From basic building blocks, generated from photosynthesis and glucose breakdown, the synthesis of Steviol Glycosides requires 15 specialized enzymes. The organisms, which have all these 15 enzymes in functioning condition within their cells can only synthesize Steviol Glycosides. All these enzymes are proteins and the necessary software code for producing these proteins should reside in the DNA of the organism capable of producing Steviol Glycosides.

How genetic code is translated into proteins/enzymes

For translation of a piece of genetic code into a protein/enzyme in cells involves an interesting mechanism. The nucleotide sequence code on the DNA is first transcribed into a RNA fragment. Each bit of genetic code is transcribed into that particular RNA. DNA and RNA use different alphabets (nucleotides), and for that a “transcription” is necessary. This operation is done in the nucleus of the plant cell. This “Transcripted” RNA fragment is called “Messenger RNA”.

 

Plant and animal DNA sometimes contain short non-coding and nonsense stretches of DNA even within the large DNA stretch coding a specific protein. This non-coding stretches within a gene are called “Introns”. During transcription of Messenger RNA or mRNA, all those non-coding parts are also transcribed into it, but, are later removed, to make a clean representation of the genetic code.

The “cleaned up” mRNA is then migrates out of the nucleus into the cytoplasm. Then it binds to an organelle – “Ribosome”. Then the ribosome organizes “Translation” of the nucleotide code on the mRNA into proteins. An amino acid chain is then made according to the code on the mRNA. The chain of amino acid, after its formation, folds into a three dimensional structure and becomes ready for its designated role.

Genetic Engineering of Yeast to Train Them to Produce Steviol glycosides :

Yeast is a very common micro-organism which is being used in bread leavening and beer making for quite a long time. It has been reported that yeast has been subjected to targeted genetic modification to incorporate all the steviol glycoside bio-synthesis machinery in it. There are several reasons for selecting yeast as the organism of choice, which are –

  1. Yeast is easy to grow at industrial scale in simple and cheap liquid nutrient medium

  2. It is a robust organism and can be trained to grow under various stressed conditions

  3. Genetic manipulation of yeast is cheap and easy

  4. The entire genomic DNA sequence for yeast is known. We have complete information about its 6692 genes

  5. It is a genetically stable organisms and does not get genetically altered during its large scale culture

  6. It can produce the target compounds in high concentration and its growth are not inhibited by such high concentration of a specific metabolite

 

 

The major objectives of genetic modification of Yeast to develop steviol glycoside producing strains are as follows –

  1. Increase the precursor pool (isopentenyl diphosphate and dimethylallyl diphosphate) in the yeast cells

  2. Expression of enzymes for synthesis of poly-isoprenoid precursor geranylgeranyl diphosphate and production of committed precursor steviol

  3. Expression of enzymes of addition of glucose molecules to steviol for conversion into steviol glycosides

 

Now let us discuss what it takes to achieve the individual objectives –

Increase in the precursor pool

Isopentenyl diphosphate (IPP) and it isomer dimethylallyl diphosphate (DMAPP) is the precursor of all the isoprenoid molecules in their biosynthetic pathway. Thus, these two are essential precursor for steviol glycoside biosynthesis. High concentration of these two precursors is an essential prerequisite for high production of steviol glycosides.

MVA or MEP ?

There are two major pathways through which these precursors are formed. One is the methyl-D-erythritol phosphate (MEP) pathway, which has already been discussed here. The other pathway is the mevalonate pathway, in which, IPP and DMAPP is synthesized in a different process. In stevia, the major mechanism of biosynthesis of IPP and DMAPP is the MEP pathway, though MVA Pathway may have some minor contribution*.

Theoretically, the MEP pathway is more efficient in terms of pathway yield, largely due to the fact that using this pathway loses less carbon as CO2 as compared to the MVA pathway (MEP pathway: 1 CO2 molecules per molecule of IPP produced; MVA pathway: 4 CO2 molecules per molecule of IPP produced; sugar as carbon source).          Reference

Unfortunately, yeast only has the MVA pathway. All the efforts to introduce the MEP pathway into yeast through incorporation of the necessary genes was unsuccessful because the last two enzymes of the pathway (1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase and 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase) are both metalloproteins which require reduced iron-sulfur clusters to function. Yeast does not have the necessary cellular mechanism for trafficking the reduced iron-sulfur clusters. So, in yeast, for production of the precursors IPP and DMAPP, we need to use the rather inefficient MVA pathway.

References :

Totte´, N., Charon, L., Rohmer, M., Compernolle, F., Baboeuf, I., Geuns,J.M.C., 2000. Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway. Tetrahedron Lett. 41, 6407–6410.

Totte´, N., Van den Ende, W., Van Damme, E.J.M., Compernolle, F., Baboeuf, I., Geuns, J.M.C., 2003. Cloning and heterologous expression of early genes in gibberellin and steviol biosynthesis via the methylerythritol phosphate pathway in Stevia rebaudiana. Can. J. Bot. 81, 517–522.

Brandle, J.E. and Telmer, P.G. (2007), "Steviol glycoside biosynthesis." Phytochemistry, volume 68, issue 14, pages 1855-1863.

De-regulation of MVA pathway

Due to the variety of essential compounds produced in the MVA pathway, the activity of many enzymes of this pathway is strictly regulated at different levels by the cellular mechanisms. In yeast, the production of IPP and DMAPP is mainly regulated by an enzyme of the pathway : 3-Hydroxy-3-MethylGlutaryl-coenzyme a reductase or HMGCR. The production and activity of this enzyme is regulated by the concentrations of downstream products of MVA pathway. Generally, high concentration of end-products suppresses this enzyme activity…to enforce a metabolic control on the process, and to ensure that there is no production of any end products of this pathway in quantities in excess to the demand. This feedback control also ensures that there is no overspending of cellular metabolic energy to any particular process. Under high concentrations of the products of the process catalyzed by HMCGR, the production of this enzyme by decoding the genes by transcription process is suppressed. The products of the MVA process can also exert negative feedback control on the enzyme by blocking the active site of the enzyme. 

To ensure high production flux through the MVA, this feedback control system is to be hacked.  The approach needs to be two pronged. Firstly, the transcriptional level regulation is to be removed my modification of the HMCGR gene. There is also scope for altering the HMCGR structure by protein engineering to make it immune to the feedback control without any compromise to its activity.

Hydroxymethylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme of mevalonate pathway, has been involved in the tumorigenesis of several tumor types. HMGCR gene is thus considered as a candidate metabolic oncogene (genes associated with cancer) for humans

Reference

 In all animals high HMCGR activity is associated with high cholesterol levels. The “Statin” drugs actually inhibits the HMCGR activity to control cholesterols

Reference

Interesting Facts

Expression of enzymes for synthesis of poly-isoprenoid precursor


Isoprenoids constitute one of the most diverse families of natural products. The total number of these compounds is in the order of 20 000–35 000 and their structures and functions are overwhelmingly varied. They serve numerous biochemical functions: as quinones in electron transport chains, as components of membranes (prenyl-lipids in archaebacteria and sterols in eubacteria and eukaryotes), in subcellular targeting and regulation (prenylation of proteins), as photosynthetic pigments (carotenoids, side chain of chlorophyll), as hormones (gibberellins, brassinosteroids, abscisic acid), and as plant defense compounds (monoterpenes, sesquiterpenes, diterpenes).


For efficient production of steviol glycosides, the genetically modified yeast should have all the necessary enzymes for conversion of IPP and DMAPP into steviol, viz. geranylgeranyl diphosphate synthase (GGDPS), copalyl diphosphate synthase (CPS), kaurene synthase (KS), kaurene oxidase (KO), kaurenoic acid 13-hydroxylase (KAH). Wild yeast does not have all these enzymes, hence genes for these enzymes are to introduced into yeast genome from other organisms (heterologous genes). Moreover, to ensure availability of IPP and DMAPP precursors for steviol biosynthesis pathway, its diversion to other isoprenoids production is to be suppressed.


Expression of enzymes of addition of glucose molecules to steviol for conversion into steviol glycosides


The last step for steviol glycoside biosynthesis involves glycosylation of steviol. For these processes, three Uridine Diphosphate-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1 are required. Yeast does not possess these enzymes. Genes for these enzymes are also to be introduced to the genome of the production strain.
 

Isoprenoid biosynthetic pathways in the plant cell. 

Objectives of Genetic Modification of Yeasts for Steviol Glycoside Production