An artificial chromosome ylAC enables efficient assembly of multiple genes in Yarrowia lipolytica for biomanufacturing.

The efficient use of the yeast Yarrowia lipolytica as a cell factory is hampered by the lack of powerful genetic engineering tools dedicated for the assembly of large DNA fragments and the robust expression of multiple genes. Here we describe the design and construction of artificial chromosomes (ylAC) that allow easy and efficient assembly of genes and chromosomal elements. We show that metabolic pathways can be rapidly constructed by various assembly of multiple genes in vivo into a complete, independent and linear supplementary chromosome with a yield over 90%.

Changes in lipid metabolism convey acid tolerance in .

Background: The yeast plays an essential role in the fermentation of lignocellulosic hydrolysates. Weak organic acids in lignocellulosic hydrolysate can hamper the use of this renewable resource for fuel and chemical production. Plasma-membrane remodeling has recently been found to be involved in acquiring tolerance to organic acids, but the mechanisms responsible remain largely unknown.

Developing cellulolytic as a platform for the production of valuable products in consolidated bioprocessing of cellulose.

Background: Both industrial biotechnology and the use of cellulosic biomass as feedstock for the manufacture of various commercial goods are prominent features of the bioeconomy. In previous work, with the aim of developing a consolidated bioprocess for cellulose bioconversion, we conferred cellulolytic activity of , one of the most widely studied "nonconventional" oleaginous yeast species. However, further engineering this strain often leads to the loss of previously introduced heterologous genes due to the presence of multiple sites when using -recombinase to remove previously employed selection markers.

Expressing accessory proteins in cellulolytic to improve the conversion yield of recalcitrant cellulose.

Background: A recently constructed cellulolytic is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from in order to enhance the degradation of recalcitrant substrate.

Results: The production of EG I was enhanced using a promoter engineering strategy.

Conferring cellulose-degrading ability to to facilitate a consolidated bioprocessing approach.

Background: , one of the most widely studied "nonconventional" oleaginous yeast species, is unable to grow on cellulose. Recently, we identified and overexpressed two endogenous β-glucosidases in , thus enabling this yeast to use cello-oligosaccharides as a carbon source for growth. Using this engineered yeast platform, we have now gone further toward building a fully cellulolytic for use in consolidated bioprocessing of cellulose.

Physiological responses to acid stress by Saccharomyces cerevisiae when applying high initial cell density.

High initial cell density is used to increase volumetric productivity and shorten production time in lignocellulosic hydrolysate fermentation. Comparison of physiological parameters in high initial cell density cultivation of Saccharomyces cerevisiae in the presence of acetic, formic, levulinic and cinnamic acids demonstrated general and acid-specific responses of cells. All the acids studied impaired growth and inhibited glycolytic flux, and caused oxidative stress and accumulation of trehalose.

Expression of aspartic protease from Neurospora crassa in industrial ethanol-producing yeast and its application in ethanol production.

In order to further improve the utilization rate of raw materials and increase ethanol productivity, the gene Asp, encoding aspartic protease in Neurospora crassa was cloned and expressed in industrial ethanol-producing yeast. To promote secretion of the acid protease, the gene was fused to signal sequence of the yeast α-factor gene and constitutively expressed under transcriptional control of the Saccharomyces cerevisiae PGK1 promoter. The resultant recombinant enzyme was characterized with respect to pH and temperature optimum.

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose.

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source.

Improving the ethanol yield by reducing glycerol formation using cofactor regulation in Saccharomyces cerevisiae.

To increase ethanol yield and decrease glycerol production in Saccharomyces cerevisiae, the strategies of direct cofactor-regulation were explored. During anaerobic batch fermentations, the yeast expressing Bacillus cereus gapN gene, encoding non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrognease, produced 73.8 g ethanol l(-1), corresponding to 96% of theoretical maximum yield compared to 92% for the wild type.

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance.

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S.

Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast.

The GPD2 gene, encoding NAD(+)-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P (PGK)-gapN) exhibited a 48.

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production.

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD(+)-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Delta and gpd2Delta were studied under alcoholic fermentation conditions. gpd1Delta mutants exhibited a 4.