The GRAS notice filed by Cargill is available............ here
Since the GRAS notice is available in public domain, I understand that it will not be unethical to review some of the data presented in that document.
I am citing the following table from that GRAS Notice.
It seems that they have not made any change in the MVA pathway and did not try any MVA pathway deregulation. They did not try to introduce the MEP pathway also in their production strain also. The biosynthesis of steviol glycoside is evidently dependant on original built-in IPP and DMAPP biosynthesis system.
Deletion of mating type switching gene
Along with vegetative reproduction by budding, yeast can also reproduce sexually. Any yeast population has two types of cells….either a type or α type. When both the types are present, mating between them may occur. Yeast cells can also switch their mating types. In this genetic modification work, the production strain has been rendered HO-(haploid) by deletion of a specific gene to prevent switching of mating types during the production of steviol glycosides so that they can not undergo mating events and bring about unwanted genetic rearrangement.
The GRAS notice mentioned – “The growth rate of the strain is slightly altered due to expression of a large number of heterologous genes”. The obvious effect of expression of large number of heterologous genes should be diversion of significant portion of metabolic resources for assembling of the products of the heterologous genes, which in turn should adversely affect the growth vigour.
Yield and Production Cost
Evolva and Cargill did not disclose the yield of steviol glycoside and the final concentration of SG that could be achieved in the fermentation medium. The following lines are cited from Evolva website.
"EverSweet™ production costs are currently above where we and Cargill want them to be for launch. This is as a result of a combination of factors, including strain characteristics; fermentation and downstream processing costs; facility conversion costs, production scale, and current customer indications on pricing. We have initiated further development work to resolve the identified bottlenecks and ensure EverSweet™ can be launched as soon as possible. Currently this is estimated to be sometime after 2016" Link
I shall be glad to know about any update from Evolva in this regard.
In December 2015, Swiss headquartered biotech company Evolva announces that it has achieved a technical milestone in its partnership with Cargill, Inc. to commercialise EverSweet™, the next-generation fermentation based, zero-calorie stevia sweetener. Reaching this milestone triggers the payment of USD 0.5 million by Cargill to Evolva. EverSweet™ is made with the best-tasting sweetness components found in the stevia leaf, Reb M and Reb D, which deliver a great taste with better sweetness intensity, faster sweetness onset and improved sweetness quality.
The following lines have been taken from EverSweet™ website –
“By adding a few genes to the specially crafted baker’s yeast, we enable our baker’s yeast to produce the same type of enzymes used by stevia plants. We feed our yeast some simple sugars and the yeast transforms the simple sugar into the very sweetest steviol glycosides, just like a stevia plant.
The yeast is then filtered away and the remaining sweet steviol glycosides are concentrated and purified. These sweet steviol glycosides are identical to certain steviol glycosides that are naturally formed in very small quantities in the stevia leaf.”