Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol

Use of agricultural biomass, other than corn-starch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87-94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.     

基于酶电极系统的葡萄糖浓度在线控制

补料分批技术在发酵工业中被广泛应用,其物料流加方式有3类,其中恒流速和指数补料属无反馈控制操作,靠经验或预设的数学模型决定补料速度,但由于发酵过程的复杂性,实际过程往往偏离预设的模型;恒底物浓度流加属反馈控制,通过对特定参数的检测,根据参数的变化情况反馈控制物料的流加,可控制菌生长在最佳条件下,从而获得高浓度的目的产物。反馈控制分直接控制和间接控制。间接     

Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts

The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N-terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three-fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (micro(+)/micro(-)) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met-ala-glu-hGH (MAE-hGH) was obtained. The fermentation process comprised a glucose-limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE-hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE-hGH--the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells--i.e. with or without formation of acetate--was not critical per se in order to obtain a high specific yield of MAE-hGH. Therefore it is unlikely that formation of MAE-hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE-hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context. From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.     

Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation

Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum.     

改造大肠杆菌合成海藻糖途径以高效合成海藻糖

海藻糖是相容性溶质的一种,因其具有多种生物学功能,在食品、化妆品、药品以及器官移植等方面均有很广泛应用。然而近几年生产海藻糖主要集中在使用酶催化的方法,虽然这种方法的转化效率高,但是却存在着副产物的问题,难以得到高纯度的海藻糖产品,严重制约了海藻糖的应用。本文通过基因工程技术在大肠杆菌Escherichia coli中构建了海藻糖高效合成新途径,通过全细胞催化合成海藻糖。利用PCR技术在哈氏噬纤维菌Cytophaga hutchinsonii中克隆获得海藻糖双功能合成酶基因(tpsp),采用E.coli pTac-HisA高效表达载体,实现海藻糖双功能合成酶基因(tpsp)高效表达,利用高效表达菌株进行全细胞催化,将葡萄糖高效转化为海藻糖。结果表明C.hutchinsonii海藻糖合成酶基因(tpsp)在E.coli中成功实现表达,该酶能够在胞内将葡萄糖高效转化为海藻糖,并将其转运到胞外,实现海藻糖的高效率合成,海藻糖的产量提高到1.2 g/L,相对转化率为21%。当将此高产菌株在发酵罐中进行转化时,海藻糖的产量达到13.3 g/L,葡萄糖的相对转化率达到48.6%。采用C.hutchinsonii海藻糖合成酶基因高效表达并且应用于海藻糖全细胞合成催化在国内外尚属首次报道,海藻糖的转化率及产率都已达到文献报道最高水平,本研究为开拓海藻糖生产新技术奠定了基础。     

Proposed mechanism of acetate accumulation in two recombinant Escherichia coli strains during high density fermentation

The productivity of Escherichia coli as a producer of recombinant proteins is affected by its metabolic properties, especially by acetate production. Two commercially used E. coli strains, BL21 (lambdaDE3) and JM109, differ significantly in their acetate production during batch fermentation at high initial glucose concentrations. E. coli BL21 grows to an optical density (OD, 600 nm) of 100 and produces no more than 2 g/L acetate, while E. coli JM109 grows to an OD (600 nm) of 80 and produces up to 14 g/L acetate. Even in fed-batch fermentation, when glucose concentration is maintained between 0.5 and 1.0 g/L, JM109 accumulates 4 times more acetate than BL21. To investigate the difference between the two strains, metabolites and enzymes involved in carbon utilization and acetate production were analyzed (isocitrate, ATP, phosphoenolpyruvate, pyruvate, isocitrate lyase, and isocitrate dehydrogenase). The results showed that during batch fermentation isocitrate lyase activity and isocitrate concentration were higher in BL21 than in JM109, while pyruvate concentration was higher in JM109. The activation of the glyoxylate shunt pathway at high glucose concentrations is suggested as a possible explanation for the lower acetate accumulation in E. coli BL21. Metabolic flux analysis of the batch cultures supports the activity of the glyoxylate shunt in E. coli BL21.     

Enhanced trehalose production improves growth of Escherichia coli under osmotic stress

The biosynthesis of trehalose has been previously shown to serve as an important osmoprotectant and stress protectant in Escherichia coli. Our results indicate that overproduction of trehalose (integrated lacI-Ptac-otsBA) above the level produced by the native regulatory system can be used to increase the growth of E. coli in M9-2% glucose medium at 37 degrees C to 41 degrees C and to increase growth at 37 degrees C in the presence of a variety of osmotic-stress agents (hexose sugars, inorganic salts, and pyruvate). Smaller improvements were noted with xylose and some fermentation products (ethanol and pyruvate). Based on these results, overproduction of trehalose may be a useful trait to include in biocatalysts engineered for commodity chemicals.     

Kinetics of ethanol production by recombinant Escherichia coli KO11

The fermentation kinetics for separate as well as simultaneous glucose and xylose fermentation with recombinant ethanologenic Escherichia coli KO11 are presented. Glucose and xylose were consumed simultaneously and exhibited mutual inhibition. The glucose exhibited 15 times stronger inhibition in xyclose fermentation than vice versa. The fermentation of condensate from steampretreated willow (Salix) was investigated. The kinetics were studied in detoxified as well as in nondetoxified condensate. The fermentation of the condensate followed two phases: First the glucose and some of the pentoses (xylose in addition to small amounts of arabinose) were fermented simultaneously, and then the remaining part of the pentoses were fermented. The rate of the first phase was independent of the detoxification method used, whereas the rate of the second phase was found to be strongly dependent. When the condensate was detoxified with overliming in combination with sulfite, which was the best detoxification method investigated, the sugars in the condensate, 9 g/L, were fermented in 11 h. The same fermentation took 150 h in nondetoxified condensate. The experimental data were used to develop an empirical model, describing the batch fermentation of recombinant E. coli KO11 in the condensate. The model is based on Monod kinetics including substrate and product inhibition and the sum of the inhibition exerted by the rest of the inhibitors, lumped together. (c) 1995 John Wiley & Sons, Inc.     

Aerobic fermentation of D-glucose by an evolved cytochrome oxidase-deficient Escherichia coli strain

Fermentation of glucose to D-lactic acid under aerobic growth conditions by an evolved Escherichia coli mutant deficient in three terminal oxidases is reported in this work. Cytochrome oxidases (cydAB, cyoABCD, and cbdAB) were removed from the E. coli K12 MG1655 genome, resulting in the ECOM3 (E. coli cytochrome oxidase mutant) strain. Removal of cytochrome oxidases reduced the oxygen uptake rate of the knockout strain by nearly 85%. Moreover, the knockout strain was initially incapable of growing on M9 minimal medium. After the ECOM3 strain was subjected to adaptive evolution on glucose M9 medium for 60 days, a growth rate equivalent to that of anaerobic wild-type E. coli was achieved. Our findings demonstrate that three independently adaptively evolved ECOM3 populations acquired different phenotypes: one produced lactate as a sole fermentation product, while the other two strains exhibited a mixed-acid fermentation under oxic growth conditions with lactate remaining as the major product. The homofermenting strain showed a D-lactate yield of 0.8 g/g from glucose. Gene expression and in silico model-based analyses were employed to identify perturbed pathways and explain phenotypic behavior. Significant upregulation of ygiN and sodAB explains the remaining oxygen uptake that was observed in evolved ECOM3 strains. E. coli strains produced in this study showed the ability to produce lactate as a fermentation product from glucose and to undergo mixed-acid fermentation during aerobic growth.     

Enrichment of fermentation media and optimization of expression conditions for the production of EAK(16) peptide as fusions with SUMO

EAK(16) (AEAEAKAKAEAKAEAK) belongs to a novel class of self-assembling peptides, which is being investigated in research and industry. SUMO belongs to the ubiquitin class of proteins and is a promising fusion partner currently in use. In this study, EAK(16) peptide fusions with hexa-histidine tagged SUMO have been constructed using Escherichia coli based pET expression vector. Intracellular expression of the SUMO-EAK(16) fusion using LB media has been optimized. Low-cost complex media (fungal autolysates, wheat and gluten hydrolysates) produced via a novel wheat-based biorefinery have been used as alternative fermentation media to LB. Shake flask cultures using either enriched LB or complex wheat-derived media containing 2 g/L of glucose resulted in intracellular SUMO-EAK(16) fusion protein production of approximately 250 mg/L fermentation volume which corresponded to 30-35% of the total bacterial protein expressed being the fusion protein. Fusion protein productivities up to five times higher were achieved when using a bioreactor.     

Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0. 5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB(glc). Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.     

Study of two-stage processes for the microbial production of 1,3-propanediol from glucose

The microbial production of 1,3-propanediol (1,3-PD) from glucose was studied in a two-stage fermentation process on a laboratory scale. In the first stage, glucose was converted to glycerol either by the osmotolerant yeast Pichia farinosa or by a recombinant Escherichia coli strain. In the second stage, glycerol in the broth from the first stage was converted to 1,3-PD by Klebsiella pneumoniae. The culture broth from P. farinosa was shown to contain toxic metabolites that strongly impair the growth of K. pneumoniae and the formation of 1,3-PD. Recombinant E. coli is more suitable than P. farinosa for producing glycerol in the first stage. The fermentation pattern from glycerol can be significantly altered by the presence of acetate, leading to a significant reduction of PD yield in the second stage. However, in the recombinant E. coli culture acetate formation can be prevented by fed-batch cultivation under limiting glucose supply, resulting in an effective production of 1,3-PD in the second stage with a productivity of 2.0 g l(-1) h(-1) and a high yield (0.53 g/g) close to that of glycerol fermentation in a synthetic medium. The overall 1,3-PD yield from glucose in the two stage-process with E. coli and K. pneumoniae reached 0.17 g/g.     

Process development with nitrogenase-producing Escherichia coli C-M74 (pUS1) CellS

A process for the continuous fermentation of the genetically modified, nitrogenase-producing Escherichia coli C-M74 (pUS1)-strain has been developed. This strain, which is able to fix molecular nitrogen, has the nifgenes of the bacterium Klebsiella pneumoniae. Cell growth and nitrogenase activity of the enzyme have been optimized both in batch and continuous fermentations. For the fermentations, trial runs were performed by cultivating the E. coli cells in 50-ml culture bottles. The medium composition was varied in order to provide high biomass production and nitrogenase activity. For an effective fermentation control, an on-line analysis was built up for the substrates ammonium and glucose. Other medium components such as ampicillin, citric acid, acetic acid, nitrogenase activity, and protein were measured by using different off-line methods. Modern optical methods like in-line microfluorometry for monitoring the culture fluorescence and laser flow cytometry for the estimation of DNA and protein content were also employed. Plasmid stability was also determined.     

大肠杆菌突变株QQS101发酵生产琥珀酸初步研究

琥珀酸是一种具有重要应用价值的生物基平台化合物。对大肠杆菌focA-pflB ldhA突变株QQS101在严格厌氧条件下生长和葡萄糖代谢能力进行了考察,比较分析了葡萄糖与大肠杆菌混合酸发酵产物的单位碳的还原程度,认为非严格厌氧条件有利于QQS101发酵葡萄糖积累琥珀酸,进一步对有氧生长碳源进行了对比试验的结果表明,以木糖支持有氧生长,QQS101摇瓶发酵39 h消耗葡萄糖37.6 g/L,琥珀酸的产量达到31.01 g/L,摩尔产率为1.258 mol Succinate/mol Glucose。发酵过程中,丙氨酸的添加能够提高琥珀酸的摩尔产率。     

Kinetics of inclusion body production in batch and high cell density fed-batch culture of Escherichia coli expressing ovine growth hormone.

A process for maximizing the volumetric productivity of recombinant ovine growth hormone (r-oGH) expressed in Escherichia coli during high cell density fermentation process has been devised. Kinetics of r-oGH expression as inclusion bodies and its effect on specific growth rates of E. coli cells were monitored during batch fermentation process. It was observed that during r-oGH expression in E. coli, the specific growth rate of the culture became an intrinsic property of the cells which reduced in a programmed manner upon induction. Nutrient feeding during protein expression phase of the fed-batch process was designed according to the reduction in specific growth rate of the culture. By feeding yeast extract along with glucose during fed-batch operation, high cell growth with very little accumulation of acetic acid was observed. Use of yeast extract helped in maintaining high specific cellular protein yield which resulted in high volumetric productivity of r-oGH. In 16 h of fed-batch fermentation, 3.2 g l-1 of r-oGH were produced at a cell OD of 124. This is the highest concentration of r-oGH reported to date using E. coli expression system. The volumetric productivity of r-oGH was 0.2 g l-1 h-1, which is also the highest value reported for any therapeutic protein using IPTG inducible expression system in a single stage fed-batch process.     

Change in proteomic profiles of genetically modified 1,3-propanediol-producing recombinant E. coli

The recombinant E. coli Delta6 mutant (galR, glpK, gldA, IdhA, lacI, tpiA) was used to produce 1,3-propanediol (PD) from glucose. The 1,3-PD production increased with feedback control of the glucose concentration using fed-batch fermentation. The maximum 1,3-PD concentration produced was 43 g/l after 60 h of fermentation. Glycerol production was minimized when controlling the glucose concentration at less than 1 g/l. The expression levels of seven enzymes related to the 1,3-PD production metabolism were compared during the cell growth phase and 1,3-PD production phase, and their expression levels all increased during 1,3-PD production, with the exception of alcohol dehydrogenase.     

The application of a novel heat flux calorimeter for studying growth of Escherichia coli W in aerobic batch culture

The modification and principle of a novel heat flux calorimeter for the in situ, on-line measurement of the heat generated during microbial growth is described. Data concerning the physical characterization of the calorimeter as a fermentor, including stability and sensitivity of the heat signal, are presented. The calorimeter has been successfully applied to the study of the aerobic batch culture of Escherichia coli W on glucose under carbon and nitrogen limitation. A direct correlation between growth and heat evolution was obtained. Quantitative analysis of the data suggests that the new calorimetric technique could be used for monitoring growth and specific metabolic events, for convenient medium optimization, and as a basis for a novel fermentation process control system.     

An autoclavable glucose biosensor for microbial fermentation monitoring and control

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation. (c) 1995 John Wiley & Sons, Inc.     

Overproduction of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus sp. strain PCC6301 in glucose-controlled high-cell-density fermentations by Escherichia coli K-12.

A predictive and feedback glucose feed controller, previously developed for nutrient-sufficient growth of Escherichia coli to high cell densities, was used to produce large quantities of a heterologous, cyanobacterial recombinant hexadecameric (L8S8) protein, ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in E. coli. Culture and plasmid stability conditions were optimized to yield the production of approximately 1 g of soluble, active recombinant RubisCO per liter. Recombinant RubisCO also was produced in lactose-induced high-cell-density fermentation of E. coli K-12.