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Yarrowia lipolytica is an oleaginous yeast that produces high titers of fatty acid-derived biofuels and biochemicals. It can grow on hydrophobic carbon sources and lignocellulosic hydrolysates. The genome sequence of Y. lipolytica NRRL Y-64008 is reported to aid in its development as a biotechnological chassis for producing biofuels and bioproducts.

Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

The halotolerant and osmotolerant yeast Zygosaccharomyces rouxii can produce multiple volatile compounds and has the ability to grow on lignocellulosic hydrolysates. We report the annotated genome sequence of Z. rouxii NRRL Y-64007 to support its development as a platform organism for biofuel and bioproduct production.

Background: Sugarcane plant engineered to accumulate lipids in its vegetative tissue is being developed as a new bioenergy crop. The new crop would be a source of juice, oil, and cellulosic sugars. However, limited tolerance of industrially recognized yeasts towards inhibitors generated during the processing of lignocellulosic biomass to produce fermentable sugars is a major challenge in developing scalable processes for second-generation drop-in fuel production. To this end, hydrolysates generated from engineered sugarcane—'oilcane' bagasse contain added phenolics and fatty acids that further restrict the growth of fermenting microorganisms and necessitate nutrient supplementation and/or detoxification of hydrolysate which makes the fermentation process expensive. Herein, we propose a resourceful and economical approach for growing lab and commercial strains of S. cerevisiae on unrefined cellulosic sugars aerobically and fermentatively. Results: An equal ratio of hydrolysate and juice was found optimum for growth and fermentation by lab and commercial strains of Saccharomyces cerevisiae engineered for xylose fermentation. The industrial strain grew and fermented efficiently under low aeration conditions having an ethanol titer, yield, specific and volumetric productivities of 46.96 ± 0.19 g/l, 0.51 ± 0.00 g/g, 0.27 ± 0.02 g/g.h and 1.95 ± 0.01 g/l.h, respectively, while the lab strain grew better under higher aeration conditions having the ethanol titer, yield, specific and volumetric productivities of 24.93 ± 0.09, 0.27 ± 0.00 g/g, 0.17 ± 0.00 g/g.h and 1.04 ± 0.00 g/l.h, respectively. Acclimation of cultures in a blended medium significantly improved the performance of the yeast strains. Conclusions: The addition of transgenic oilcane juice, which is inedible and rich in amino acids, to the hydrolysate averted the need for expensive nutrient supplementation and detoxification steps of hydrolysate. The approach provides an economical solution to reduce the cost of fermentation at an industrial scale for second-generation drop-in fuel production. [ABSTRACT FROM AUTHOR]

The fermentative production of valuable chemicals from lignocellulosic feedstocks has attracted considerable attention. Although Saccharomyces cerevisiae is a promising microbial host, it lacks the ability to efficiently metabolize xylose, a major component of lignocellulosic feedstocks. The xylose oxidative pathway offers advantages such as simplified metabolic regulation and fewer enzymatic steps. Specifically, the pathway involves the conversion of xylose into 2-keto-3-deoxy-xylonate, which can be channeled into two distinct pathways, the Dahms pathway and the Weimberg pathway. However, the growth of yeast on xylose as the sole carbon source through the xylose oxidative pathway has not been achieved, limiting its utilization. We successfully engineered S. cerevisiae to metabolize xylose as its sole carbon source via the xylose oxidative pathways, achieved by enhancing enzyme activities through iron metabolism engineering and rational enzyme selection. We found that increasing the supply of the iron-sulfur cluster to activate the bottleneck enzyme XylD by BOL2 disruption and tTYW1 overexpression facilitated the growth of xylose and the production of ethylene glycol at 1.5 g/L via the Dahms pathway. Furthermore, phylogenetic analysis of xylonate dehydratases led to the identification of a highly active homologous enzyme. A strain possessing the Dahms pathway with this highly active enzyme exhibited reduced xylonate accumulation. Furthermore, the introduction of enzymes based on phylogenetic tree analysis allowed for the utilization of xylose as the sole carbon source through the Weimberg pathway. This study highlights the potential of iron metabolism engineering and phylogenetic enzyme selection for the development of non-native metabolic pathways in yeast. Key points: • A 1.5 g/L ethylene glycol was produced via the Dahms pathway in S. cerevisiae. • Enzyme activation enabled growth on xylose via both the Dahms and Weimberg pathways. • Tested enzymes in this study may expand the application of xylose oxidative pathway. [ABSTRACT FROM AUTHOR]

Cellulosic biomass hydrolysates are rich in glucose and xylose, but most microorganisms, including Komagataella phaffii, are unable to utilize xylose effectively. To address this limitation, we engineered a K. phaffii strain optimized for xylose metabolism through the xylose oxidoreductase pathway and promoter optimization. A promoter library with varying strengths was used to fine-tune the expression levels of the XYL1, XYL2, and XYL3 genes, resulting in a strain with a strong promoter for XYL2 and weaker promoters for XYL1 and XYL3. This engineered strain exhibited superior growth, achieving 14 g cells/L and a maximal growth rate of 0.4 g cells/L-h in kenaf hydrolysate, outperforming a native strain by 17%. This study is the first to report the introduction of the xylose oxidoreductase pathway into K. phaffii, demonstrating its potential as an industrial platform for producing yeast protein and other products from cellulosic biomass. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) are complex sugars highly abundant in human milk but currently not present in infant formula. Rapidly accumulating evidence from in vitro and in vivo studies, combined with epidemiological associations and correlations, suggests that HMOs benefit infants through multiple mechanisms and in a variety of clinical contexts. Until recently, however, research on HMOs has been limited by an insufficient availability of HMOs. Most HMOs are found uniquely in human milk, and thus far it has been prohibitively tedious and expensive to isolate and synthesize them. This article reviews new strategies to overcome this lack of availability by generating HMOs through chemoenzymatic synthesis, microbial metabolic engineering, and isolation from human donor milk or dairy streams. Each approach has its advantages and comes with its own challenges, but combining the different methods and acknowledging their limitations creates new opportunities for research and application with the goal of improving maternal and infant health.

UnlabelledEfficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering.ImportanceIn order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering.