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Industrial 20-m3-scale and laboratory-scale aerobic fed-batch processes with Escherichia coli were compared. In the large-scale process the observed overall biomass yield was reduced by 12% at a cell density of 33 g/l
and formate accumulated to 50 mg/l during the later constant-feeding stage of the process. Though the dissolved oxygen signal
did not show any oxygen limitation, it is proposed that the lowered yield and the formate accumulation are caused by mixed-acid
fermentation in local zones where a high glucose concentration induced oxygen limitation. The hypothesis was further investigated
in a scale-down reactor with a controlled oxygen-limitation compartment. In this scale-down reactor similar results were obtained:
i.e. an observed yield lowered by 12% and formate accumulation to 238 mg/l. The dynamics of glucose uptake and mixed-acid
product formation (acetate, formate, d-lactate, succinate and ethanol) were investigated within the 54 s of passage time through the oxygen-limited compartment.
Of these, all except succinate and ethanol were formed; however, the products were re-assimilated in the oxygen-sufficient
reactor compartment. Formate was less readily assimilated, which accounts for its accumulation. The total volume of the induced-oxygen-limited
zones was estimated to be 10% of the whole liquid volume in the large bioreactor. It is also suggested that repeated excretion
and re-assimilation of mixed-acid products contribute to the reduced yield during scale-up and that formate analysis is useful
for detecting local oxygen deficiency in large-scale E. coli processes.
Received: 7 November 1998 / Received revision: 4 February 1999 / Accepted: 5 February 1999 相似文献
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F. Bylund F. Guillard S.-O. Enfors C. Trägårdh G. Larsson 《Bioprocess and biosystems engineering》1999,20(5):377-389
A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m3 bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor. 相似文献
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Kar T Delvigne F Masson M Destain J Thonart P 《Journal of industrial microbiology & biotechnology》2008,35(9):1053-1059
The influence of three extracellular factors (namely, the methyl oleate dispersion in the broth, the dissolved oxygen variations, and the pH fluctuation) on the lipase production by Y. lipolytica in batch bioreactor has been investigated in different scale-down apparatus. These systems allow to reproduce the hydrodynamic phenomena encountered in large-scale equipments for the three specified factors. The effects of the extracellular factors have been observed at three distinct levels: the microbial growth, the extracellular lipase production, and the induction of the gene LIP2 encoding for the main lipase of Y. lipolytica. Among the set of environmental factors investigated, the dissolved oxygen fluctuations generated in a controlled scale-down reactor (C-SDR) have led to the more pronounced physiological effect by decreasing the LIP2 gene expression level. The other environmental factors observed in a partitioned scale-down reactor, i.e., the methyl oleate dispersion and the pH fluctuations, have led to a less severe stress traduced only by a decrease of the microbial yield and thus of the extracellular lipase specific production rate. 相似文献
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DNA microarray analysis was used to profile gene expression in a commercial isolate of Saccharomyces cerevisiae grown in a synthetic grape juice medium under conditions mimicking a natural environment for yeast: High-sugar and variable nitrogen conditions. The high nitrogen condition displayed elevated levels of expression of genes involved in biosynthesis of macromolecular precursors across the time course as compared to low-nitrogen. In contrast, expression of genes involved in translation and oxidative carbon metabolism were increased in the low-nitrogen condition, suggesting that respiration is more nitrogen-conserving than fermentation. Several genes under glucose repression control were induced in low-nitrogen in spite of very high (17%) external glucose concentrations, but there was no general relief of glucose repression. Expression of many stress response genes was elevated in stationary phase. Some of these genes were expressed regardless of the nitrogen concentration while others were found at higher levels only under high nitrogen conditions. A few genes, FSP2, RGS2, AQY1, YFL030W, were expressed more strongly with nitrogen limitation as compared to other conditions. 相似文献
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Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis 总被引:3,自引:0,他引:3
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Presecan-Siedel E Galinier A Longin R Deutscher J Danchin A Glaser P Martin-Verstraete I 《Journal of bacteriology》1999,181(22):6889-6897
In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase. 相似文献
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In large-scale bioprocesses microbes are exposed to heterogeneous substrate availability reducing the overall process performance. A series of deletion strains was constructed from E. coli MG1655 aiming for a robust phenotype in heterogeneous fermentations with transient starvation. Deletion targets were hand-picked based on a list of genes derived from previous large-scale simulation runs. Each gene deletion was conducted on the premise of strict neutrality towards growth parameters in glucose minimal medium. The final strain of the series, named E. coli RM214, was cultivated continuously in an STR-PFR (stirred tank reactor – plug flow reactor) scale-down reactor. The scale-down reactor system simulated repeated passages through a glucose starvation zone. When exposed to nutrient gradients, E. coli RM214 had a significantly lower maintenance coefficient than E. coli MG1655 (Δms = 0.038 gGlucose/gCDW/h, p < 0.05). In an exemplary protein production scenario E. coli RM214 remained significantly more productive than E. coli MG1655 reaching 44% higher eGFP yield after 28 h of STR-PFR cultivation. This study developed E. coli RM214 as a robust chassis strain and demonstrated the feasibility of engineering microbial hosts for large-scale applications. 相似文献
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为研究金丝桃苷对高糖诱导的人神经母细胞瘤(SH-SY5Y)细胞氧化损伤的保护作用及机制,用含100mmo L/L葡萄糖和分别为20、50、100μmo L/L金丝桃苷的培养基共同孵育SH-SY5Y细胞36 h,检测细胞活力、细胞培养液中乳酸脱氢酶(LDH)水平及半胱氨酸天冬氨酸蛋白酶-3(caspase-3)活性,细胞内活性氧(ROS)水平、丙二醛(MDA)、还原型谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性及SIRT1和NF-кB基因的mRNA水平和蛋白含量。结果显示金丝桃苷可提高高糖诱导后SH-SY5Y细胞的存活率,抑制细胞LDH释放,清除ROS,降低MDA含量与caspase-3活性,增强SOD、CAT活性和GSH含量;同时,金丝桃苷还能提高SIRT1基因的mR-NA表达及蛋白含量,降低NF-кB基因的mRNA水平和蛋白含量。结果表明金丝桃苷能通过激活SIRT1基因,抑制NF-кB基因保护高糖所致SH-SY5Y细胞的氧化损伤。 相似文献
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Overproduction of acetate kinase activates the phosphate regulon in the absence of the phoR and phoM functions in Escherichia coli. 总被引:7,自引:4,他引:3
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A DNA fragment of Escherichia coli cloned on pBR322 elevated the production of alkaline phosphatase and phosphate-binding protein in a phoR phoM strain. Nucleotide sequence analysis and enzyme assays revealed that the DNA fragment contained the ackA gene, which codes for acetate kinase. A high gene dosage of ackA was needed to induce the production of alkaline phosphatase and phosphate-binding protein in this strain. Overexpression of ackA elevated the intracellular ATP concentration, an effect that might be related to activation of the phosphate regulon in the phoR phoM strain. 相似文献
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Annick Lejeune F. Delvigne P. Thonart 《Journal of industrial microbiology & biotechnology》2010,37(3):225-236
Yeast is a widely used microorganism at the industrial level because of its biomass and metabolite production capabilities.
However, due to its sensitivity to the glucose effect, problems occur during scale-up to the industrial scale. Hydrodynamic
conditions are not ideal in large-scale bioreactors, and glucose concentration gradients can arise when these bioreactors
are operating in fed-batch mode. We have studied the effects of such gradients in a scale-down reactor, which consists of
a mixed part linked to a non-mixed part by a recirculation pump, in order to mimic the hydrodynamic conditions encountered
at the large scale. During the fermentation tests in the scale-down reactor, there was a drop in both biomass yield (ratio
between the biomass produced and the glucose added) and trehalose production and an increase in both fermentation time (time
between inoculation and beginning of stationary phase) and ethanol production. We have developed a stochastic model which
explains these effects as the result of an induction process determined mainly by the hydrodynamic conditions. The concentration
profiles experienced by the microorganisms during the scale-down tests were expressed and linked to the biomass yields of
the scale-down tests. 相似文献
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The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared. In addition to the parameters of product yield and quality, also cell yield, respiration, overflow, mixed acid fermentation, glucose concentration, and cell lysis were studied and compared. The results show that oxygen limitation following glucose overflow was the critical parameter and not the glucose overflow itself. This was verified by the pattern of byproduct formation where formate was the dominating factor and not acetic acid. A correlation between the accumulation of formate, the degree of heterogeneity, and cell lysis was also visualized when recombinant protein was expressed. The production pattern could be mimicked in the SDR reactor for all parameters, except for product quantity and quality, where 30% fewer rhGH-degraded forms were present and where about 80% higher total yield was achieved, resulting in 10% greater accumulation of properly formed rhGH monomer. 相似文献
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Cheng DW Jiang Y Shalev A Kowluru R Crook ED Singh LP 《Archives of physiology and biochemistry》2006,112(4-5):189-218
Renal mesangial cells play an important role in the development of diabetic kidney disease. We have previously demonstrated that some of the effects of high glucose on mesangial extracellular matrix (ECM) protein expression are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 oligochips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on mRNA expression of a mouse mesangial cell line (MES-13). We sought to determine the portion of mRNA expression in MES-13 cells, which is mediated both by high glucose and glucosamine, i.e., via the HBP. Of the 34,000 genes on the chip, approximately 55.7 - 60.8% genes are detected in MES-13 cells. Culturing MES-13 cells for 48 h with HG alters the expression of approximately 389 genes at our preset threshold levels (at least 2-fold change) where 263 genes are up-regulated and 126 genes are down-regulated. GlcN also increases the expression of 106 genes and decreases 94 genes during the same period of incubation. Seventy-two genes in the chip are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The mRNA level of thioredoxin interacting protein (TXNIP), an inhibitor of thioredoxin activity, is maximally increased approximately 18.8 and 9.9-fold respectively by HG and GlcN. The differential expression of several genes found in the microarray analysis is further validated by real-time quantitative PCR. Significant biological processes commonly targeted by HG and GlcN are the TXNIP-thioredoxin system, oxidative stress, endoplasmic reticulum (ER) stress, extracellular matrix genes, and interferon-inducible genes. Stable overexpression of TXNIP in MES-13 cells increases glucose and glucosamine-mediated ECM gene expression and oxidative stress. We conclude from these results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes reactive oxygen species generation to cause cellular oxidative stress, ECM gene expression and apoptosis. 相似文献
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Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance. 总被引:11,自引:8,他引:3
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The proU locus in Escherichia coli encodes an important osmoregulatory function which mediates the growth-promoting effect of L-proline and glycine betaine in high-osmolarity media. This locus was cloned, in contiguity with a closely linked Tn10 insertion, onto a multicopy plasmid directly from the E. coli chromosome. For a given level of osmotic stress, the magnitude of osmoresponsive induction of a single-copy proU::lac fusion was reduced in strains with multiple copies of the proU+ genes; in comparison with haploid proU+ strains, strains with the multicopy proU+ plasmids also exhibited enhanced osmotolerance in media supplemented with 1 mM L-proline or glycine betaine. Experiments involving subcloning, Tn1000 mutagenesis, and interplasmid complementation in a deletion mutant provided evidence for the presence at this locus of two cistrons, both of which are necessary for the expression of ProU function. We propose the designations proU for the gene originally identified by the proU224::Mu d1(lac Ap) insertion and proV for the gene upstream (that is, counterclockwise) of proU. 相似文献
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Jantama K Haupt MJ Svoronos SA Zhang X Moore JC Shanmugam KT Ingram LO 《Biotechnology and bioengineering》2008,99(5):1140-1153
Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622-733 mM of succinate with molar yields of 1.2-1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized. 相似文献