Metabolic engineering of robust industrial strains for de novo resveratrol production from sole carbon sources for application in process-like conditions

For industrial applications, the search for robust microorganisms is essential to design sustainable processes. In addition to the wide range of uses in the food industry, S. cerevisiae is used as a robust cell factory to produce many chemicals of interest to society, from biofuels to high-value natural products. Resveratrol is a phenolic antioxidant compound, usually extracted by a complex and low-efficiency process from grapes or the roots of Japanese knotweed, and therefore being dependent on the supply of plant resources, as well as environmental factors. It can also be synthesized chemically through a complex and polluting method. In this sense, its production through microbial biosynthesis can side these drawbacks, being however mainly produced at the cost of expensive substrates as pcoumaric acid. De novo resveratrol production from glucose has been recently reported in a laboratory strain1. In this work, multiple robust industrial diploid strains were successfully engineered with resveratrol central pathway (PAL2/C4H/CL2/VST1) using CRISPR/Cas9 system for its de novo production through phenylalanine using glucose as sole carbon source. Top-producing strains revealed a resveratrol titre ca. 25% higher when compared to a laboratory strain using the same glucose concentration. Strains were screened in both batch and fed-batch conditions, revealing promising performance for scale-up application. This work aims for the construction of a fully engineered robust yeast strain for resveratrol production for industrial application.This study was supported by the Portuguese Foundation for Science and Technology (FCT) by the strategic funding of UID/BIO/04469/2013 unit, Ph.D. grant SFRH/BD/130739/2017 to Carlos E. Costa, COMPETE 2020 (POCI-01-0145- FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004), YeasTempTation (ERA-IB-2-6/0001/2014), and MultiBiorefinery project (POCI-01-0145-FEDER-016403).info:eu-repo/semantics/publishedVersio

Engineered industrial yeast for resveratrol de novo production from wine waste

The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce several chemicals of interest, from biofuels to high-value natural products. Resveratrol is a polyphenolic compound with strong antioxidant activity, naturally present in plants such as grapes, that can be synthesized by fermentation. Its microbial biosynthesis is a valuable alternative to its plant extraction or chemical synthesis, both complex and unsustainable. Nevertheless, its biosynthesis is mostly attained from expensive substrates such as p-coumaric acid. Therefore, there is a demand for cheaper substrates, namely carbon sources. Portugal and Spain are two of the largest wine manufacturers in the world, and this vast production generates a substantial amount of wine wastes like grape pomace or wine lees. Wine lees, consisting of residual fermentative microorganisms and other particles, have high organic content and ethanol, among other components. They have been previously proposed as economic nutrients for microbial production of biochemical products but could also be used as substrate. Furthermore, there is a significant surplus of grape must that is not used for wine production, which could serve as a carbon source for biotechnological processes that generate value-added products. Here, a robust industrial diploid strain engineered with the resveratrol biosynthetic pathway[1] via CRISPR/Cas9 was used for resveratrol production from carbon sources. Initially, overexpression of genes from the Pentose Phosphate Pathway coupled with the expression of AtATR2 and ScCYB5 for enhancement of cytochrome P450 activity increased resveratrol titre by 55%. Subsequently, resveratrol production using wine waste as substrate was assessed. Grape must fermentation, using glucose and fructose as carbon sources, led to a production of 259 mg/L of resveratrol. In addition, fermentation of wine lees achieved a titre of 264 mg/L of resveratrol using ethanol as sole carbon source. To the best of our knowledge, this is the first report of resveratrol production exclusively from ethanol. The use of wine wastes expands the palette of feedstocks available for the establishment of sustainable processes in a circular bioeconomy.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit; Ph.D. grant SFRH/BD/130739/2017 to Carlos E. Costa, and BIOVINO (0688_BIOVINO_6_E)info:eu-repo/semantics/publishedVersio

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