Lipid production through simultaneous utilization of glucose, xylose, and L-arabinose by Pseudozyma hubeiensis: a comparative screening study.
ABSTRACT: Co-fermentation of glucose, xylose and L-arabinose from lignocellulosic biomass by an oleaginous yeast is anticipated as a method for biodiesel production. However, most yeasts ferment glucose first before consuming pentoses, due to glucose repression. This preferential utilization results in delayed fermentation time and lower productivity. Therefore, co-fermentation of lignocellulosic sugars could achieve cost-effective conversion of lignocellulosic biomass to microbial lipid. Comprehensive screening of oleaginous yeasts capable of simultaneously utilizing glucose, xylose, and L-arabinose was performed by measuring the concentration of sugars remaining in the medium and of lipids accumulated in the cells. We found that of 1189 strains tested, 12 had the ability to co-ferment the sugars. The basidiomycete yeast Pseudozyma hubeiensis IPM1-10, which had the highest sugars consumption rate of 94.1 %, was selected by culturing in a batch culture with the mixed-sugar medium. The strain showed (1) simultaneous utilization of all three sugars, and (2) high lipid-accumulating ability. This study suggests that P. hubeiensis IPM1-10 is a promising candidate for second-generation biodiesel production from hydrolysate of lignocellulosic biomass.
Project description:The use of lignocellulosic biomass as a feedstock for microbial fermentation processes presents an opportunity for increasing the yield of bioproducts derived directly from glucose. Lignocellulosic biomass consists of several fermentable sugars, including glucose, xylose, and arabinose. In this study, we investigate the ability of an E. coli ?pgi ?zwf mutant to consume alternative carbon sources (xylose, arabinose, and glycerol) for growth while reserving glucose for product formation. Deletion of pgi and zwf was found to eliminate catabolite repression as well as the ability of E. coli to consume glucose for biomass formation. In addition, the yield from glucose of the bioproduct D-glucaric acid was significantly increased in a ?pgi ?zwf strain.
Project description:Glucose, xylose and arabinose are the three most abundant monosaccharide found in lignocellulosic biomass. Effectively and simultaneously utilization of these sugars by microorganisms for production of the biofuels and bio-chemicals is essential toward directly fermentation of the lignocellulosic biomass. In our previous study, the recombinant Bacillus subtilis 168ARSRCP?acoA?bdhA strain was already shown to efficiently utilize xylose for production of acetoin, with a yield of 0.36 g/g xylose. In the current study, the Bacillus subtilis168ARSRCP?acoA?bdhA strain was further engineered to produce acetoin from a glucose, xylose, and arabinose mixtures. To accomplish this, the endogenous xylose transport protein AraE, the exogenous xylose isomerase gene xylA and the xylulokinase gene xylB from E. coli were co-overexpressed in the Bacillus subtilis 168ARSRCP?acoA?bdhA strain, which enabled the resulting strain, denoted ZB02, to simultaneously utilize glucose and xylose. Unexpectedly, the ZB02 strain could simultaneously utilize glucose and arabinose also. Further results indicated that the transcriptional inhibition of the arabinose transport protein gene araE was the main limiting factor for arabinose utilization in the presence of glucose. Additionally, the arabinose operon in B. subtilis could be activated by the addition of arabinose, even in the presence of glucose. Through fed-batch fermentation, strain ZB02 could simultaneously utilize glucose, xylose, and arabinose, with an average sugar consumption rate of 3.00 g/l/h and an average production of 62.2 g/l acetoin at a rate of 0.864 g/l/h. Finally, the strain produced 11.2 g/l acetoin from lignocellulosic hydrolysate (containing 20.6g/l glucose, 12.1 g/l xylose and 0.45 g/l arabinose) in flask cultivation, with an acetoin yield of 0.34 g/g total sugar. The result demonstrates that this strain has good potential for the utilization of lignocellulosic hydrolysate for production of acetoin.
Project description:Lignocellulosic biomass offers a sustainable source for biofuel production that does not compete with food-based cropping systems. Importantly, two critical bottlenecks prevent economic adoption: many industrially relevant microorganisms cannot ferment pentose sugars prevalent in lignocellulosic medium, leaving a significant amount of carbon unutilized. Furthermore, chemical biomass pretreatment required to release fermentable sugars generates a variety of toxins, which inhibit microbial growth and metabolism, specifically limiting pentose utilization in engineered strains. Here we dissected genetic determinants of anaerobic xylose fermentation and stress tolerance in chemically pretreated corn stover biomass, called hydrolysate. We previously revealed that loss-of-function mutations in the stress-responsive MAP kinase HOG1 and negative regulator of the RAS/Protein Kinase A (PKA) pathway, IRA2, enhances anaerobic xylose fermentation. However, these mutations likely reduce cells' ability to tolerate the toxins present in lignocellulosic hydrolysate, making the strain especially vulnerable to it. We tested the contributions of Hog1 and PKA signaling via IRA2 or PKA negative regulatory subunit BCY1 to metabolism, growth, and stress tolerance in corn stover hydrolysate and laboratory medium with mixed sugars. We found mutations causing upregulated PKA activity increase growth rate and glucose consumption in various media but do not have a specific impact on xylose fermentation. In contrast, mutation of HOG1 specifically increased xylose usage. We hypothesized improving stress tolerance would enhance the rate of xylose consumption in hydrolysate. Surprisingly, increasing stress tolerance did not augment xylose fermentation in lignocellulosic medium in this strain background, suggesting other mechanisms besides cellular stress limit this strain's ability for anaerobic xylose fermentation in hydrolysate.
Project description:Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.
Project description:BACKGROUND:Efficient bioconversion of lignocellulosic biomass to bioethanol is one of key challenges in the situation of increasing bioethanol demand. The ethanologenic microbes for such conversion are required to possess abilities of utilization of various sugars including xylose and arabinose in lignocellulosic biomass. As required additional characteristics, there are a weak or no glucose repression that allows cells to simultaneously utilize various sugars together with glucose and thermotolerance for fermentation at high temperatures, which has several advantages including reduction of cooling cost. Spathaspora passalidarum ATCC MYA-4345, a type strains, isolated previously have mainly of these abilities or characteristics but its thermotolerance is not so strong and its glucose repression on xylose utilization is revealed. RESULTS:Newly isolated S. passalidarum CMUWF1-2 was found to have a high ability to produce ethanol from various sugars included in lignocellulosic biomass at high temperatures. The strain achieved ethanol yields of 0.43 g, 0.40 g and 0.20 g ethanol/g xylose at 30 °C, 37 °C and 40 °C, respectively. Interestingly, no significant glucose repression was observed in experiments with mixed sugars, being consistent with the strong resistance to 2-deoxyglucose, and antimycin A showed no effect on its growth in xylose medium. Moreover, the strain was tolerant to glucose and ethanol at concentrations up to 35.0% (w/v) and 8.0% (v/v), respectively. CONCLUSIONS:S. passalidarum CMUWF1-2 was shown to achieve efficient production of ethanol from various sugars and a high ethanol yield from xylose with little accumulation of xylitol. The strain also exhibited stress-resistance including thermotolerance and no detectable glucose repression as beneficial characteristics. Therefore, S. passalidarum CMUWF1-2 has remarkable potential for conversion of lignocellulosic biomass to bioethanol.
Project description:BACKGROUND:Hydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations. RESULTS:Here we described the cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations. CONCLUSIONS:Using a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations.
Project description:Bioethanol production processes with Saccharomyces cerevisiae using lignocellulosic biomass as feedstock are challenged by the simultaneous utilization of pentose and hexose sugars from biomass hydrolysates. The pentose uptake into the cell represents a crucial role for the efficiency of the process. The focus of the here presented study was to understand the uptake and conversion of the pentose l-arabinose in S. cerevisiae and reveal its regulation by d-glucose and d-galactose. Gal2p-the most prominent transporter enabling l-arabinose uptake in S. cerevisiae wild-type strains-has an affinity for the transport of l-arabinose, d-glucose, and d-galactose. d-Galactose was reported for being mandatory for inducing GAL2 expression. GAL2 expression is also known to be regulated by d-glucose-mediated carbon catabolite repression, as well as catabolite inactivation. The results of the present study demonstrate that l-arabinose can be used as sole carbon and energy source by the recombinant industrial strain S. cerevisiae DS61180. RT-qPCR and RNA-Seq experiments confirmed that l-arabinose can trigger its own uptake via the induction of GAL2 expression. Expression levels of GAL2 during growth on l-arabinose reached up to 21% of those obtained with d-galactose as sole carbon and energy source. l-Arabinose-induced GAL2 expression was also subject to catabolite repression by d-glucose. Kinetic investigations of substrate uptake, biomass, and product formation during growth on a mixture of d-glucose/l-arabinose revealed impairment of growth and ethanol production from l-arabinose upon d-glucose depletion. The presence of d-glucose is thus preventing the fermentation of l-arabinose in S. cerevisiae DS61180. Comparative transcriptome studies including the wild-type and a precursor strain delivered hints for an increased demand in ATP production and cofactor regeneration during growth of S. cerevisiae DS61180 on l-arabinose. Our results thus emphasize that cofactor and energy metabolism demand attention if the combined conversion of hexose and pentose sugars is intended, for example in biorefineries using lignocellulosics.
Project description:BACKGROUND: Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation. RESULTS: A new recombinant S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized. CONCLUSION: Pentose fermenting recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar.
Project description:Bioethanol production from lignocellulosic biomass has received increasing attention over the past decade. Many attempts have been made to reduce the cost of bioethanol production by combining the separate steps of the process into a single-step process known as consolidated bioprocessing. This requires identification of organisms that can efficiently decompose lignocellulose to simple sugars and ferment the pentose and hexose sugars liberated to ethanol. There have been many attempts in engineering laboratory strains by adding new genes or modifying genes to expand the capacity of an industrial microorganism. There has been less attention in improving bioethanol-related processes utilizing natural variation existing in the natural ecotypes. In this study, we sought to identify genomic loci contributing to variation in saccharification of cellulose and fermentation of glucose in the fermenting cellulolytic fungus Neurospora crassa through quantitative trait loci (QTL) analysis. We identified one major QTL contributing to fermentation of glucose and multiple putative QTL's underlying saccharification. Understanding the natural variation of the major QTL gene would provide new insights in developing industrial microbes for bioethanol production.
Project description:Anaerobic fermentation using lignocellulosic hydrolysates as co-substrates is an economically attractive method to enhance 1,3-propanediol (1,3-PD) production by increasing the conversion yield from glycerol. Lignocellulosic hydrolysates contain the mixed sugars that are primarily glucose, xylose, and arabinose. Therefore, these three individual sugars were used, separately, as co-substrates with glycerol, in 1,3-PD production by a Clostridium diolis strain DSM 15410, resulting in an 18%-28% increase in the 1,3-PD yield. Co-fermentation of the mixed sugars and glycerol obtained a higher intracellular NADH/NAD(+) ratio and increased the 1,3-PD yield by 22% relative to fermentation of glycerol alone. Thereafter, two kinds of lignocellulosic hydrolysates, corn stover hydrolysate and corncob molasses, were individually co-fermented with glycerol. The maximum 1,3-PD yield from glycerol reached 0.85 mol/mol. Fed-batch co-fermentation was also performed, improving the 1,3-PD yield (from 0.62 mol/mol to 0.82 mol/mol). These results demonstrate that the co-fermentation strategy is an efficient and economical way to produce 1,3-PD from glycerol.