Background: 2 0 -Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5 0 -diphosphate (GDP)-L-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP-L-fucose biosynthetic enzymes and heterologous fucosyltransferase.

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, one of the most abundant human milk oligosaccharides (HMOs), has received much attention due to its health-promoting activities, such as stimulating the growth of beneficial gut microorganisms, inhibiting pathogen infection, and enhancing the host immune system. Consequently, large quantities of 2-FL are on demand for food applications as well as in-depth investigation of its biological properties. Biosynthesis of 2-FL has been attempted primarily in Escherichia coli, which might not be the best option to produce food and cosmetic ingredients due to the presence of endotoxins on the cell surface. In this study, an alternative route to produce 2-FL via a de novo pathway using a food-grade microorganism, Saccharomyces cerevisiae, has been devised. Specifically, heterologous genes, which are necessary to achieve the production of 2-FL from a mixture of glucose and lactose, were introduced into S. cerevisiae. When the lactose transporter (Lac12), de novo GDP-L-fucose pathway (consisting of GDP-Dmannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase (WcaG)), and α1,2-fucosyltransferase (FucT2) were introduced, the resulting engineered strain (D452L-gwf) produced 0.51 g/L of 2-FL from a batch fermentation. In addition, 0.41 g/L of L-fucose was produced when α-L-fucosidase was additionally expressed in the 2-FL producing strain (D452L-gwf). To our knowledge, this is the first report of 2-FL and L-fucose production in engineered S. cerevisiae via the de novo pathway. This study provides the possibility of producing HMOs by a food-grade microorganism S. cerevisiae and paves the way for more HMO production in the future.

Lactose is an inevitable byproduct of the dairy industry. In addition to cheese manufacturing, the growing Greek yogurt industry generates excess acid whey, which contains lactose. Therefore, rapid and efficient conversion of lactose to fuels and chemicals would be useful for recycling the otherwise harmful acid whey. Saccharomyces cerevisiae, a popular metabolic engineering host, cannot natively utilize lactose. However, we discovered that an engineered S. cerevisiae strain (EJ2) capable of fermenting cellobiose can also ferment lactose. This finding suggests that a cellobiose transporter (CDT-1) can transport lactose and a β-glucosidase (GH1-1) can hydrolyze lactose by acting as a β-galactosidase. While the lactose fermentation by the EJ2 strain was much slower than the cellobiose fermentation, a faster lactose-fermenting strain (EJ2e8) was obtained through serial subcultures on lactose. The EJ2e8 strain fermented lactose with a consumption rate of 2.16g/Lh. The improved lactose fermentation by the EJ2e8 strain was due to the increased copy number of cdt-1 and gh1-1 genes. Looking ahead, the EJ2e8 strain could be exploited for the production of other non-ethanol fuels and chemicals from lactose through further metabolic engineering.

Background: Succinic acid is a crucial platform chemical for production of various industrially significant compounds. For a sustainable and eco-friendly process, succinic acid synthesis has been shifted towards the fermentative route using renewable biomass substrates. Pineapple consumption and processing generate an immense amount of waste from its non-edible peel portion. As a carbon source, pineapple peel can be valorized for succinic acid bioproduction. Results: The hydrothermal pretreatment (121°C, 15 min) of pineapple peel waste resulted in the highest sugar release of 35.22 g/L (18 g/L glucose and 17 g/L fructose). The subsequent fermentation of pineapple peel hydrolysate was performed by a natural succinic acid producer, Actinobacillus succinogenes TISTR 1994. When the non-detoxified hydrolysate was used as a sole carbon source, 6.21 g/L of succinic acid was produced from 26.16 g/L of sugars. Additional supplementation of 9 g/L mixed nitrogen source enhanced the formation of succinic acid to 9.96 g/L from roughly the same amount of sugar. The current production conditions using mainly hydrolysate-based medium gave the succinic acid yield of 0.39 g/g sugar suggesting feasibilities for further improvement. Conclusion: Bio-based succinic acid production was attempted for the first time using the solid pineapple waste as a main starting material. Results demonstrated a proof of concept that the abundant pineapple peel waste can serve as a renewable substrate for a low-cost, value-added bioconversion to succinic acid. Optimization of nutritional composition in hydrolysate is necessary to enhance the yield of succinic acid in future studies.