scholarly journals Combinatorial Design of a Highly Efficient Xylose-Utilizing Pathway in Saccharomyces cerevisiae for the Production of Cellulosic Biofuels

2012 ◽  
Vol 79 (3) ◽  
pp. 931-941 ◽  
Author(s):  
Byoungjin Kim ◽  
Jing Du ◽  
Dawn T. Eriksen ◽  
Huimin Zhao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Flux ◽  
Value Added ◽  
Major Product ◽  
Combinatorial Design ◽  
Biofuel Production ◽  
Pathway Engineering ◽  
Full Potential ◽  
Xylose Utilization ◽  
Highly Efficient

ABSTRACTBalancing the flux of a heterologous metabolic pathway by tuning the expression and properties of the pathway enzymes is difficult, but it is critical to realizing the full potential of microbial biotechnology. One prominent example is the metabolic engineering of aSaccharomyces cerevisiaestrain harboring a heterologous xylose-utilizing pathway for cellulosic-biofuel production, which remains a challenge even after decades of research. Here, we developed a combinatorial pathway-engineering approach to rapidly create a highly efficient xylose-utilizing pathway for ethanol production by exploring various combinations of enzyme homologues with different properties. A library of more than 8,000 xylose utilization pathways was generated using DNA assembler, followed by multitiered screening, which led to the identification of a number of strain-specific combinations of the enzymes for efficient conversion of xylose to ethanol. The balancing of metabolic flux through the xylose utilization pathway was demonstrated by a complete reversal of the major product from xylitol to ethanol with a similar yield and total by-product formation as low as 0.06 g/g xylose without compromising cell growth. The results also suggested that an optimal enzyme combination depends on not only the genotype/phenotype of the host strain, but also the sugar composition of the fermentation medium. This combinatorial approach should be applicable to any heterologous pathway and will be instrumental in the optimization of industrial production of value-added products.

scholarly journals Combined Biosynthetic Pathway Engineering and Storage Pool Expansion for High-Level Production of Ergosterol in Industrial Saccharomyces cerevisiae

2021 ◽  
Vol 9 ◽  
Author(s):  
Zhi-Jiao Sun ◽  
Jia-Zhang Lian ◽  
Li Zhu ◽  
Yi-Qi Jiang ◽  
Guo-Si Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biosynthetic Pathway ◽  
Metabolic Flux ◽  
Feeding Strategy ◽  
Pathway Engineering ◽  
Ergosterol Biosynthesis ◽  
Sterol Esters ◽  
Storage Pool ◽  
Ergosterol Content ◽  
Dry Cell Weight

Ergosterol, a terpenoid compound produced by fungi, is an economically important metabolite serving as the direct precursor of steroid drugs. Herein, ergsosterol biosynthetic pathway modification combined with storage capacity enhancement was proposed to synergistically improve the production of ergosterol in Saccharomyces cerevisiae. S. cerevisiae strain S1 accumulated the highest amount of ergosterol [7.8 mg/g dry cell weight (DCW)] among the wild-type yeast strains tested and was first selected as the host for subsequent metabolic engineering studies. Then, the push and pull of ergosterol biosynthesis were engineered to increase the metabolic flux, overexpression of the sterol acyltransferase gene ARE2 increased ergosterol content to 10 mg/g DCW and additional overexpression of a global regulatory factor allele (UPC2-1) increased the ergosterol content to 16.7 mg/g DCW. Furthermore, considering the hydrophobicity sterol esters and accumulation in lipid droplets, the fatty acid biosynthetic pathway was enhanced to expand the storage pool for ergosterol. Overexpression of ACC1 coding for the acetyl-CoA carboxylase increased ergosterol content from 16.7 to 20.7 mg/g DCW. To address growth inhibition resulted from premature accumulation of ergosterol, auto-inducible promoters were employed to dynamically control the expression of ARE2, UPC2-1, and ACC1. Consequently, better cell growth led to an increase of ergosterol content to 40.6 mg/g DCW, which is 4.2-fold higher than that of the starting strain. Finally, a two-stage feeding strategy was employed for high-density cell fermentation, with an ergosterol yield of 2986.7 mg/L and content of 29.5 mg/g DCW. This study provided an effective approach for the production of ergosterol and other related terpenoid molecules.

scholarly journals Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Yanfei Zhang ◽  
Stephan Lane ◽  
Jhong-Min Chen ◽  
Sarah K. Hammer ◽  
Jake Luttinger ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biosynthetic Pathway ◽  
Xylose Isomerase ◽  
Biofuel Production ◽  
Mitochondrial Activity ◽  
Xylose Utilization ◽  
Gene Deletions ◽  
Theoretical Yield ◽  
Byproduct Formation ◽  
Branched Chain

Abstract Background Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose. Results In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast Saccharomyces cerevisiae. We then increased the flux through these pathways by making gene deletions of BAT1, ALD6, and PHO13, to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared to glucose. Conclusions The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.

scholarly journals Simulating Extracellular Glucose Signals Enhances Xylose Metabolism in Recombinant Saccharomyces cerevisiae

2020 ◽  
Vol 8 (1) ◽  
pp. 100 ◽  
Author(s):  
Meiling Wu ◽  
Hongxing Li ◽  
Shan Wei ◽  
Hongyu Wu ◽  
Xianwei Wu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Networks ◽  
Glucose Sensing ◽  
Biofuel Production ◽  
Xylose Utilization ◽  
High Concentration ◽  
Xylose Metabolism ◽  
Camp Phosphodiesterase ◽  
Xylose Consumption ◽  
Recombinant Saccharomyces Cerevisiae

Efficient utilization of both glucose and xylose from lignocellulosic biomass would be economically beneficial for biofuel production. Recombinant Saccharomyces cerevisiae strains with essential genes and metabolic networks for xylose metabolism can ferment xylose; however, the efficiency of xylose fermentation is much lower than that of glucose, the preferred carbon source of yeast. Implications from our previous work suggest that activation of the glucose sensing system may benefit xylose metabolism. Here, we show that deleting cAMP phosphodiesterase genes PDE1 and PDE2 increased PKA activity of strains, and consequently, increased xylose utilization. Compared to the wild type strain, the specific xylose consumption rate (rxylose) of the pde1Δ pde2Δ mutant strains increased by 50%; the specific ethanol-producing rate (rethanol) of the strain increased by 70%. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. Deletion of either RGT2 or SNF3 reduced expression of HXT1 in strains cultured in 1 g L−1 xylose, which suggests that xylose can bind both Snf3 and Rgt2 and slightly alter their conformations. Deletion of SNF3 significantly weakened the expression of HXT2 in the yeast cultured in 40 g L−1 xylose, while deletion of RGT2 did not weaken expression of HXT2, suggesting that S. cerevisiae mainly depends on Snf3 to sense a high concentration of xylose (40 g L−1). Finally, we show that deletion of Rgt1, increased rxylose by 24% from that of the control. Our findings indicate how S. cerevisiae may respond to xylose and this study provides novel targets for further engineering of xylose-fermenting strains.

scholarly journals Effects of Ultrasound on Fermentation of Glucose to Ethanol by Saccharomyces cerevisiae

Fermentation ◽  
2019 ◽  
Vol 5 (1) ◽  
pp. 16 ◽  
Author(s):  
Luis Huezo ◽  
Ajay Shah ◽  
Frederick Michel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mass Transfer ◽  
Glucose Uptake ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Biofuel Production ◽  
Inhibitory Effects ◽  
Negative Effects ◽  
Intraparticle Mass Transfer ◽  
Improve Mass Transfer

Previous studies have shown that pretreatment of corn slurries using ultrasound improves starch release and ethanol yield during biofuel production. However, studies on its effects on the mass transfer of substrates and products during fermentation have shown that it can have both beneficial and inhibitory effects. In this study, the effects of ultrasound on mass transfer limitations during fermentation were examined. Calculation of the external and intraparticle observable moduli under a range of conditions indicate that no external or intraparticle mass transfer limitations should exist for the mass transfer of glucose, ethanol, or carbon dioxide. Fermentations of glucose to ethanol using Saccharomyces cerevisiae were conducted at different ultrasound intensities to examine its effects on glucose uptake, ethanol production, and yeast population and viability. Four treatments were compared: direct ultrasound at intensities of 23 and 32 W/L, indirect ultrasound (1.4 W/L), and no-ultrasound. Direct and indirect ultrasound had negative effects on yeast performance and viability, and reduced the rates of glucose uptake and ethanol production. These results indicate that ultrasound during fermentation, at the levels applied, is inhibitory and not expected to improve mass transfer limitations.

Biohydrogen production from used diapers: Evaluation of effect of temperature and substrate conditioning

2017 ◽  
Vol 35 (3) ◽  
pp. 267-275 ◽  
Author(s):  
PX Sotelo-Navarro ◽  
HM Poggi-Varaldo ◽  
SJ Turpin-Marion ◽  
A Vázquez-Morillas ◽  
M Beltrán-Villavicencio ◽  
...  
Keyword(s):  
Value Added ◽  
Clean Energy ◽  
Solid Substrate ◽  
Biofuel Production ◽  
Biohydrogen Production ◽  
Effect Of Temperature ◽  
Batch Reactors ◽  
Disposable Diapers ◽  
Batch Bioreactors ◽  
H2 Yield

This research assessed the viability to use disposable diapers as a substrate for the production of biohydrogen, a valuable clean-energy source. The important content of cellulose of disposable diapers indicates that this waste could be an attractive substrate for biofuel production. Two incubation temperatures (35 °C and 55 °C) and three diaper conditioning methods (whole diapers with faeces, urine, and plastics, WD; diapers without plastic components, with urine and faeces, DWP; diapers with urine but without faeces and plastic, MSD) were tested in batch bioreactors. The bioreactors were operated in the solid substrate anaerobic hydrogenogenic fermentation with intermittent venting mode (SSAHF-IV). The batch reactors were loaded with the substrate at ca. 25% of total solids and 10% w/w inoculum. The average cumulative bioH2 production followed the order WD > MSD > DWP. The bio-H2 production using MSD was unexpectedly higher than DWP; the presence of plastics in the first was expected to be associated to lower degradability and H2 yield. BioH2 production at 55 °C was superior to that of 35 °C, probably owing to a more rapid microbial metabolism in the thermophilic regime. The results of this work showed low yields in the production of H2 at both temperatures compared with those reported in the literature for municipal and agricultural organic waste. The studied process could improve the ability to dispose of this residue with H2 generation as the value-added product. Research is ongoing to increase the yield of biohydrogen production from waste disposable diapers.

scholarly journals Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhuo Liu ◽  
Shih-Hsin Ho ◽  
Kengo Sasaki ◽  
Riaan den Haan ◽  
Kentaro Inokuma ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofuel Production ◽  
Cellulosic Biofuel

scholarly journals Recombinant Diploid Saccharomyces cerevisiae Strain Development for Rapid Glucose and Xylose Co-Fermentation

Fermentation ◽  
2018 ◽  
Vol 4 (3) ◽  
pp. 59 ◽  
Author(s):  
Tingting Liu ◽  
Shuangcheng Huang ◽  
Anli Geng
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alcoholic Fermentation ◽  
Ethanol Yield ◽  
Xylose Isomerase ◽  
Cost Effective ◽  
Xylose Utilization ◽  
Yeast Strains ◽  
Multiple Copies ◽  
Biomass Sugars ◽  
Sugar Conversion

Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L−1 glucose and 95 g L−1 xylose generated 120.6 g L−1 ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g−1, and ethanol productivity of 5.26 g L−1·h−1.

Sign in / Sign up

Export Citation Format

Share Document