Revision as of 02:47, 28 September 2013

Caffeine

Caffeine (1,3,7-trimethylxanthine) is a powerful stimulant consumed in large quantities worldwide. Originally found in just coffee and tea beverage, caffeine has found its way into many different consumer products, like energy drinks (1). Following the trend of increased consumption, more and more research into the health effects of caffeine and more economical methods of production is being performed.

Currently, the most common method for the isolation of caffeine is the decaffeination of commercially harvested plants, such as green tea leaves and Coffea arabica beans. The ecological and financial burden of current caffeine production methods makes an alternative approach, such as microbial synthesis achieved through synthetic biology, much more desirable. While significant gains have been made with the microbial degradation of caffeine, its synthesis in a biological chassis is far less successful (4,5).

Rationale

The great demand for caffeine in food products and beverages makes this compound an exciting one to target for synthesis and incorporation in our ultrabiotic. The caffeine biosynthetic pathway serves as part of a proof-of-concept showing that the CRISPR system could be used to alter population dynamics in bioreactors through immunity to phage. By providing CRISPR immunity to a common phage and not to a certain rare phage, the system would allow the removal of caffeine-producing bacteria in a bioreactor through the addition of that rare phage. This has the potential to create a caffeinated or "decaffeinated" product. The mechanism of tunable populations is demonstrated in the “population control” section of our Wiki.

Objective

Our goal was to implement the caffeine biosynthetic pathway in E. coli to enable the production of caffeine in a bioreactor. This work would largely build on the previous efforts of the 2012 TU Munich team, which successfully obtained caffeine precursors when expressing caffeine’s biosynthetic machinery in yeast.

Chemistry

Caffeine biosynthesis occurs by successive modifications to purine nucleotide precursors such as AMP and GMP. Starting with xanthosine as substrate, the pathway below depicts the final stage of caffeine synthesis in organisms such as Coffee arabica. Xanthosine methyltransferase (XMT) starts by methylating xanthosine with the ubiquitous methyl donor S-adenosyl-L-Met (SAM), which acts as methyl donor for all subsequent methylations in the pathway. XMT also catalyzes the second step of the pathway, the removal of the ribose group.

Characterization

We obtained the three genes required for caffeine biosynthesis from the 2012 TU Munich. This included CaXMT, CaMXMT, and CaDXMT, which had previously been based on the natural plant genes and codon-optimized for expression in yeast. While not codon-optimized for expression in bacteria, believed that we could express them first in a rare codon-supplemented bacteria such as Rosetta Cells™. That way we could determine if it was possible for these three enzymes to be well expressed and obtained in an active form in bacteria before proceeding to the more costly step of codon-optimizing them for bacterial expression.

We proceeded by removing the yeast ribosome-binding site (rbs) and replacing it with a bacterial Shine-Dalgarno sequence. Each gene was cloned separately under an arabinose promoter and a constitutive promoter, in addition to a construct with all three under the same promoter. Expression was tested in a variety of conditions in E. cloni® 10G cells, but no expression was detected (Figure 1). This was expected as the genes are not optimized for normal bacterial expression. Unfortunately, Rosetta Cells™ already contain a chloramphenicol plasmid, pRARE, and as such, these caffeine constructs could not yet be transformed into the Rosetta Cells for further expression testing. Efforts are now under way for a kanamycin resistance marker to be cloned into each caffeine enzyme construct to allow for compatibility with the Rosetta Cells™.

Figure 1. DESCRIPTION

Sources

(1) http://link.springer.com/chapter/10.1007/978-1-4419-0338-9_26
(2) http://www.plantphysiol.org/content/132/1/372
(3) http://www.tandfonline.com/doi/full/10.1080/15422119.2013.771127
(4) http://connection.ebscohost.com/c/articles/79461823/biodegradation-caffeine-by-trichosporon-asahii-isolated-from-caffeine-contaminated-soil

(5) https://www.jstage.jst.go.jp/article/bbb1992/57/8/57_8_1286/_article

@@ Line 4: / Line 4: @@
 <p>
-Caffeine (1,3,7-trimethylxanthine) is arguably the most popular stimulant and psychoactive substance consumed in recent times. This is evident from the huge demand for coffee and tea beverages and increasingly energy drinks, all of which are common dietary sources of caffeine (1). Following the trend of increasing consumption, much research into caffeine’s effects on health and cost-effective methods of production have been done.</p>
+Caffeine (1,3,7-trimethylxanthine) is a powerful stimulant consumed in large quantities worldwide. Originally found in just coffee and tea beverage, caffeine has found its way into many different consumer products, like energy drinks (1). Following the trend of increased consumption, more and more research into the health effects of caffeine and more economical methods of production is being performed. </p>
-<p>
-Currently, methods of isolating caffeine for use as an additive include the decaffeination of commercially harvested plants, such as green tea leaves and <i>Coffea arabica</i> beans. The main enzymes for caffeine biosynthesis, and thus caffeine itself, accumulate in seeds, cotyledons, and leaves of these plants (2). This is an expensive process involving large quantities of organic solvents or water, or specialized techniques such as supercritical fluid extraction with carbon dioxide (3).</p>
 <p>
-The ecological and financial burden of current caffeine production methods makes an alternative approach (microbial synthesis via synthetic biology) desirable. While there has been significant research done on the degradation of caffeine using microbes (4,5), synthesizing caffeine in a biological chassis is less common in the literature. Our goal was to implement the caffeine biosynthetic pathway (using xanthine as substrate) in E. coli to enable the production of caffeine in a bioreactor. This is largely building on the work of the [https://2012.igem.org/Team:TU_Munich 2012 TU Munich team], which successfully got caffeine precursors expressed in yeast.</p>
+Currently, the most common method for the isolation of caffeine is the decaffeination of commercially harvested plants, such as green tea leaves and <i>Coffea arabica</i> beans. The ecological and financial burden of current caffeine production methods makes an alternative approach, such as microbial synthesis achieved through synthetic biology, much more desirable. While significant gains have been made with the microbial degradation of caffeine, its synthesis in a biological chassis is far less successful (4,5). </p>
@@ Line 16: / Line 14: @@
 The great demand for caffeine in food products and beverages makes this compound an exciting one to target for synthesis and incorporation in our ultrabiotic. The caffeine biosynthetic pathway serves as part of a proof-of-concept showing that the CRISPR system could be used to alter population dynamics in bioreactors through immunity to phage. By providing CRISPR immunity to a common phage and not to a certain rare phage, the system would allow the removal of caffeine-producing bacteria in a bioreactor through the addition of that rare phage. This has the potential to create a caffeinated or "decaffeinated" product. The mechanism of tunable populations is demonstrated in the “population control” section of our Wiki.
+==Objective==
+<p>
+Our goal was to implement the caffeine biosynthetic pathway in <i>E. coli</i> to enable the production of caffeine in a bioreactor. This work would largely build on the previous efforts of the [https://2012.igem.org/Team:TU_Munich 2012 TU Munich team], which successfully obtained caffeine precursors when expressing caffeine’s biosynthetic machinery in yeast. </p>
@@ Line 31: / Line 36: @@
 ==Characterization==
+<p>We obtained the three genes required for caffeine biosynthesis from the [https://2012.igem.org/Team:TU_Munich 2012 TU Munich]. This included CaXMT, CaMXMT, and CaDXMT, which had previously been based on the natural plant genes and codon-optimized for expression in yeast. While not codon-optimized for expression in bacteria, believed that we could express them first in a rare codon-supplemented bacteria such as Rosetta Cells™. That way we could determine if it was possible for these three enzymes to be well expressed and obtained in an active form in bacteria before proceeding to the more costly step of codon-optimizing them for bacterial expression. </p>
+<p>
+We proceeded by removing the yeast ribosome-binding site (rbs) and replacing it with a bacterial Shine-Dalgarno sequence. Each gene was cloned separately under an arabinose promoter and a constitutive promoter, in addition to a construct with all three under the same promoter. Expression was tested in a variety of conditions in <i>E. cloni</i>® 10G cells, but no expression was detected (Figure 1). This was expected as the genes are not optimized for normal bacterial expression. Unfortunately, Rosetta Cells™ already contain a chloramphenicol plasmid, pRARE, and as such, these caffeine constructs could not yet be transformed into the Rosetta Cells for further expression testing. Efforts are now under way for a kanamycin resistance marker to be cloned into each caffeine enzyme construct to allow for compatibility with the Rosetta Cells™.
 <html>
 <div id="gelpic">

Team:British Columbia/Project/Caffeine

From 2013.igem.org