Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> IR-2 for high-temperature ethanol production

The pentose phosphate pathway (PPP) plays an important role in the efficiency of xylose fermentation during cellulosic ethanol production. In simultaneous saccharification and co-fermentation (SSCF), the optimal temperature for cellulase hydrolysis of lignocellulose is much higher than that of fermentation. Successful use of SSCF requires optimization of the expression of PPP genes at elevated temperatures. This study examined the combinatorial expression of PPP genes at high temperature. The results revealed that over-expression of TAL1 and TKL1 in Saccharomyces cerevisiae (S. cerevisiae) at 30 °C and over-expression of all PPP genes at 36 °C resulted in the highest ethanol productivities. Furthermore, combinatorial over-expression of PPP genes derived from S. cerevisiae and a thermostable yeast Kluyveromyces marxianus allowed the strain to ferment xylose with ethanol productivity of 0.51 g/L/h, even at 38 °C. These results clearly demonstrate that xylose metabolism can be improved by the utilization of appropriate combinations of thermostable PPP genes in high-temperature production of ethanol.     

Raw starch conversion by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> amylases

Background

Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.

Results

The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l^-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l^-1 (0.038 and 0.028 g l^-1 h^-1), respectively, after 10 days. With a substrate load of 200 g l^-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l^-1 ethanol (0.58 g l^-1 h^-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l^-1 ethanol (0.36 g l^-1 h^-1).

Conclusions

In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l^-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

     

Metabolic engineering for high glycerol production by the anaerobic cultures of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is used by the cosmetic, paint, automotive, food, and pharmaceutical industries and for production of explosives. Currently, glycerol is available in commercial quantities as a by-product from biodiesel production, but the purity and the cost of its purification are prohibitive. The industrial production of glycerol by glucose aerobic fermentation using osmotolerant strains of the yeasts Candida sp. and Saccharomyces cerevisiae has been described. A major drawback of the aerobic process is the high cost of production. For this reason, the development of yeast strains that effectively convert glucose to glycerol anaerobically is of great importance. Due to its ability to grow under anaerobic conditions, the yeast S. cerevisiae is an ideal system for the development of this new biotechnological platform. To increase glycerol production and accumulation from glucose, we lowered the expression of TPI1 gene coding for triose phosphate isomerase; overexpressed the fused gene consisting the GPD1 and GPP2 parts coding for glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, respectively; overexpressed the engineered FPS1 gene that codes for aquaglyceroporin; and overexpressed the truncated gene ILV2 that codes for acetolactate synthase. The best constructed strain produced more than 20 g of glycerol/L from glucose under micro-aerobic conditions and 16 g of glycerol/L under anaerobic conditions. The increase in glycerol production led to a drop in ethanol and biomass accumulation.     

Inactivation of the transcription factor <Emphasis Type="Italic">mig1</Emphasis> (<Emphasis Type="Italic">YGL035C</Emphasis>) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves tolerance towards monocarboxylic weak acids: acetic,formic and levulinic acid

Toxic concentrations of monocarboxylic weak acids present in lignocellulosic hydrolyzates affect cell integrity and fermentative performance of Saccharomyces cerevisiae. In this work, we report the deletion of the general catabolite repressor Mig1p as a strategy to improve the tolerance of S. cerevisiae towards inhibitory concentrations of acetic, formic or levulinic acid. In contrast with the wt yeast, where the growth and ethanol production were ceased in presence of acetic acid 5 g/L or formic acid 1.75 g/L (initial pH not adjusted), the m9 strain (Δmig1::kan) produced 4.06?±?0.14 and 3.87?±?0.06 g/L of ethanol, respectively. Also, m9 strain tolerated a higher concentration of 12.5 g/L acetic acid (initial pH adjusted to 4.5) without affecting its fermentative performance. Moreover, m9 strain produced 33% less acetic acid and 50–70% less glycerol in presence of weak acids, and consumed acetate and formate as carbon sources under aerobic conditions. Our results show that the deletion of Mig1p provides a single gene deletion target for improving the acid tolerance of yeast strains significantly.     

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for second-generation bioethanol production

Background

Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance.

Results

Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse.

Conclusions

This study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and promising substrate for the isolation of S. cerevisiae strains showing enhanced inhibitor, temperature, and osmotic tolerance compared with established industrial strains. This integrated approach of selecting multiple resistant yeasts from a single source demonstrates the potential of obtaining yeasts that are able to withstand a number of fermentation-related stresses. The yeast strains isolated and selected in this study represent strong candidates for bioethanol production from lignocellulosic hydrolysates.

     

Engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase,xylosidase and a bacterial xylose isomerase

Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes.     

Gene expression profiles of the thermotolerant yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain KKU-VN8 during high-temperature ethanol fermentation using sweet sorghum juice

Objectives

To investigate gene expression profiles of the thermotolerant yeast Saccharomyces cerevisiae strain KKU-VN8, a potential high-ethanol producer, in response to various stresses during high-temperature ethanol fermentation using sweet sorghum juice (SSJ) under optimal conditions.

Results

The maximal ethanol concentration obtained by S. cerevisiae KKU-VN8 using SSJ at 40 °C was 66.6 g/l, with a productivity of 1.39 g/l/h and a theoretical ethanol yield of 81%. Quantitative RT-PCR assays were performed to investigate the gene expression profiles of S. cerevisiae KKU-VN8. Differential expression of genes encoding heat-shock proteins (HSP82, HSP104, SSA4), genes involved in trehalose metabolism (TPS1, TPS2, NTH1) and genes involved the glycolytic pathway (ADH1, ADH2, CDC19) at various time points during fermentation was observed. The expression levels of HSP82, HSP104, SSA4, ADH1 and CDC19 were significantly higher than those of the controls (10.2-, 4-, 8-, 8.9- and 5.9-fold higher, respectively). In contrast, the expression levels of TPS1, TPS2, NTH1 and ADH2 were approx. 2-fold less than those of the controls.

Conclusions

The highly expressed genes encoding heat-shock proteins, HSP82 and SSA4, potentially play an important role in helping S. cerevisiae KKU-VN8 cope with various stresses that occur during high-temperature fermentation, leading to higher ethanol production efficiency.

     

Identification of alcohol stress tolerance genes of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 using adaptive laboratory evolution

Background

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.

Results

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Conclusions

Novel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.

     

Ethanol production from acid- and alkali-pretreated corncob by endoglucanase and β-glucosidase co-expressing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> subject to the expression of heterologous genes and nutrition added

Low-cost technologies to overcome the recalcitrance of cellulose are the key to widespread utilization of lignocellulosic biomass for ethanol production. Efficient enzymatic hydrolysis of cellulose requires the synergism of various cellulases, and the ratios of each cellulase are required to be regulated to achieve the maximum hydrolysis. On the other hand, engineering of cellulolytic Saccharomyces cerevisiae strains is a promising strategy for lignocellulosic ethanol production. The expression of cellulase-encoding genes in yeast would affect the synergism of cellulases and thus the fermentation ability of strains with exogenous enzyme addition. However, such researches are rarely reported. In this study, ten endoglucanase and β-glucosidase co-expressing S. cerevisiae strains were constructed and evaluated by enzyme assay and fermentation performance measurement. The results showed that: (1) maximum ethanol titers of recombinant strains exhibited high variability in YPSC medium (20 g/l peptone, 10 g/l yeast extract, 100 g/l acid- and alkali-pretreated corncob) within 10 days. However, they had relatively little difference in USC medium (100 g/l acid- and alkali-pretreated corncob, 0.33 g/l urea, pH 5.0). (2) Strains 17# and 19#, with ratio (CMCase to β-glucosidase) of 7.04 ± 0.61 and 7.40 ± 0.71 respectively, had the highest fermentation performance in YPSC. However, strains 11# and 3# with the highest titers in USC medium had a higher ratio of CMCase to β-glucosidase, and CMCase activities. These results indicated that nutrition, enzyme activities and the ratio of heterologous enzymes had notable influence on the fermentation ability of cellulase-expressing yeast.     

Measurement of individual cell strength of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> in cell culture

Botryococcus braunii is a microalga considered for biofuel production and may require physical disruption of cells/colonies for efficient hydrocarbon extraction. In this study, the strength of individual cells of B. braunii was measured using a nanoindenter. From the load and cell size, the pressure for bursting the cell was calculated to be 56.9 MPa. This value is 2.3–10 times those of Saccharomyces cerevisiae and Chlorella vulgaris found in another research, because B. braunii has two types of cell walls with different thicknesses. The energy required to disrupt 1 g of dry B. braunii cells, estimated by load-displacement curves, is 3.19 J g^?1 which is 0.19–1.2 times higher than those of S. cerevisiae and C. vulgaris. When using a high-pressure homogenizer for disrupting B. braunii cells, the cell disruption degree increased with the treatment pressure at above 30 MPa, and 70% of cells were disrupted at 80 MPa.     

Evaluation of ethanol production and bioadsorption of heavy metals by various red seaweeds

The objective of this study was to evaluate ethanol production and bioadsorption with four red seaweeds, Gelidium amansii, Gracilaria verrucosa, Kappaphycus alvarezii and Eucheuma denticulatum. To produce ethanol, thermal acid hydrolysis, enzymatic saccharification and fermentation was carried out. After pretreatment, 38.5, 39.9, 31.0 and 27.5 g/L of monosaccharides were obtained from G. amansii, G. verrucosa, K. alvarezii and E. denticulatum, respectively. Ethanol fermentation was performed with Saccharomyces cerevisiae KCCM 1129 adapted to 80 g/L galactose. The ethanol productions by G. amansii, G. verrucosa, K. alvarezii and E. denticulatum were 18.8 g/L with Y _EtOH = 0.49, 19.1 g/L with Y _EtOH = 0.48, 14.5 g/L with Y _EtOH = 0.47 and 13.0 g/L with Y _EtOH = 0.47, respectively. The waste seaweed slurries after the ethanol fermentation were reused to adsorb Cd(II), Pb(II) and Cu(II). Using langmuir isotherm model, Cu(II) had the highest affinity for waste seaweeds with the highest q _max and electronegativity values among three heavy metals.     

The effect of lyophilization and storage time on the survival rate and hydrolytic activity of <Emphasis Type="Italic">Trichoderma</Emphasis> strains

The study evaluates the survivability and storage stability of seven Trichoderma strains belonging to the species: T. harzianum (1), T. atroviride (4), and T. virens (2) after the lyophilization of their solid state cultures on wheat straw. Biomass of Trichoderma strains was freeze-dried with and without the addition of maltodextrin. Furthermore, in order to determine the ability of tested Trichoderma strains to preserve selected technological features, the biosynthesis of extracellular hydrolases (cellulases, xylanases, and polygalacturonases) after a 3-month storage of lyophilizates was investigated. Strains of T. atroviride (except TRS40) and T. harzianum TRS85 showed the highest viability after lyophilization process (up to 100%). After 3 months of storage, T. atroviride TRS14 exhibited the highest stability (95.23%); however, the number of active conidia remained at high level of 10⁶–10⁷ cfu/g for all tested T. atroviride strains and T. harzianum TRS85. Interestingly, after a 3-month storage of lyophilized formulations, most of the tested Trichoderma strains exhibited higher cellulolytic and xylanolytic activities compared to the control, i.e., before freeze-drying process. The highest activities of these enzymes exhibited the following: T. atroviride TRS14–2.37 U/g and T. atroviride TRS25–21.47 U/g, respectively, whereas pectinolytic activity was weak for all tested strains, with the highest value of 0.64 U/g registered for T. virens TRS109.     

Screening and Mutation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> UV-20 with a High Yield of Second Generation Bioethanol and High Tolerance of Temperature,Glucose and Ethanol

A wild-type strain was isolated from slightly rotted pears after three rounds of enrichment culture, identified as Saccharomyces cerevisiae 3308, and evaluated for its fermentation capability of second generation bioethanol and tolerance of temperature, glucose and ethanol. S. cerevisiae 3308 was mutated by using the physical and chemical mutagenesis methods, ultraviolet (UV) and diethyl sulfate (DES), respectively. Positive mutated strains were mainly generated by the treatment of UV, but numerous negative mutations emerged under the treatment of DES. A positive mutated strain, UV-20, produced ethanol from 62.33?±?1.34 to 122.22?±?2.80 g/L at 30–45 °C, and had a maximum yield of ethanol at 37 °C. Furthermore, UV-20 produced 121.18?±?2.51 g/L of second generation bioethanol at 37 °C. Simultaneously, UV-20 exhibited superior tolerance to 50% of glucose and 21% of ethanol. In a conclusion, all of these results indicated that UV-20 has a potential industrial application value.     

Effects of Probiotics on Survival,Growth and Digestive Enzymes Activities in Freshwater Prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> (De Man 1879)

A study was conducted to examine the effects of three probiotics, Lactobacillus sporogenes, Bacillus subtilis and Saccharomyces cerevisiae on the survival, growth and digestive enzymes activities of the freshwater prawn Macrobrachium rosenbergii post larvae (PL). The probiotics, L. sporogenes (4 %), B. subtilis (3 %) and S. cerevisiae (4 %) were taken and mixed with basal diet. Diet without probiotics served as control. These probiotics diets were fed to M. rosenbergii PL for a period of 60 days. After the feeding trail, the growth parameters such as survival, weight gain, specific growth rate and protein efficiency rate were found to be significantly (P < 0.05) higher in 4 % S. cerevisiae incorporated diet fed PL when compared with control. In the case of feed conversion rate just the reverse was seen (P < 0.05) at this concentration. This indicates its superior quality among different concentrations of probiotics tested. Activities of digestive enzymes, such as protease, amylase and lipase were significantly (P < 0.05) higher at this concentration (4 % S. cerevisiae). Some of essential and non-essential amino acids also significantly elevated in probiotics supplemented diet fed prawns. This study indicated that probiotics, S. cerevisiae incorporated diets were beneficial for M. rosenbergii in terms of increasing growth, enzyme and amino acid production.     

Acetaldehyde kinetics of enological yeast during alcoholic fermentation in grape must

Acetaldehyde strongly binds to the wine preservative SO₂ and, on average, causes 50–70 mg l^?1 of bound SO₂ in red and white wines, respectively. Therefore, a reduction of bound and total SO₂ concentrations necessitates knowledge of the factors that affect final acetaldehyde concentrations in wines. This study provides a comprehensive analysis of the acetaldehyde production and degradation kinetics of 26 yeast strains of oenological relevance during alcoholic fermentation in must under controlled anaerobic conditions. Saccharomyces cerevisiae and non-Saccharomyces strains displayed similar metabolic kinetics where acetaldehyde reached an initial peak value at the beginning of fermentations followed by partial reutilization. Quantitatively, the range of values obtained for non-Saccharomyces strains greatly exceeded the variability among the S. cerevisiae strains tested. Non-Saccharomyces strains of the species C. vini, H. anomala, H. uvarum, and M. pulcherrima led to low acetaldehyde residues (<10 mg l^?1), while C. stellata, Z. bailii, and, especially, a S. pombe strain led to large residues (24–48 mg l^?1). Acetaldehyde residues in S. cerevisiae cultures were intermediate and less dispersed (14–34 mg l^?1). Addition of SO₂ to Chardonnay must triggered significant increases in acetaldehyde formation and residual acetaldehyde. On average, 0.33 mg of residual acetaldehyde remained per mg of SO₂ added to must, corresponding to an increase of 0.47 mg of bound SO₂ per mg of SO₂ added. This research demonstrates that certain non-Saccharomyces strains display acetaldehyde kinetics that would be suitable to reduce residual acetaldehyde, and hence, bound-SO₂ levels in grape wines. The acetaldehyde formation potential may be included as strain selection argument in view of reducing preservative SO₂ concentrations.     

Functional analysis of alcohol dehydrogenase (<Emphasis Type="Italic">ADH</Emphasis>) genes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To characterize the genes responsible for ethanol utilization in Pichia pastoris.

Results

ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.

Conclusion

The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

     

Genome sequence of <Emphasis Type="Italic">Candida versatilis</Emphasis> and comparative analysis with other yeast

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.     

Changes in intracellular metabolism underlying the adaptation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to ethanol stress

Saccharomyces cerevisiae is often stressed by the ethanol which accumulates during the production of bioethanol by the fermentation process. The study of ethanol-adapted S. cerevisiae strains provide an opportunity to clarify the molecular mechanism underlying the adaptation or tolerance of S. cerevisiae to ethanol stress. The aim of this study was to clarify this molecular mechanism by investigating the ethanol adaptation-associated intracellular metabolic changes in S. cerevisiae using a gas chromatography–mass spectrometry-based metabolomics strategy. A partial least-squares-discriminant analysis between the parental strain and ethanol-adapted strains identified 12 differential metabolites of variable importance with a projection value of >1. The ethanol-adapted strains had a more activated glycolysis pathway and higher energy production than the parental strain, suggesting the possibility that an increased energy production and energy requirement might be partly responsible for an increased ethanol tolerance. An increased glycine content also partly contributed to the higher ethanol tolerance of the ethanol-adapted strains. The decreased oleic acid content may be a self-protection mechanism of ethanol-adapted strains to maintain membrane integrity through decreasing membrane fluidity. We suggest that while being exposed to ethanol stress, ethanol-adapted S. cerevisiae cells may remodel their metabolic phenotype and the composition of their cell membrane to adapt to ethanol stress and acquire higher ethanol tolerance.     

Improving isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with over-expressing NADPH-specific glucose-6-phosphate dehydrogenase (Zwf1)

Isobutanol is a more promising biofuel than ethanol due to its higher energy density and lower hygroscopicity. Saccharomyces cerevisiae, as a model eukaryote, has the potential advantage to produce isobutanol because of its greater tolerance to higher alcohols. NADPH is a key cofactor for isobutanol synthesis, and glucose-6-phosphate dehydrogenase (Zwf1) is one of the main NADPH-supplying sources in S. cerevisiae. In this study, we investigated the effects of over-expressing ZWF1 on isobutanol titers. Our results showed that engineered strain HZAL-7023 produced 6.22 mg isobutanol per g glucose, which increased by 6.64-fold compared with the parent strain, while engineered strain HZAL-7023 22-ZWF1 produced 11.46 mg isobutanol per g glucose, which increased by 1.82-fold compared with engineered strain HZAL-7023. These results suggested that improvement of NADPH supply through over-expressing ZWF1 contributed to isobutanol biosynthesis in S. cerevisiae. These results also verified the proposed concept of increasing isobutanol titers in S. cerevisiae by resolving cofactor imbalance. Finally, this study provides a new strategy for enhancing isobutanol biosynthesis.