPairs.xls]]: Enzyme pairs that possess a common metabolite that is known to be toxic.
 
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*Gene fusion or multicomplex proteins:
 
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**[[Media:RosettaEnzymePairs.xls|RosettaEnzymePairs.xls]]: The enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis.
 
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**[[Media:SwissProtPairs.xls|SwissProtPairs.xls]]: The enzyme pairs annotated to form multifunctional enzymes in SwissProt database.
 
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By looking up their EC numbers in BRENDA database, the enzymes that are not immediate adjacent to each other in the pathway are eliminated. The enzymes with molecular weight well beyond microcompartment range (100 kDa) are also eliminated from the final table. The remaining enzyme pairs are considered as primary candidates for enzymatic channeling (Fig.2).
 
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For example:
 
{| border="1" cellpadding="5" cellspacing="0"
{| border="1" cellpadding="5" cellspacing="0"
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| align="center" |'''Enzyme1 EC'''
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| align="center" |'''Enzyme1 Size (kDa)'''
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| align="center" |'''Enzyme2 EC'''
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| align="center" |'''Enzyme2 Size (kDa)'''
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| align="center" |'''Source'''
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| align="center" |'''Pathway'''
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| align="center" |'''Major Product'''
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| align="center" |'''Applications'''
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|-
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|'''EC 1'''
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| 2.1.3.2||32-125||6.3.4.4||47-96||Rosette||Alanine and aspartate metabolism||adenylosuccinate||
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|'''Molecular Weight 1'''
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|'''EC 2'''
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|'''Molecular Weight 2'''
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|'''Sources'''
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|'''Pathway Involved'''
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|'''Final Product'''
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|-
|-
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|2.4.1.15
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| 1.1.1.1||25-57||1.2.1.10||96.7-520||Swiss Prot||Butanoate metabolism ||Butanoyl-coA||
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|45-630
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|-
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|3.1.3.12
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| 1.1.1.95||35-250||2.6.1.52||35-96||Rosette||Glycine, serine and threonine metabolism ||Phosphoserine||
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|25-973
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|-
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|Rosette
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| 2.3.3.9||52-81||4.1.3.1||14-65||Swiss Prot &Rosette||Glyoxylate and dicarboxylate metabolism||Isocitrate||
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|Starch and sucrose metabolism
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|-
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|a,a trehalose
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| 1.5.1.15||32-34||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||
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|-
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| 1.5.1.5||30-150||3.5.4.9||30-45||Swiss Prot||One carbon pool by folate||Formyl-THF||
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|-
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| 1.1.1.57||||5.3.1.12||||Rosette||Pentose and glucuronate interconversions||D-Glucuronate||
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|-
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| 2.2.1.1||100-141||5.1.3.1||45-200||Rosette||Pentose phosphate pathway||D-ribolose-5P||
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|-
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| 2.2.1.2||35-75||5.3.1.9||27-67||Rosette||Pentose phosphate pathway||a-glucose-6P||
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|-
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| 1.3.1.13||52-210||4.2.1.51||12-137||Rosette||Phenylalanine, tyrosine and tryptophan biosynthesis||Phenyl-pyruvate||
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|-
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| 2.3.1.8||35-71||2.7.2.15||43||Rosette||Propanoate metabolism||Propanoate||Food preservatives
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|-
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| 2.3.1.8||35-70||2.7.2.1||12 to 66||Rosette||Propanoate metabolism/Pyruvate metabolism||Propanoate/acetate||Energy
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|-
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| 2.4.2.10||39-140||4.1.1.23||14-64||Swiss Prot||Pyrimidine metabolism||UMP||
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|-
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| 2.4.2.1||45-86||2.4.2.3||32-160||Rosette||Pyrimidine metabolism||Uridine||
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|-
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| 2.4.1.15||45-630||3.1.3.12||25-973||Rosette||Starch and sucrose metabolism||a,a trehalose||biotechnology application
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|-
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| 1.2.1.41||41-189||2.7.2.11||47-354||Swiss Prot &Rosette||Urea cycle and metabolism of amino groups||Glutamate||Flavor enhancer, Plant growth
|}
|}
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===b) Alternative microcompartments===
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[[image:TMDT_Pathway.jpg|right|thumb|Figure 3:  Complete pathway map obtained from BRENDA database with the first enzyme colored in red:.]]
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==2. What alternative microcompartment chassis could be explored?==
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Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.
Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.
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One of the putative microcompartment systems in Clostridium kluyveri associated with the oxidation of ethanol, however, shows great promises as an alternative microcompartment chassis because:
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One of the putative microcompartment systems in ''Clostridium kluyveri'' associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:
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#The gene cluster involved contains only seven genes: including two genes for two almost identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of Salmonella typhimurium [9].
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#The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of ''Salmonella typhimurium'' [9].
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#From cell extracts of C. kluyveri a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].
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#From cell extracts of ''C. kluyveri'' a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].
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In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted.  To search for a consensus targeting sequence we obtained the ''C. kluyveri'' compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria.  We used the following two query sequences:  
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===Microcompartment protein gene sequences retrieved from NCBI database:===
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{| border="1"  cellspacing="0" width="50%"
{| border="1"  cellspacing="0" width="50%"
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PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the [[Media:1072_3 BLAST.pdf|BLAST results]] and [[Media:taxonomy_report.txt|taxonomy report]].A [[Media:MUSCLE_alignment.pdf|multiple alignment]] of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence.
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Position Specific Iterative (PSI) BLAST was conducted on these two sequences respectively. PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top 50 results were saved after 5 iterations. (please add linkout pages to the following documents)
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Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter ''et al''. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif.  We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.
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CKL_1072 Hit Table
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==Conclusions==
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CKL_1072 Taxonomy Report
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We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues.  In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the ''Clostridium kluyveri'' microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes.  The ''Clostridium kluyveri'' system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment.  However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.
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CKL_1073 Hit Table
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==Future Work==
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CKL_1073 Taxonomy Report
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In order to find the targeting sequence for the ''Clostridium kluyveri'' microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames.  In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.
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Later, the top 50 hit genes and their adjacent genetic environment (10 genes 5’ upstream and 3’ downstream of the hit gene) were aligned using MUSCLE alignment. This is mainly used to identify any potential conserved sequences as targeting sequences to translocate enzymes into the microcompartment.  
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==References==
 +
1.Cheng, S., ''et al''., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.
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2.Yeates, T.O., ''et al''., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.
-
=References=
+
-
1.Cheng, S., et al., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.
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-
 
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2.Yeates, T.O., et al., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.
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3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.
3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.
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8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.
8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.
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9.Seedorf, H., et al., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.
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9.Seedorf, H., ''et al''., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.
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10.Wackett, L.P., et al., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.
+
10.Wackett, L.P., ''et al''., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.
11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.
-
12.Yeates, T.O., et al., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.
+
12.Yeates, T.O., ''et al''., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.
 +
 
 +
13.Tanaka, S., ''et al''., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.
-
13.Tanaka, S., et al., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.
+
14.Sutter, M. ''et al''. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).

Latest revision as of 03:03, 22 October 2009

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The diversity of bacterial microcompartments in nature and their potential for biotechnological applications

Contents

Summary

Spatial segregation is widely believed to be a defining organizational feature of eukaryotic cells: proteins, nucleic acids and small molecules are contained within and often actively transported between the many membrane-bound, subcellular organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, it has recently been found that a number of bacteria conditionally express proteinaceous microcompartments. These polyhedral organelles are usually 100-150 nm in cross section [1] and consist of proteinaceous outer shells, reminiscent of viral capsids, surrounding a core of enzymes[2]. It is thought that microcompartments allow bacteria to sequester specific metabolic enzymes and their substrates to enhance enzymatic efficiency (enzyme channeling) and protect cells from the toxic effects of certain intermediates. While several examples of these compartments have been reported their diversity has not been fully explored. We wish to design new microcompartments with modified properties or novel enzymatic activities, which could result in potentially useful applications in biotechnology. With this goal in mind, we attempted to address the following two questions using a bioinformatics approach (1) What enzymes will benefit the most from enzymatic channeling? and (2) Are there any other alternative microcompartments that could be explored for enzymatic channeling engineering?

Background

Most of our knowledge on bacterial microcompartments has been derived from the three well-studied microcompartment systems in nature.

Figure 1: The carbon concentrating mechanism in carboxysome.
  1. Carboxysomes
    • First reported in 1956, carboxysomes were the first bacteria microcompartments to be discovered. They are often present in cyanobacteria and other chemoautotrophic bacteria [3]. They are known to play a key role in enhancing autotrophic carbon fixation in the Calvin cycle. The shell of the carboxysome encodes the enzymes carbonic anhydrase (CA) and ribulose bis-phosphate carboxylase monooxygenase (RuBisCO). CA converts bicarbonate ions into carbon dioxide, which is then converted into 3-phosphoglycerate (3-PGA) by RuBisCO. Carboxysome not only allows for co-localization of CA and RuBisCO, but also acts as a diffusion barrier to retain carbon dioxide in the immediate vicinity of RuBisCO and thus catalyzes the conversion [2]. See Figure 1.
  2. Pdu microcompartment
    • In 1994, homologues of carboxysome shell proteins were reported in S. enterica. They are amongst a cluster of genes that are involved in coenzyme B12-dependent metabolism of 1,2 propanediol [4]. The gene cluster was later termed the pdu operon and the microcompartment formed was later termed propanediol utilization microcompartment. The proposed fuction of the pdu microcompartment is to encapsulate the enzymes that are necessary for cell to degrade propanediol and most importantly, to protect the cell from the toxic effects of propionaldehyde, an intermediate formed during the process [5].
  3. Eut microcompartment
    • Later, similar structures were also found in E.coli and S. enterica when they were grown using ethanolamine as energy source. These structures were named ethanolamine utilization microcompartment (encoded by eut operon) and they often display very high genetic similarity with pdu microcompartment. Eut microcompartments contain enzymes involved in the degradation of ethanolamine and protect cell from acetaldehyde [6-8].
    • Generally speaking, both pdu and eut microcompartments are less uniform in size and more irregular in shape than carboxysomes[2].

These compartments are generally unsuitable for our purposes because a) Their shell proteins consist of several different types whose functions are not well characterized and b) It is not known how the associated enzymes are targeted to the microcompartment. However, several other putative microcompartments have been inferred by sequence similarity. These include

  • A microcompartment was suggested to be involved in the oxidation of ethanol by Clostridium kluyveri. [9].
  • A microcompartment associated with a puryvate-formate lyase homolog is proposed to be involved in the production of ethanol from pyruvate [10].
  • A putative microcompartment in Rhodopirellula baltica is proposed to be associated with a lactate dehydrogenase homologue [1].
  • A putative microcompartment in Carboxydothermus hydrogenoformans is associated with an isochorismatase-family protein [1].
  • A putative microcompartment Solibacter usitatus can be associated with a dihydrdipicolinate synthase homologue [1].

The observation that diverse microcompartment structures are composed of proteins with homologous sequences led to the identification of a protein domain in the shell of all polyhedral bacterial microcompartments. This conserved protein domain is known as the Bacterial Microcompartment (BMC) domain ([http://pfam.sanger.ac.uk/family?acc=PF00936 Pfam00936]). It is approximately 84 amino acids long and can be either found as a part of a large protein or in tandem copies within the same operon. This domain is found to be present in 189 bacterial species to date. [2]

Microcompartments typically promote the catalysis of a particular metabolic reaction by sequestering or co-localizing functionally related enzymes. The product of one enzyme will be delivered to the next enzyme at high concentration since it cannot readily diffuse. This greatly enhances enzyme efficiency. Microcompartments can also occlude toxic intermediates that cannot be degraded by normal bacterial machinery. This was shown to be true in pdu and eut microcompartments. The degradation pathway of 1,2-propanediol and ethanolamine both proceed through aldehyde intermediates (propionaldehyde and acetaldehyde respectively). The toxicity of these aldehyde intermediates was later shown in growth assays in which cells accumulating large amounts of propionaldehyde underwent growth arrest due to propionaldehyde toxicity [5, 11]. Therefore, it is reasonable to propose that bacteria use microcompartments to mitigate the toxicity of aldehyde by encapsulating these poisonous intermediates.

Methods/Results

a) Search for enzyme pairs

Figure 2: Complete pathway map obtained from KEGG database with the first enzyme colored in red:.

In order to identify candidate enzyme pairs likely to display an observable benefit from enzyme channeling we made the following assumptions:

  1. Enzyme pairs with specific thermodynamic properties will likely benefit. Specifically, for two sequential reactions the second one will be thermodynamically more favorable than the first reaction. Consequently, the products from the first reaction be consumed at a faster rate than being generated, hence driving the reaction to completion.
    • Predictions based on free energy values: Scored.xls
  2. Enzymes involved in reactions with toxic intermediates will likely benefit from enzymatic channeling since the toxin will be less likely to interact with cellular components.
    • Enzyme pairs that possess a common metabolite that is known to be toxic: ToxicPairs.xls
  3. Enzymes that were identified as gene fusion products in other organisms will likely benefit from enzyme channeling since fusion of multi-functional enzymes is an extreme example of colocalization.
    • Enzyme pairs predicted to form multifunctional enzymes by the Prolinks Rosetta Stone analysis: RosettaEnzymePairs.xls
    • Enzyme pairs annotated to form multifunctional enzymes in SwissProt database: SwissProtPairs.xls

These results provided us with a combined list that was further refined by removing enzymes that are not immediately adjacent to each other in their biochemical pathway based on the KEGG database (Figure 2). In addition, enzymes with a molecular weight well beyond the microcompartment capacity (100 kDa) were also eliminated. The remaining enzyme pairs meeting these criteria were considered candidates for enzyme channeling and have been summarized in the following table:


Enzyme1 EC Enzyme1 Size (kDa) Enzyme2 EC Enzyme2 Size (kDa) Source Pathway Major Product Applications
2.1.3.232-1256.3.4.447-96RosetteAlanine and aspartate metabolismadenylosuccinate
1.1.1.125-571.2.1.1096.7-520Swiss ProtButanoate metabolism Butanoyl-coA
1.1.1.9535-2502.6.1.5235-96RosetteGlycine, serine and threonine metabolism Phosphoserine
2.3.3.952-814.1.3.114-65Swiss Prot &RosetteGlyoxylate and dicarboxylate metabolismIsocitrate
1.5.1.1532-343.5.4.930-45Swiss ProtOne carbon pool by folateFormyl-THF
1.5.1.530-1503.5.4.930-45Swiss ProtOne carbon pool by folateFormyl-THF
1.1.1.575.3.1.12RosettePentose and glucuronate interconversionsD-Glucuronate
2.2.1.1100-1415.1.3.145-200RosettePentose phosphate pathwayD-ribolose-5P
2.2.1.235-755.3.1.927-67RosettePentose phosphate pathwaya-glucose-6P
1.3.1.1352-2104.2.1.5112-137RosettePhenylalanine, tyrosine and tryptophan biosynthesisPhenyl-pyruvate
2.3.1.835-712.7.2.1543RosettePropanoate metabolismPropanoateFood preservatives
2.3.1.835-702.7.2.112 to 66RosettePropanoate metabolism/Pyruvate metabolismPropanoate/acetateEnergy
2.4.2.1039-1404.1.1.2314-64Swiss ProtPyrimidine metabolismUMP
2.4.2.145-862.4.2.332-160RosettePyrimidine metabolismUridine
2.4.1.1545-6303.1.3.1225-973RosetteStarch and sucrose metabolisma,a trehalosebiotechnology application
1.2.1.4141-1892.7.2.1147-354Swiss Prot &RosetteUrea cycle and metabolism of amino groupsGlutamateFlavor enhancer, Plant growth

b) Alternative microcompartments

Upon further examination, most of our known microcompartments tend to consist of a large number of protein subunits with very complex structures. Because of their complicated regulatory patterns and stoichiometry, these structures are not ideal to be used in enzymatic channeling engineering.

  • Carboxysome: 80-150 nm in cross section. Composed of several thousands polypeptide of 10-15 different types. Contain as many as 250 RuBisCO per carboxysome molecule[12, 13].
  • Pdu Microcomparment: 100-150 nm in cross section. Composed of about 18000 individual polypeptides of about 14-18 different types [1].
  • Eut Microcomparment: Similar to pdu microcompartment in terms of both size and protein composition.


One of the putative microcompartment systems in Clostridium kluyveri associated with the oxidation of ethanol, however, shows promise as an alternative microcompartment for use in our system because:

  1. The gene cluster involved contains only seven genes: including two genes for a pair of nearly identical acetaldehyde dehydrogenases, three genes for highly similar ethanol dehydrogenases, and two genes for microcompartment proteins, which are orthologs of ethanolamine using genes (eutML) of Salmonella typhimurium [9].
  2. From cell extracts of C. kluyveri a macromolecular complex of ethanol dehydrogenase and acetaldehyde dehydrogenase can be purified by differential manganese sulfate precipitation, indicating the presence of a functional and intact microcompartment enclosing these two enzymes[9].

In order to co-opt this stucture for our own use it is necessary to understand how the associated enzymes are targeted to the compartment so that alternative enzymes of our choosing can be substituted. To search for a consensus targeting sequence we obtained the C. kluyveri compartment sequence from NCBI and performed a position specific iterative Blast search for related sequences among bacteria. We used the following two query sequences:

CKL_1072
153953697|ref|YP_001394462.1| microcompartment protein [Clostridium kluyveri DSM 555]
MGQEALGMIETKGLVGAIEAADSMVKAANVALIGYEKIGSGLVTVMVRGDVGAVKAATDAGAASAKRVGE

VISVHVIPRPHTDVEKILPNIG


CKL_1073
153953698|ref|YP_001394463.1| microcompartment protein [Clostridium kluyveri DSM 555]
MNNELIEKVLGEVRKSLDLKNFDQEKLNKVVESTTEKLSDSKKEEAIKEAKPDVKVAEESKQAVVEQKAN

DVKTAPTMTEFVGTAGGDTVGLVIANVDSLLHKHLGLDNTCRSIGIISARVGAPAQMMAADEAVKGTNTE VATIELPRDTKGGAGHGIFIVLKAADVSDARRAVEIALKQTDKYLGNVYLCDAGHLEVQYTARASLIFEK AFGAPSGQAFGIMHAAPAGVGMIVADTALKTADVKLITYGSPTNGVLSYTNEILITISGDSGAVLQSLTA ARKAGLSILRSMGQDPVSMSKPTF

PSI-BLAST constructs and modifies the Position Specific Scoring Matrix constantly and uses it during the next round of search. This iterative searching strategy results in increased sensitivity. The top results were saved after 5 iterations. Download the BLAST results and taxonomy report.A multiple alignment of these sequences was constructed using CLCBio Genomic Workbench to assess the level of conservation between species and identify consensus sequence.

Since microcompartment proteins are known to be found adjacent to their corresponding enzymes, we searched the top hits for adjacent genes 5’ upstream and 3’ downstream of the hit gene. We then attempted to apply multiple sequence alignment to these sequences in an effort to identify conserved motifs corresponding to a potential targeting sequence similar to the method employed by Suter et al. A targeting sequence, if found, would allow translocation of enzymes into the microcompartment. Due to the incomplete annotation of certain genomes, the alignments did not yield a statistically significant targeting motif. We are attempting further analyses using different reading frames to refine the protein candidates used during the multiple alignment.

Conclusions

We have short-listed a number of enzymes which might benefit from channeling according to several properties such as free energy, toxicity and the occurrence of multi-functional homologues. In addition, while concluding that several classic examples of bacterial microcompartment are unsuitable for experimental manipulation, we have conducted a sequence-based search for additional protein family members corresponding to the Clostridium kluyveri microcompartment and searched the corresponding upstream and downstream genes for conserved motifs in an effort to find the targetting sequence for these enzymes. The Clostridium kluyveri system remains under consideration as a potentially viable alternative to the encapsulin-based nanocompartment. However, its suitability will depend on the results of further efforts to identify a targeting sequence for this compartment.

Future Work

In order to find the targeting sequence for the Clostridium kluyveri microcompartment, it will be necessary to fine tune the enzyme queries and potentially check out other alternative reading frames. In addition, enzymes will be selected from those short-listed above to proceed with a proof of concept experiment and potentially commercially viable application.

References

1.Cheng, S., et al., Bacterial microcompartments: their properties and paradoxes. Bioessays, 2008. 30(11-12): p. 1084-95.

2.Yeates, T.O., et al., Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol, 2008. 6(9): p. 681-91.

3.Shively, J.M., Inclusion bodies of prokaryotes. Annu Rev Microbiol, 1974. 28(0): p. 167-87.

4.Chen, P., D.I. Andersson, and J.R. Roth, The control region of the pdu/cob regulon in Salmonella typhimurium. J Bacteriol, 1994. 176(17): p. 5474-82.

5.Havemann, G.D., E.M. Sampson, and T.A. Bobik, PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol, 2002. 184(5): p. 1253-61.

6.Brinsmade, S.R., T. Paldon, and J.C. Escalante-Semerena, Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol, 2005. 187(23): p. 8039-46.

7.Penrod, J.T. and J.R. Roth, Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol, 2006. 188(8): p. 2865-74.

8.Stojiljkovic, I., A.J. Baumler, and F. Heffron, Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol, 1995. 177(5): p. 1357-66.

9.Seedorf, H., et al., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2128-33.

10.Wackett, L.P., et al., Genomic and biochemical studies demonstrating the absence of an alkane-producing phenotype in Vibrio furnissii M1. Appl Environ Microbiol, 2007. 73(22): p. 7192-8.

11.Sampson, E.M. and T.A. Bobik, Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. J Bacteriol, 2008. 190(8): p. 2966-71.

12.Yeates, T.O., et al., Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells. Biochem Soc Trans, 2007. 35(Pt 3): p. 508-11.

13.Tanaka, S., et al., Atomic-level models of the bacterial carboxysome shell. Science, 2008. 319(5866): p. 1083-6.

14.Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat Struct Mol Biol (2008).