2′-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has been spotlighted for its neutraceutical and pharmaceutical potentials. Microbial production of 2-FL is promising since it is efficient as compared to other production methods. In 2-FL microbial production via the salvage pathway for biosynthesis of guanosine 5′-diphosphate (GDP)-L-fucose from fucose, the conversion yield from fucose is important because of the high price of fucose. In this study, deletion of the genes (araA and rhaA) coding for arabinose isomerase (AraA) and rhamnose isomerase (RhaA) was attempted in engineered Escherichia coli for improving 2-FL production by using fucose, lactose, and glycerol. The engineered E. coli constructed previously is able to express fucokinase/GDP-L-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori and deficient in β-galactosidase (LacZ), fucose isomerase (FucI), and fuculose kinase (FucK). The additional double-deletion of the araA and rhaA genes in the engineered E. coli enhanced the product yield of 2-FL to 0.52 mole 2-FL/mole fucose, and hence the concentration of 2-FL reached to 47.0 g/L, which are 44% and two-fold higher than those (23.1 g/L and 0.36 mole 2-FL/mole fucose) of the control strain in fed-batch fermentation. Elimination of sugar isomerases exhibiting promiscuous activities with fucose might be critical in the microbial production of 2-FL through the salvage pathway of GDP-L-fucose.

Background 2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol. Results Endogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C 2 -compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae . A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h. Conclusions The microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.

Saccharomyces cerevisiae is a work horse for production of valuable biofuels and biochemicals including 2,3-butanediol (2,3-BDO), a platform chemical with wide industrial applications for synthetic rubber, biosolvents and food additives. Recently, a cutting-edge technology of metabolic engineering has enabled S. cerevisiae to produce 2,3-BDO with high yield and productivity. These include (i) amplification of the 2,3-BDO biosynthetic pathway, (ii) redirection of carbon flux from ethanol or glycerol toward 2,3-BDO, and (iii) 2,3-BDO production from sugars derived from renewable biomass. These breakthroughs enforced S. cerevisiae to become a promising microbial host for production of 2,3-BDO.

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β-Carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce β-carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a β-carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose-fermenting S. cerevisiae. The resulting strain produced β-carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and β-carotene production in yeast when glucose is used, did not further improve the production of β-carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for β-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl-CoA pool, and an upregulated expression level of rate-limiting genes in the β-carotene-producing pathway, including ACS1 and HMG1. As a result, 772.8 mg/L of β-carotene was obtained in a fed-batch bioreactor culture with xylose feeding. Considering the inevitable large scale production of xylose when cellulosic biomass-based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.

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Retinol is a fat-soluble vitamin A that is widely used in the food and pharmaceutical industries. Currently, retinol is commercially produced by chemical synthesis. Microbial production of retinol has been alternatively explored but restricted to a mixture of retinoids including retinol, retinal, and retinoic acid. Thus, we introduced heterologous retinol dehydrogenase into retinoids mixture-producing Saccharomyces cerevisiae for the selective production of retinol using xylose. Expression of human RDH10 and Escherichia coli ybbO led to increase in retinol production, but retinal remained as a major product. In contrast, S. cerevisiae harboring human RDH12 produced retinol selectively with negligible production of retinal. The resulting strain (SR8A-RDH12) produced retinol only. However, more glycerol was accumulated due to intracellular redox imbalance. Therefore, Lactococcus lactis noxE coding for H 2 Oforming NADH oxidase was additionally introduced to resolve the redox imbalance.The resulting strain produced 52% less glycerol and more retinol with a 30% higher yield than a parental strain. As the produced retinol was not stable, we examined culture and storage conditions including temperature, light, and antioxidants for the optimal production of retinol. In conclusion, we achieved selective production of retinol efficiently from xylose by introducing human RDH12 and NADH oxidase into S. cerevisiae.

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