Dimorphism of Trichosporon cutaneum and impact on its lipid production.

BACKGROUND: Compared to the oleaginous yeast Yarrowia lipolytica, Trichosporon cutaneum can metabolize pentose sugars more efficiently, and in the meantime is more tolerant to inhibitors, which is suitable for lipid production from lignocellulosic biomass. However, this species experiences dimorphic transition between yeast-form cells and hyphae during submerged fermentation, which consequently affects the rheology and mass transfer performance of the fermentation broth and its lipid production. RESULTS: The strain T. cutaneum B3 was cultured with medium composed of yeast extract, glucose and basic minerals. The experimental results indicated that yeast-form morphology was developed when yeast extract was supplemented at 1 g/L, but hyphae were observed when yeast extract supplementation was increased to 3 g/L and 5 g/L, respectively. We speculated that difference in nitrogen supply to the medium might be a major reason for the dimorphic transition, which was confirmed by the culture with media supplemented with yeast extract at 1 g/L and urea at 0.5 g/L and 1.0 g/L to maintain total nitrogen at same levels as that detected in the media with yeast extract supplemented at 3 g/L and 5 g/L. The morphological change of T. cutaneum B3 affected not only the content of intracellular lipids but also their composition, due to its impact on the rheology and oxygen mass transfer performance of the fermentation broth, and more lipids with less polyunsaturated fatty acids such as linoleic acid (C18:2) were produced by the yeast-form cells. When T. cutaneum B3 was cultured at an aeration rate of 1.5 vvm for 72 h with the medium composed of 60 g/L glucose, 3 g/L yeast extract and basic minerals, 27.1 g (dry cell weight)/L biomass was accumulated with the lipid content of 46.2%, and lipid productivity and yield were calculated to be 0.174 g/L/h and 0.21 g/g, respectively. Comparative transcriptomics analysis identified differently expressed genes for sugar metabolism and lipid synthesis as well as signal transduction for the dimorphic transition of T. cutaneum B3. CONCLUSIONS: Assimilable nitrogen was validated as one of the major reasons for the dimorphic transition between yeast-form morphology and hyphae with T. cutaneum, and the yeast-form morphology was more suitable for lipid production at high content with less polyunsaturated fatty acids as feedstock for biodiesel production.

Enhanced acetic acid stress tolerance and ethanol production in Saccharomyces cerevisiae by modulating expression of the de novo purine biosynthesis genes.

BACKGROUND: Yeast strains that are tolerant to multiple environmental stresses are highly desired for various industrial applications. Despite great efforts in identifying key genes involved in stress tolerance of budding yeast Saccharomyces cerevisiae, the effects of de novo purine biosynthesis genes on yeast stress tolerance are still not well explored. Our previous studies showed that zinc sulfate addition improved yeast acetic acid tolerance, and key genes involved in yeast stress tolerance were further investigated in this study. RESULTS: Three genes involved in de novo purine biosynthesis, namely, ADE1, ADE13, and ADE17, showed significantly increased transcription levels by zinc sulfate supplementation under acetic acid stress, and overexpression of these genes in S. cerevisiae BY4741 enhanced cell growth under various stress conditions. Meanwhile, ethanol productivity was also improved by overexpression of the three ADE genes under stress conditions, among which the highest improvement attained 158.39% by ADE17 overexpression in the presence of inhibitor mixtures derived from lignocellulosic biomass. Elevated levels of adenine-nucleotide pool "AXP" ([ATP] + [ADP] + [AMP]) and ATP content were observed by overexpression of ADE17, both under control condition and under acetic acid stress, and is consistent with the better growth of the recombinant yeast strain. The global intracellular amino acid profiles were also changed by overexpression of the ADE genes. Among the changed amino acids, significant increase of the stress protectant γ-aminobutyric acid (GABA) was revealed by overexpression of the ADE genes under acetic acid stress, suggesting that overexpression of the ADE genes exerts control on both purine biosynthesis and amino acid biosynthesis to protect yeast cells against the stress. CONCLUSION: We proved that the de novo purine biosynthesis genes are useful targets for metabolic engineering of yeast stress tolerance. The engineered strains developed in this study with improved tolerance against multiple inhibitors can be employed for efficient lignocellulosic biorefinery to produce biofuels and biochemicals.

Development of Robust Yeast Strains for Lignocellulosic Biorefineries Based on Genome-Wide Studies.

Lignocellulosic biomass has been widely studied as the renewable feedstock for the production of biofuels and biochemicals. Budding yeast Saccharomyces cerevisiae is commonly used as a cell factory for bioconversion of lignocellulosic biomass. However, economic bioproduction using fermentable sugars released from lignocellulosic feedstocks is still challenging. Due to impaired cell viability and fermentation performance by various inhibitors that are present in the cellulosic hydrolysates, robust yeast strains resistant to various stress environments are highly desired. Here, we summarize recent progress on yeast strain development for the production of biofuels and biochemical using lignocellulosic biomass. Genome-wide studies which have contributed to the elucidation of mechanisms of yeast stress tolerance are reviewed. Key gene targets recently identified based on multiomics analysis such as transcriptomic, proteomic, and metabolomics studies are summarized. Physiological genomic studies based on zinc sulfate supplementation are highlighted, and novel zinc-responsive genes involved in yeast stress tolerance are focused. The dependence of host genetic background of yeast stress tolerance and roles of histones and their modifications are emphasized. The development of robust yeast strains based on multiomics analysis benefits economic bioconversion of lignocellulosic biomass.

Production of L-alanyl-L-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase.

BACKGROUND: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln. RESULTS: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles. CONCLUSIONS: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.

Development of stress tolerant Saccharomyces cerevisiae strains by metabolic engineering: New aspects from cell flocculation and zinc supplementation.

Budding yeast Saccharomyces cerevisiae is widely studied for the production of biofuels from lignocellulosic biomass. However, economic production is currently challenged by the repression of cell growth and compromised fermentation performance of S. cerevisiae strains in the presence of various environmental stresses, including toxic level of final products, inhibitory compounds released from the pretreatment of cellulosic feedstocks, high temperature, and so on. Therefore, it is important to improve stress tolerance of S. cerevisiae to these stressful conditions to achieve efficient and economic production. In this review, the latest advances on development of stress tolerant S. cerevisiae strains are summarized, with the emphasis on the impact of cell flocculation and zinc addition. It was found that cell flocculation affected ethanol tolerance and acetic acid tolerance of S. cerevisiae, and addition of zinc to a suitable level improved stress tolerance of yeast cells to ethanol, high temperature and acetic acid. Further studies on the underlying mechanisms by which cell flocculation and zinc status affect stress tolerance will not only enrich our knowledge on stress response and tolerance mechanisms of S. cerevisiae, but also provide novel metabolic engineering strategies to develop robust yeast strains for biofuels production.

Synergistic effect of calcium and zinc on glucose/xylose utilization and butanol tolerance of Clostridium acetobutylicum.

Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L(-1) were achieved from 69.3 g L(-1) glucose with butanol/ABE productivities of 0.40 and 0.65 g L(-1) h(-1) compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L(-1) h(-1) obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L(-1) butanol). Under the same conditions, calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum.

Impact of zinc supplementation on the improved fructose/xylose utilization and butanol production during acetone-butanol-ethanol fermentation.

Lignocellulosic biomass and dedicated energy crops such as Jerusalem artichoke are promising alternatives for biobutanol production by solventogenic clostridia. However, fermentable sugars such as fructose or xylose released from the hydrolysis of these feedstocks were subjected to the incomplete utilization by the strains, leading to relatively low butanol production and productivity. When 0.001 g/L ZnSO4·7H2O was supplemented into the medium containing fructose as sole carbon source, 12.8 g/L of butanol was achieved with butanol productivity of 0.089 g/L/h compared to only 4.5 g/L of butanol produced with butanol productivity of 0.028 g/L/h in the control without zinc supplementation. Micronutrient zinc also led to the improved butanol production up to 8.3 g/L derived from 45.2 g/L xylose as sole carbon source with increasing butanol productivity by 31.7%. Moreover, the decreased acids production was observed under the zinc supplementation condition, resulting in the increased butanol yields of 0.202 g/g-fructose and 0.184 g/g-xylose, respectively. Similar improvements were also observed with increasing butanol production by 130.2 % and 8.5 %, butanol productivity by 203.4% and 18.4%, respectively, in acetone-butanol-ethanol fermentations from sugar mixtures of fructose/glucose (4:1) and xylose/glucose (1:2) simulating the hydrolysates of Jerusalem artichoke tubers and corn stover. The results obtained from transcriptional analysis revealed that zinc may have regulatory mechanisms for the sugar transport and metabolism of Clostridium acetobutylicum L7. Therefore, micronutrient zinc supplementation could be an effective way for economic development of butanol production derived from these low-cost agricultural feedstocks.

Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum.

The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum. In order to elucidate the zinc-associated response for enhanced glucose utilization and earlier solventogenesis, transcriptional analysis was performed on cells grown in glucose medium at the exponential growth phase of 16 h without/with supplementary zinc. Correspondingly, the gene glcG (CAC0570) encoding a glucose-specific PTS was significantly upregulated accompanied with the other two genes CAC1353 and CAC1354 for glucose transport in the presence of zinc. Additionally, genes involved in the metabolisms of six other carbohydrates (maltose, cellobiose, fructose, mannose, xylose and arabinose) were differentially expressed, indicating that the regulatory effect of micronutrient zinc is carbohydrate-specific with respects to the improved/inhibited carbohydrate utilization. More importantly, multiple genes responsible for glycolysis (glcK and pykA), acidogenesis (thlA, crt, etfA, etfB and bcd) and solventogenesis (ctfB and bdhA) of C. acetobutylicum prominently responded to the supplementary zinc at differential expression levels. Comparative analysis of intracellular metabolites revealed that the branch node intermediates such as acetyl-CoA, acetoacetyl-CoA, butyl-CoA, and reducing power NADH remained relatively lower whereas more ATP was generated due to enhanced glycolysis pathway and earlier initiation of solventogenesis, suggesting that the micronutrient zinc-associated response for the selected intracellular metabolisms is significantly pleiotropic.

The impact of zinc sulfate addition on the dynamic metabolic profiling of Saccharomyces cerevisiae subjected to long term acetic acid stress treatment and identification of key metabolites involved in the antioxidant effect of zinc.

The mechanisms of how zinc protects the cells against acetic acid toxicity and acts as an antioxidant are still not clear. Here we present results of the metabolic profiling of the eukaryotic model yeast species Saccharomyces cerevisiae subjected to long term high concentration acetic acid stress treatment in the presence and absence of zinc supplementation. Zinc addition decreased the release of reactive oxygen species (ROS) in the presence of chronic acetic acid stress. The dynamic changes in the accumulation of intermediates in central carbon metabolism were observed, and higher contents of intracellular alanine, valine and serine were observed by zinc supplementation. The most significant change was observed in alanine content, which is 3.51-fold of that of the control culture in cells in the stationary phase. Subsequently, it was found that 0.5 g L(-1) alanine addition resulted in faster glucose consumption in the presence of 5 g L(-1) acetic acid, and apparently decreased ROS accumulation in zinc-supplemented cells. This indicates that alanine exerted its antioxidant effect at least partially through the detoxification of acetic acid. In addition, intracellular glutathione (GSH) accumulation was enhanced by zinc addition, which is related to the protection of yeast cells from the oxidative injury caused by acetic acid. Our studies revealed for the first time that zinc modulates cellular amino acid metabolism and redox balance, especially biosynthesis of alanine and glutathione to exert its antioxidant effect.

Effect of zinc supplementation on acetone-butanol-ethanol fermentation by Clostridium acetobutylicum.

In this article, effect of zinc supplementation on acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum was studied. It was found that when 0.001 g/L ZnSO4·7H2O was supplemented into the medium, solventogenesis was initiated earlier, with 21.0 g/L ABE (12.6 g/L butanol, 6.7 g/L acetone and 1.7 g/L ethanol) produced with a fermentation time of 40 h, compared to 19.4 g/L ABE (11.7 g/L butanol, 6.4 g/L acetone and 1.3g/L ethanol) produced with a fermentation time of 64 h in the control without zinc supplementation, and correspondingly ABE and butanol productivities were increased to 0.53 and 0.32 g/L/h from 0.30 and 0.18 g/L/h, increases of 76.7% and 77.8%, respectively, but their yields were not compromised. The reason for this phenomenon was attributed to rapid acids re-assimilation for more efficient ABE production, which was in accordance with relatively high pH and ORP levels maintained during the fermentation process. The maximum cell density increased by 23.8%, indicating that zinc supplementation stimulated cell growth, and consequently facilitated glucose utilization. However, more zinc supplementation exhibited an inhibitory effect, indicating that zinc supplementation at very low levels such as 0.001 g/L ZnSO4·7H2O will be an economically competitive strategy for improving butanol production.

[Optimized culture medium and fermentation conditions for lipid production by Rhodosporidium toruloides].

Culture medium and fermentation conditions for lipid production by Rhodosporidium toruloides were optimized with single factor and uniform design experiment. The best medium recipe was found with 70 g/L glucose, 0.1 g/L (NH4)2SO4, 0.75 g/L yeast extract, 1.5 g/L MgSO4. 7H2O, 0.4g/L KH2PO4, sterilized at 121 degrees C for 15 min, and then supplemented with ZnSO4 1.91 x 10(-6) mmol/L, CaCl2 1.50 mmol/L, MnCl2 1.22 x 10(-4) mmol/L and CuSO4 1.00 x 10(-4) mmol/L. The optimal fermentation conditions were as follows: 50 mL of medium (pH 6.0) in 250 mL Erlenmeyer flask with 10% inoculum (28h) under orbital shaking at 200 r/min for 120h at 30 degrees C. Under these conditions, yeast biomass accumulated lipids up to 76.1%.

Protein amino acid composition of plasma membranes affects membrane fluidity and thereby ethanol tolerance in a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae.

A combination of three amino acids including 1.0 g/L isoleucine, 0.5 g/L methionine and 2.0 g/L phenylalanine was found to enhance ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae. When subjected to 20% (V/V) ethanol for 9 h at 30 degrees C, all cells died whereas 57% remained viable for the cells grown in the presence of the three amino acids. Based on the analysis of protein amino acid composition of plasma membranes and the determination of plasma membrane fluidity by measuring fluorescence anisotropy using diphenylhexatriene as a probe, it was found that the significantly increased ethanol tolerance of cells grown with the three amino acids was due to the incorporation of the supplementary amino acids into the plasma membranes, thus resulting in enhanced ability of the plasma membranes to efficiently counteract the fluidizing effect of ethanol when subjected to ethanol stress. This is the first time to report that plasma membrane fluidity can be influenced by protein amino acid composition of plasma membranes.