Alleviation of glucose repression of maltose metabolism by MIG1 disruption in Saccharomyces cerevisiae.

The MIG1 gene was disrupted in a haploid laboratory strain (B224) and in an industrial polyploid strain (DGI 342) of Saccharomyces cerevisiae. The alleviation of glucose repression of the expression of MAL genes and alleviation of glucose control of maltose metabolism were investigated in batch cultivations on glucose-maltose mixtures. In the MIG1-disrupted haploid strain, glucose repression was partly alleviated; i.e., maltose metabolism was initiated at higher glucose concentrations than in the corresponding wild-type strain. In contrast, the polyploid delta mig1 strain exhibited an even more stringent glucose control of maltose metabolism than the corresponding wild-type strain, which could be explained by a more rigid catabolite inactivation of maltose permease, affecting the uptake of maltose. Growth on the glucose-sucrose mixture showed that the polypoid delta mig1 strain was relieved of glucose repression of the SUC genes. The disruption of MIG1 was shown to bring about pleiotropic effects, manifested in changes in the pattern of secreted metabolites and in the specific growth rate.     

The impact of MIG1 and/or MIG2 disruption on aerobic metabolism of succinate dehydrogenase negative Saccharomyces cerevisiae

The zinc finger proteins Mig1 and Mig2 play important roles in glucose repression of Saccharomyces cerevisiae. To investigate whether the alleviation of glucose effect would result in an increase in aerobic succinate production, MIG1 and/or MIG2 were disrupted in a succinate dehydrogenase (SDH)-negative S. cerevisiae strain. Moreover, their impacts on physiology of the SDH-negative S. cerevisiae strain were studied under fully aerobic conditions when glucose was the sole carbon source. Our results showed that the succinate production for the SDH-negative S. cerevisiae was very low even under fully aerobic conditions. Furthermore, deletion of MIG1 and/or MIG2 did not result in an increase in succinate production in the SDH-negative S. cerevisiae strain. However, the synthesis of acetate was significantly affected by MIG1 deletion or in combination with MIG2 deletion. The acetate production for the mig1/mig2 double mutant BS2M was reduced by 69.72% compared to the parent strain B2S. In addition, the amount of ethanol produced by BS2M was slightly decreased. With the mig2 mutant BSM2, the concentrations of pyruvate and glycerol were increased by 26.23% and 15.28%, respectively, compared to the parent strain B2S.     

Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger

Aspergillus niger produces oxalic acid as a by-product which causes problems with downstream processing of industrial enzymes. To overcome this problem the oah gene encoding oxaloacetate hydrolase (EC 3.7.1.1) was disrupted in a glucoamylase-producing strain of A. niger and the resulting strain was incapable of producing oxalic acid. The strain with the disrupted gene was compared with the wild-type strain producing oxalic acid in batch cultivations. The specific growth rate of both strains was 0.20 h(-1). The citric acid yields were identical, but the glucoamylase yield was only 50% in the disruptant compared with the wild-type strain. Batch experiments with 13C-labeled glucose as substrate were carried out to determine the metabolic fluxes through the central metabolism. The two strains had almost identical metabolic fluxes, which suggested that it was possible to disrupt the oah gene without pleiotropic consequences. The flux through the pentose phosphate pathway was around 60% of the glucose uptake for both strains, which suggested that a sufficient supply of NADPH was available for biosynthesis.     

敲除MIG1和SNF1基因对酿酒酵母共利用葡萄糖和木糖的影响

Mig1和Snf1是酿酒酵母葡萄糖阻遏效应的两个关键调控因子。为了提高酿酒酵母工程菌同时利用葡萄糖和木糖的能力,分别对MIG1和SNF1基因进行了单敲除和双敲除,并通过摇瓶发酵实验和RNA-Seq转录组分析,初步揭示了Mig1和Snf1可能影响葡萄糖和木糖共利用表达差异基因的层级调控机制。研究结果表明,MIG1单敲除对混合糖的共利用影响不大;SNF1单敲除会加快混合糖中木糖的利用而且葡萄糖和木糖可以被同时利用,这可能归因于SNF1单敲除会解除对一些氮分解代谢阻遏基因表达的抑制,从而促进了细胞对氮源营养的利用;进一步敲除MIG1,会解除更多氮分解代谢阻遏基因表达的抑制,以及一些碳中心代谢途径基因表达上调。虽然MIG1和SNF1双敲除菌株利用葡萄糖加快而利用木糖变慢,但是葡萄糖和木糖可以被同时利用,进而加快乙醇的积累。综上所述,MIG1和SNF1的敲除导致氮分解阻遏基因表达上调,有助于促进葡萄糖和木糖的共利用;解析Mig1和Snf1对氮分解阻遏基因的层级调控作用,为进一步提高葡萄糖和木糖的共利用提供新的靶点。     

Physiological studies in aerobic batch cultivations of Saccharomyces cerevisiae strains harboring the MEL1 gene

Physiological studies of Saccharomyces cerevisiae strains harboring the MEL1 gene were carried out in aerobic batch cultivations on glucose-galactose mixtures and on the disaccharide melibiose, which is hydrolyzed by the enzyme melibiase (Mel1, EC 3.2.1.22) into a glucose and a galactose moiety. The strains examined (T200, T256, M24, and TH1) were all derived from the bakers' and distillers' strain of S. cerevisiae, DGI 342. All the strains showed a significant higher ethanol yield when growing on glucose, and half the biomass yield, compared with growth on galactose. The maximum specific uptake rates were 2.5-3.3-fold higher on glucose than on galactose for all the strains examined, and hence, ethanol production was pronounced on glucose due to respiro-fermentative metabolism. The T256 strain and the T200 strain having the MEL1 gene inserted in the HXK2 locus and the LEU2 locus, respectively, hydrolyzed melibiose with low specific hydrolysis rates of 0.03 C-mol/g/h and 0.04 C-mol/g/h, respectively. This resulted in high biomass yields on melibiose in the order of 10 g/C-mol compared with 3.7 g/C-mol for M24 and 1.6 g/C-mol for TH1. The M24 strain, constructed by classical breeding, and the mig1/gal80 disrupted and melibiase-producing strain TH1, were superior in their ability to hydrolyze melibiose into glucose and galactose showing specific melibiose hydrolysis rates of 0.17 C-mol/g/h and 0.24 C-mol/g/h, respectively. Hence, high ethanol yields on melibiose were obtained with these two strains. Growth on the glucose-galactose mixtures showed a reduction of glucose control successfully obtained in the M24 strain and the TH1 strain.     

Engineering of carbon catabolite repression in recombinant xylose fermenting<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Two xylose-fermenting glucose-derepressed Saccharomyces cerevisiae strains were constructed in order to investigate the influence of carbon catabolite repression on xylose metabolism. S. cerevisiae CPB.CR2 (mig1, XYL1, XYL2, XKS1) and CPB.MBH2 (mig1, mig2, XYL1, XYL2, XKS1) were analysed for changes in xylose consumption rate and ethanol production rate during anaerobic batch and chemostat cultivations on a mixture of 20 g l^–1 glucose and 50 g l^–1 xylose, and their characteristics were compared to the parental strain S. cerevisiae TMB3001 (XYL1, XYL2, XKS1). Improvement of xylose utilisation was limited during batch cultivations for the constructed strains compared to the parental strain. However, a 25% and 12% increased xylose consumption rate during chemostat cultivation was achieved for CPB.CR2 and CPB.MBH2, respectively. Furthermore, during chemostat cultivations of CPB.CR2, where the cells are assumed to grow under non-repressive conditions as they sense almost no glucose, invertase activity was lower during growth on xylose and glucose than on glucose only. The 3-fold reduction in invertase activity could only be attributed to the presence of xylose, suggesting that xylose is a repressive sugar for S. cerevisiae.     

A constitutive catabolite repression mutant of a recombinantSaccharomyces cerevisiae strain improves xylose consumption during fermentation

Efficient xylose utilisation by microorganisms is of importance to the lignocellulose fermentation industry. The aim of this work was to develop constitutive catabolite repression mutants in a xylose-utilising recombinantSaccharomyces cerevisiae strain and evaluate the differences in xylose consumption under fermentation conditions.S. cerevisiae YUSM was constitutively catabolite repressed through specific disruptions within theMIG1 gene. The strains were grown aerobically in synthetic complete medium with xylose as the sole carbon source. Constitutive catabolite repressed strain YCR17 grew four-fold better on xylose in aerobic conditions than the control strain YUSM. Anaerobic batch fermentation in minimal medium with glucose-xylose mixtures and N-limited chemostats with varying sugar concentrations were performed. Sugar utilisation and metabolite production during fermentation were monitored. YCR17 exhibited a faster xylose consumption rate than YUSM under high glucose conditions in nitrogen-limited chemostat cultivations. This study shows that a constitutive catabolite repressed mutant could be used to enhance the xylose consumption rate even in the presence of high glucose in the fermentation medium. This could help in reducing fermentation time and cost in mixed sugar fermentation.     

On-line monitoring of recombinant bacterial cultures using multi-wavelength fluorescence spectroscopy

Multi-wavelength fluorescence spectroscopy was evaluated as a tool for on-line monitoring of recombinant Escherichia coli cultivations expressing human basic fibroblast growth factor (hFGF-2). The data sets for the various combinations of the excitation and emission spectra from batch cultivations were analyzed using principal component analysis. Chemometric models (the partial least squares method) were developed for correlating the fluorescence data and the experimentally measured variables such as the biomass and glucose concentrations as well as the carbon dioxide production rate. Excellent correlations were obtained for these variables for the calibration cultivations. The predictability of these models was further tested in batch and fed-batch cultivations. The batch cultivations were well predicted by the PLS models for biomass, glucose concentrations and carbon dioxide production rate (RMSEPs were respectively 5%, 7%, 9%). However, when tested for biomass concentrations in fed-batch cultivations (with final biomass three times higher than the highest calibration data) the models had good predictability at high growth rates (RMSEPs were 3% and 4%, respectively for uninduced and induced fed-batch cultivations), which was as good as for the batch cultivations used for developing the models (RMSEPs were 3% and 5%, respectively for uninduced and induced batch cultivations). The fed-batch cultivations performed at low growth rates exhibited much higher fluorescence for fluorophores such as flavin and NAD(P)H as compared to fed-batch cultivations at high growth rate. Therefore, the PLS models tended to over-predict the biomass concentrations at low growth rates. Obviously the cells changed their concentration of biogenic fluorophores depending on the growth rate. Although multi-wavelength fluorescence spectroscopy is a valuable tool for on-line monitoring of bioprocess, care must be taken to re-calibrate the PLS models at different growth rates to improve the accuracy of predictions.     

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.     

Distributed control of the glycolytic flux in wild-type cells and catabolite repression mutants of Saccharomyces cerevisiae growing in carbon-limited chemostat cultures

The sensitivity of the control of glycolysis was studied in the wild-type (WT) strain CEN.PK122 and in isogenic catabolite-repression mutants growing in carbon-limited, aerobic chemostat cultures at different dilution rates, D. Based on a model of glycolysis in which the glucose transport step was considered reversible and inhibited by glucose 6-phosphate (G6P), the matrix method of metabolic control analysis was applied. In the present work, we report that the control of glycolysis was significantly distributed between the glucose uptake, hexokinase, and phosphofructokinase steps. The flux control properties were sensitive to the glucose gradient through the membrane and the extent of inhibition of the transport by G6P as parameters of the glucose-uptake kinetics in all strains tested. In the WT strain at low and high D, most of the control was exerted by the phosphofructokinase (PFK)-catalyzed step. In the cat1 mutant, the step catalyzed by PFK was the most rate controlling while in the cat3 strain, the control was shared between the PFK, hexokinase (HK), and glucose transport steps. On the other hand, the mig1 mutant exhibited high control by the glucose transporter depending on the glucose gradient across the membrane. The results obtained are discussed in terms of the dependence upon the type of metabolism displayed by yeast and the kinetics of the sugar transport step.     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Derepression of galactose metabolism in melibiase producing bakers' and distillers' yeast

Beet molasses is widely used as a growth substrate for bakers' and distillers' yeast in the production of biomass and ethanol. Most commercial yeasts do not fully utilise the carbohydrates in molasses since they are incapable of hydrolysing the disaccharide melibiose to glucose and galactose. Also, expression of genes encoding enzymes for the utilisation of carbon sources that are alternatives to glucose is tightly regulated, sometimes rates of yeast growth and/or ethanol production. The GAL genes are regulated by specific induction by galactose and repression during growth on glucose. In an industrial distillers' yeast, two genes interacting synergistically in glucose repression of galactose utilization, MIG1 and GAL80, have been disrupted with MEL1, encoding melibiase. The physiology of the wild-type strain and the recombinant strains was investigated on mixtures of glucose and galactose and on molasses. The recombinant strain started to ferment galactose when 9.7 g 1(-1) glucose was still present during a batch fermentation, whereas the wild-type strain did not consume any galactose in the presence of glucose. The ethanol yield in the recombinant strain was 0.50 g ethanol g sugar (-1) in an ethanol fermentation on molasses, compared with 0.48 g ethanol g sugar (-1) for the wild-type strain. The increased ethanol yield was due to utilization of melibiose in the molasses.