Microbial production of next-generation stevia sweeteners

BACKGROUND: The glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana. RESULTS: In the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C (13)- and C (19)-bound glucoses. Four of these reactions lead to Reb D and Reb M while the other four result in formation of side-products unwanted for production. In this work, side-product formation was reduced by targeted optimization of UGT76G1 towards 1,3 glucosylation of steviol glucosides that are already 1,2-diglucosylated. The optimization of UGT76G1 was based on homology modelling, which enabled identification of key target amino acids present in the substrate-binding pocket. These residues were then subjected to site-saturation mutagenesis and a mutant library containing a total of 1748 UGT76G1 variants was screened for increased accumulation of Reb D or M, as well as for decreased accumulation of side-products. This screen was performed in a Saccharomyces cerevisiae strain expressing all enzymes in the rebaudioside biosynthesis pathway except for UGT76G1. CONCLUSIONS: Screening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1(Thr146Gly) and UGT76G1(His155Leu), which diminished accumulation of unwanted side-products and gave increased specific accumulation of the desired Reb D or Reb M sweeteners. This improvement in a key enzyme of the Stevia sweetener biosynthesis pathway represents a significant step towards the commercial production of next-generation stevia sweeteners. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0609-1) contains supplementary material, which is available to authorized users

Production of steviol glycosides in recombinant hosts

Properties of nitrogen cluster plasmas produced by an intense, ultrashort laser pulse have been investigated numerically and experimentally. The classical dynamics simulations show that on increasing the cluster size a plasma with residual electron energy above 1 keV can be created due to collisional heating, which is considerably higher than the value obtained with a conventional low-density gas target. Experimentally, nitrogen gas jets created by two types of nozzles were irradiated with a laser pulse of 55 fs, up to 1.2× 1017 / cm2. A seeded gas jet consisting of nitrogen and helium was also employed to promote the production of large clusters. The influences of the shape of nozzle, the seeded gas, and the gas jet stagnation pressure on the properties of plasmas were examined by spectroscopic observations. K -shell emissions showed that for the gas jet using the conical nozzle the electrons underwent intense collisional heating within the large clusters, resulting in the production of highly charged ions. In contrast, the emissions observed with the capillary nozzle exhibited the characteristics of a cold plasma without suffering substantial electron heating, indicating the absence of large clusters. That is, the differences between the two types of nozzles in the efficiency of electron heating and subsequent residual energies after the passage of the laser pulse, which are strongly dependent upon the cluster size, drastically changed the properties of the produced plasmas. The reason that for the capillary gas jet the plasma density deduced from the recombination spectra was significantly higher than the value obtained using the conical nozzle is also given by the difference in residual electron energy

Metabolic Engineering of Valine- and Isoleucine-Derived Glucosinolates in Arabidopsis Expressing CYP79D2 from Cassava

Glucosinolates are amino acid-derived natural products that, upon hydrolysis, typically release isothiocyanates with a wide range of biological activities. Glucosinolates play a role in plant defense as attractants and deterrents against herbivores and pathogens. A key step in glucosinolate biosynthesis is the conversion of amino acids to the corresponding aldoximes, which is catalyzed by cytochromes P450 belonging to the CYP79 family. Expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in Arabidopsis resulted in the production of valine (Val)- and isoleucine-derived glucosinolates not normally found in this ecotype. The transgenic lines showed no morphological phenotype, and the level of endogenous glucosinolates was not affected. The novel glucosinolates were shown to constitute up to 35% of the total glucosinolate content in mature rosette leaves and up to 48% in old leaves. Furthermore, at increased concentrations of these glucosinolates, the proportion of Val-derived glucosinolates decreased. As the isothiocyanates produced from the Val- and isoleucine-derived glucosinolates are volatile, metabolically engineered plants producing these glucosinolates have acquired novel properties with great potential for improvement of resistance to herbivorous insects and for biofumigation