Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol formation in Saccharomyces cerevisiae

Gpd1 and Gpd2 are the two isoforms of glycerol 3-phosphate dehydrogenase (GPDH), which is the rate-controlling enzyme of glycerol formation in Saccharomyces cerevisiae. The two isoenzymes play crucial roles in osmoregulation and redox balancing. Past approaches to increase ethanol yield at the cost of reduced glycerol yield have most often been based on deletion of either one or two isogenes (GPD1 and GPD2). While single deletions of GPD1 or GPD2 reduced glycerol formation only slightly, the gpd1Δ gpd2Δ double deletion strain produced zero glycerol but showed an osmosensitive phenotype and abolished anaerobic growth. Our current approach has sought to generate "intermediate" phenotypes by reducing both isoenzyme activities without abolishing them. To this end, the GPD1 promoter was replaced in a gpd2Δ background by two lower-strength TEF1 promoter mutants. In the same manner, the activity of the GPD2 promoter was reduced in a gpd1Δ background. The resulting strains were crossed to obtain different combinations of residual GPD1 and GPD2 expression levels. Among our engineered strains we identified four candidates showing improved ethanol yields compared to the wild type. In contrast to a gpd1Δ gpd2Δ double-deletion strain, these strains were able to completely ferment the sugars under quasi-anaerobic conditions in both minimal medium and during simultaneous saccharification and fermentation (SSF) of liquefied wheat mash (wheat liquefact). This result implies that our strains can tolerate the ethanol concentration at the end of the wheat liquefact SSF (up to 90 g liter(-1)). Moreover, a few of these strains showed no significant reduction in osmotic stress tolerance compared to the wild type.     

Increasing ethanol titer and yield in a gpd1Δ gpd2Δ strain by simultaneous overexpression of GLT1 and STL1 in Saccharomyces cerevisiae

We have investigated whether simultaneous modification of cofactor metabolism and glycerol in a strain of Saccharomyces cerevisiae can eliminate glycerol synthesis during ethanol production. Two strains, S812 (gpd1Δ gpd2Δ PGK1p-GLT1) and LE17 (gpd1Δ gpd2Δ PGK1p-GLT1 PGKp-STL1) were generated that showed a 8 and 8.2 % increase in the ethanol yield, respectively, compared to the wild type KAM-2 strain. The ethanol titer was improved from 90.4 g/l for KAM-2 to 97.6 g/l for S812 and 97.8 g/l for LE17, respectively. These results provide a new insight into rationalization of metabolic engineering strategies for improvement of ethanol yield through elimination of glycerol production.     

Increased ethanol production from glycerol by Saccharomyces cerevisiae strains with enhanced stress tolerance from the overexpression of SAGA complex components

During the industrial production of ethanol using yeast, the cells are exposed to stresses that affect their growth and productivity; therefore, stress-tolerant yeast strains are highly desirable. To increase ethanol production from glycerol, a greater tolerance to osmotic and ethanol stress was engineered in yeast strains that were impaired in endogenous glycerol production by the overexpression of both SPT3 and SPT15, components of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex. The engineered strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupSpt3.15Cas) formed significantly more biomass compared to the strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas), and both engineered strains displayed increased biomass when compared to the control YPH499 fps1Δgpd2Δ (pESC-TRP) strain. The trehalose accumulation and ergosterol content of these strains were 2.3-fold and 1.6-fold higher, respectively, than the parent strains, suggesting that levels of cellular membrane components were correlated with the enhanced stress tolerance of the engineered strains. Consequently, the ethanol production of the engineered strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupSpt3.15Cas) was 1.8-fold more than that of strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas), with about 8.1g/L ethanol produced. In conclusion, we successfully established that the co-expression of SPT3 and SPT15 that improved the fermentation performance of the engineered yeast strains which produced higher ethanol yields than stress-sensitive yeast strains.     

Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺), was expressed in the gpd1Δ gpd2Δ strain, anaerobic growth was restored by supplementation with 2.0 g liter⁻¹ acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).Bioethanol production by Saccharomyces cerevisiae is currently, by volume, the single largest fermentation process in industrial biotechnology. A global research effort is under way to expand the substrate range of S. cerevisiae to include lignocellulosic hydrolysates of nonfood feedstocks (e.g., energy crops and agricultural residues) and to increase productivity, robustness, and product yield (for reviews see references 20 and 35). A major challenge relating to the stoichiometry of yeast-based ethanol production is that substantial amounts of glycerol are invariably formed as a by-product (24). It has been estimated that, in typical industrial ethanol processes, up to 4% of the sugar feedstock is converted into glycerol (24). Although glycerol also serves as a compatible solute at high extracellular osmolarity (10), glycerol production under anaerobic conditions is primarily linked to redox metabolism (34).During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation. In this process, the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD⁺-dependent alcohol dehydrogenase. The fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD⁺ to NADH occurs elsewhere in the metabolism. Such a net production of NADH occurs in assimilation when yeast biomass is synthesized from glucose and ammonia (34). Under anaerobic conditions, NADH reoxidation in S. cerevisiae is strictly dependent on reduction of sugar to glycerol (34). Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol-3-phosphate, a reaction catalyzed by NAD⁺-dependent glycerol-3-phosphate dehydrogenase. Subsequently, the glycerol-3-phosphate formed in this reaction is hydrolyzed by glycerol-3-phosphatase to yield glycerol and inorganic phosphate.The importance of glycerol production for fermentative growth of yeasts was already observed in the 1960s during studies of non-Saccharomyces yeasts that exhibit a so-called “Custers effect.” In such yeast species, which are naturally unable to produce glycerol, fermentative growth on glucose is possible only in the presence of an external electron acceptor that can be reduced via an NADH-dependent reaction (e.g., the reduction of acetoin to butanediol via NAD⁺-dependent butanediol dehydrogenase) (29). It was later shown that gpd1Δ gpd2Δ strains of S. cerevisiae, which are also unable to produce glycerol, are similarly unable to grow under anaerobic conditions unless provided with acetoin as an external electron acceptor (8).In view of its large economic significance, several metabolic engineering strategies have been explored to reduce or eliminate glycerol production in anaerobic cultures of S. cerevisiae. Nissen et al. (25) changed the cofactor specificity of glutamate dehydrogenase, the major ammonia-fixing enzyme of S. cerevisiae, thereby increasing NADH consumption in biosynthesis. This approach significantly reduced glycerol production in anaerobic cultures grown with ammonia as the nitrogen source. Attempts to further reduce glycerol production by expression of a heterologous transhydrogenase, with the aim to convert NADH and NADP⁺ into NAD⁺ and NADPH, were unsuccessful (24) because intracellular concentrations of these pyridine nucleotide cofactor couples favor the reverse reaction (23).The goal of the present study was to investigate whether the engineering of a linear pathway for the NADH-dependent reduction of acetic acid to ethanol can replace glycerol formation as a redox sink in anaerobic, glucose-grown cultures of S. cerevisiae and thus provide a stoichiometric basis for elimination of glycerol production during industrial ethanol production. Significant amounts of acetic acid are released upon hydrolysis of lignocellulosic biomass, and, in fact, acetic acid is studied as an inhibitor of yeast metabolism in lignocellulosic hydrolysates (5, 7, 26). The S. cerevisiae genome already contains genes encoding acetyl coenzyme A (acetyl-CoA) synthetase (32) and NAD⁺-dependent alcohol dehydrogenases (ADH1-5 [12]). To complete the linear pathway for acetic acid reduction, we expressed an NAD⁺-dependent, acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) from Escherichia coli into a gpd1Δ gpd2Δ strain of S. cerevisiae. This enzyme, encoded by the E. coli mhpF gene (15), catalyzes the reaction acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺. Growth and product formation of the engineered strain were then compared in the presence and absence of acetic acid and compared to those of a congenic reference strain.     

Effects of oxygen supply on yeast growth and metabolism in continuous fermentation

The yield changes in cell mass and metabolites with changes in the oxygen supply rate were investigated in continuous ethanol fermentation. With increases in oxygen concentration in the purging gas from 5.3 to 39.3 %, the specific oxygen uptake rate (qO₂) increased from 0.158 to 1.24 mmol/g/h. With this change, cell mass increased from 13.2 to 14.9 g/l and glycerol decreased from 4.8 to 0.99 g/l, although little change in ethanol yield was observed. At a higher oxygen concentration and/or at a lower respiratory quotient (RQ), glycerol disappeared, acetaldehyde, acetoin and 2,3-butanediol increased, and ethanol started to decrease. The yields of iso-butylalcohol and iso-amylalcohol also increased with increases in the oxygen supply rate when RQ was lower than approximately 10. Reduction in the redox balance (NADH/NAD) in the cells by qO₂, appeared to reduce initially the rate of glycerol-3-phosphate formation and next the rate of ethanol formation, resulting in the accumulation of acetaldehyde and formation of 2,3-butanediol through acetoin. Fatty acid composition changed with changes in the oxygen supply rate. The value for unsaturation, Δ mol⁻¹, increased from 0.745 to 0.836 with the increase in qO₂ from 0.158 to 1.79 mmol/g/h. Increases in oleic acid (C18:1) and decreases in palmitic acid (C16:0) were the major changes with the increases in Δ mol⁻¹.     

Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O<Subscript>2</Subscript> deprivation

Objective

To explore the glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O₂ deprivation.

Result

Overexpression of a glycerol facilitator, glycerol dehydrogenase and dihydroxyacetone kinase from Escherichia coli K-12 in C. glutamicum led to recombinant strains NC-3G diverting glycerol utilization towards succinate production under O₂ deprivation. Under these conditions, strain NC-3G efficiently consumed glycerol and produced succinate without growth. The recombinant C. glutamicum utilizing glycerol as the sole carbon source showed higher intracellular NADH/NAD⁺ ratio compare with utilizing glucose. The mass conversion of succinate increased from 0.64 to 0.95. Using an anaerobic fed-batch fermentation process, the final strain produced 38.4 g succinate/l with an average yield of 1.02 g/g.

Conclusions

The metabolically-engineered strains showed an efficient succinate production using glycerol as sole carbon source under O₂ deprivation.

     

Effects of GPD1 Overexpression in Saccharomyces cerevisiae Commercial Wine Yeast Strains Lacking ALD6 Genes

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP⁺-dependent Mg²⁺-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.     

Elimination of glycerol and replacement with alternative products in ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2–3% of the sugar fermented is converted to glycerol. Replacing the NAD⁺-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD⁺ by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD⁺ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.     

Effect of alternative NAD<Superscript>+</Superscript>-regenerating pathways on the formation of primary and secondary aroma compounds in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> glycerol-defective mutant

Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD⁺-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD⁺ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD⁺/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.     

A metabolic engineering strategy for producing free fatty acids by the Yarrowia lipolytica yeast based on impairment of glycerol metabolism

In recent years, bio‐based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L^?1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L^?1. Next, an isolated oleate‐resistant strain produced 382.8 mg L^?1 of free fatty acids. Finely, acyl‐CoA carboxylase gene (ACC1) over‐expression resulted to production of 1436.7 mg L^?1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L^?1 during test tube cultivation.

     

Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing

2,3-Butanediol is a promising valuable chemical that can be used in various areas as a liquid fuel and a platform chemical. Here, 2,3-butanediol production in Saccharomyces cerevisiae was improved stepwise by eliminating byproduct formation and redox rebalancing. By introducing heterologous 2,3-butanediol biosynthetic pathway and deleting competing pathways producing ethanol and glycerol, metabolic flux was successfully redirected to 2,3-butanediol. In addition, the resulting redox cofactor imbalance was restored by overexpressing water-forming NADH oxidase (NoxE) from Lactococcus lactis. In a flask fed-batch fermentation with optimized conditions, the engineered adh1Δadh2Δadh3Δadh4Δadh5Δgpd1Δgpd2Δ strain overexpressing Bacillus subtilis α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD), S. cerevisiae 2,3-butanediol dehydrogenase (Bdh1), and L. lactis NoxE from a single multigene-expression vector produced 72.9 g/L 2,3-butanediol with the highest yield (0.41 g/g glucose) and productivity (1.43 g/(L·h)) ever reported in S. cerevisiae.     

Enhanced production of ethanol from glycerol by engineered Hansenula polymorpha expressing pyruvate decarboxylase and aldehyde dehydrogenase genes from Zymomonas mobilis

To improve production of ethanol from glycerol, the methylotrophic yeast Hansenula polymorpha was engineered to express the pdc and adhB genes encoding pyruvate decarboxylase and aldehyde dehydrogenase II from Zymomonas mobilis, respectively, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The ethanol yield was 3.3-fold higher (2.74 g l^?1) in the engineered yeast compared with the parent strain (0.83 g l^?1). Further engineering to stimulate glycerol utilization in the recombinant strain via expression of dhaD and dhaKLM genes from Klebsiella pneumoniae encoding glycerol dehydrogenase and dehydroxyacetone kinase, respectively, resulted in a 3.7-fold increase (3.1 g l^?1) in ethanol yield.     

Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase

The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol.     

产甘油假丝酵母与酿酒酵母胞浆3-磷酸甘油脱氢酶基因的功能比较

胞浆3-磷酸甘油脱氢酶(GPD)是酿酒酵母细胞甘油合成过程中的关键限速酶．尽管高产甘油菌株产甘油假丝酵母基因组中编码该酶的基因CgGPD已经被克隆出来,但是具体的功能,特别是与酿酒酵母GPD1和GPD2基因的功能比较值得进一步研究．以酿酒酵母渗透压敏感型的gpd1/gpd2和gpd1突变株为宿主,分别导入CgGPD、GPD1和GPD2基因,比较分析了CgGPD、GPD1和GPD2基因在高渗透压胁迫条件下和厌氧环境中的表达调控,及其对细胞甘油合成能力的影响．研究发现,GPD1基因受到渗透压诱导表达,GPD2基因在细胞厌氧条件下起着氧化还原平衡调节作用,而CgGPD基因不仅能够在渗透压胁迫条件下通过过量快速合成甘油调节渗透压平衡,而且能够在厌氧培养环境中互补GPD2基因的缺失,使gpd1/gpd2缺失突变株能够正常生长,同时提高了突变株的甘油合成能力．结果表明,CgGPD基因在gpd1/gpd2缺失突变株中既具有GPD1基因的功能,又能发挥GPD2基因的功能．     

Flow rate and bead size as critical parameters for immobilized-yeast reactors

The productivity of immobilized yeast cell reactors varies with a number of parameters, including flow, amount and growth rate of yeast, bead size and type of medium. Variation of these parameters has a pronounced effect on reaction rate. This paper presents typical ranges for these productivities and demonstrates the patterns of changes that take place when bead size, flow and reaction medium are varied. Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for the production of ethanol. The productivity of immobilized yeast in a batch reactor (0.2 g ethanol/g yeast · h) was only two-thirds that of free cells suspended at an equivalent cell density (0.3 g ethanol/g yeast · h). Different flow rates and bead sizes were used to ‘optimize’ the productivity. The productivity of 3.34 mm beads at a flow rate of 8.8 litre h^?1(superficial velocity: 0.12 cm s^?1) was 95% higher than that at 1.0 l h^?1. Maximum productivities of 0.34, 0.27, 0.22 g/g yeast· h were obtained (at a flow rate of 8.8 l h^?1) for 9.2% yeast-immobilized beads of 3.34, 4.45 and 5.65 mm in diameter, respectively.     

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.