碳源对重组大肠杆菌发酵生产GLP-1融合蛋白的影响

以一株表达人胰高血糖素样肽-1融合蛋白的重组大肠杆菌为研究对象,首先通过摇瓶实验对碳源种类进行了初步选择,发现葡萄糖和甘油对菌体生长以及GLP-1融合蛋白表达较为适宜。进一步在5 L反应器上对初始葡萄糖及甘油浓度进行了考察,发现高浓度碳源有利于菌体生长却抑制GLP-1融合蛋白表达,但能提高GLP-1融合蛋白的体积得率。在0.25%初始葡萄糖或甘油存在的条件下,在培养过程中流加葡萄糖或甘油维持其在发酵液中的浓度,比较了两者对菌体生长以及GLP-1融合蛋白表达的影响,结果发现,以甘油为碳源时,菌体生长以及GLP-1融合蛋白的表达量均高于以葡萄糖为碳源的结果,最终发酵液的菌浓(OD_(600))可达到25.4,较葡萄糖为碳源时19.1提高了33.0%,GLP-1融合蛋白表达水平和体积得率分别可达到22.4%和1.051 g/L,较葡萄糖为碳源的15.8%和0.504 g/L分别提高41.8%和108.5%。该结果对GLP-1融合蛋白表达菌株发酵条件的进一步优化提供了依据。     

Strategy for the Improvement of Prodigiosin Production by a Serratia marcescens Mutant through Fed-Batch Fermentation

A Serratia marcescens mutant for prodigiosin production was obtained by u.v. mutation with rational screening methods and a two-step feeding strategy was used to increase its productivity. In flasks, the mutant strain B6 gave a 2.8-fold higher prodigiosin production than that of the parent strain with glycerol as a carbon source. In a 5-l bioreactor, with a two-step feeding strategy in which glucose was selected as the initial carbon source in the fermentation media and glycerol was fed as a ‘prodigiosin inducer’, it gave a 7.8 times higher prodigiosin production (583 mg/l) than the parent stain with the original cultivation mode.     

甘油影响红曲菌产色素的碳源选择性探究

红曲菌(Monascus spp.)是我国重要的药食同源微生物,红曲色素(Monascus pigments,MPs)是其主要次级代谢产物之一。有研究表明,甘油可促进红曲菌产MPs,但作用机制不明。以丛毛红曲菌(Monascus pilosus)MS-1为实验菌株,考察甘油与葡萄糖或蔗糖复合对红曲菌产MPs的影响。在不含碳源的合成培养基中,将甘油与葡萄糖或蔗糖复合,采用分光光度法和高效液相色谱法等分析MPs的产量和组分、生物量及发酵液pH。当甘油与葡萄糖复合,添加甘油后发酵液pH、生物量无显著变化(P0.05),总色价显著降低(P0.05)。当2 g/L或40 g/L甘油与蔗糖复合,发酵液pH显著降低而生物量及总色价显著增加(P0.05)。当40 g/L甘油与蔗糖复合时,总色价是仅以蔗糖为碳源时的16.5倍,且MPs同系物数量明显增多(P0.05)。在合成培养基条件下,甘油促进红曲菌产MPs具有碳源种类的选择性。该结果可为研究甘油影响红曲菌产MPs的作用机制提供参考,为甘油用于MPs生产提供依据。     

Heterotrophic growth and lipid production of Chlorella protothecoides on glycerol

During the production of biodiesel, a significant amount of glycerol is generated which currently has little commercial value. A study on the growth and lipid production of Chlorella protothecoides using glycerol as the carbon source was performed to demonstrate the utility of recycling crude glycerol created during biodiesel production. Glycerol was examined as both the sole carbon source and in combination with glucose. Algae cultures grown on only glycerol in shake flasks showed a specific growth rate and final lipid yield of 0.1/h and 0.31 g lipid/g substrate, respectively. The values were similar to those observed on pure glucose, 0.096/h and 0.24 g lipid/g substrate. When the media contained a mixture of glycerol and glucose, simultaneous uptake of the two substrates was observed. Due to the difference in rates of lipid storage, lipid production was 0.077 g lipid/(l h) during growth on glycerol, while growth on glucose had a productivity of 0.096 g lipid/(l h). During growth on the 9:1 mixture of both glucose and glycerol, lipid productivity was 0.098 g lipid/(l h). In order to simulate the use of waste glycerol from biodiesel production the experiments were repeated and similar growth rates, yields, and lipid productivities were achieved. Therefore, we have demonstrated the promise for simultaneous high growth rates and lipid yields of C. protothecoides heterotrophically grown on mixtures of glycerol.     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Enhanced cellulase production by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> by cultivation on glycerol followed by induction on cellulosic substrates

The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.     

Synergetic utilization of glucose and glycerol for efficient myo-inositol biosynthesis

myo-Inositol (MI) as a dietary supplement can provide various health benefits. One major challenge to its efficient biosynthesis is to achieve proper distribution of carbon flux between growth and production. Herein, this challenge was overcome by synergetic utilization of glucose and glycerol. Specifically, glycerol was catabolized to support cell growth while glucose was conserved as the building block for MI production. Growth and production were coupled via the phosphotransferase system, and both modules were optimized to achieve efficient production. First, the optimal enzyme combination was established for the production module. It was observed that enhancing the production module resulted in both increased MI production and better cell growth. In addition, glucose was shown to inhibit glycerol utilization via carbon catabolite repression and the inhibition was released by over-expressing glycerol kinase. Furthermore, the inducible promoter was replaced by strong constitutive promoters to avoid inducer use. With these efforts, the final strain produced MI with both high titer and yield. In fed-batch cultivation, 76 g/L of MI was produced, showing scale-up potential. This study provides a promising strategy to achieve rational distribution of carbon flux.     

以葡萄糖为生长相基质的重组毕赤酵母表达植酸酶发酵

甲醇营养型毕赤酵母表达外源蛋白是在醇氧化酶（alcohol oxidase,AOX）启动子（PAOXI）严格调控下进行的,然而这种启动子在转录水平受到葡萄糖的阻遏。本文研究了毕赤酵母在葡萄糖替代甘油为生长相碳源时表达重组植酸酶蛋白的发酵特征。结果表明：初始葡萄糖浓度为20dL的细胞得率高,为0．39g[DCW]／g。通过基于实时参数（溶氧和呼吸商）调控的葡萄糖补料策略,生长相40h后细胞密度达到100g[DCW]／L,甲醇诱导100h后植酸酶产量达到2200FTUphytase／mL,甲醇得率系数为0．25FTU phytase／gmethnol。因此,在毕赤酵母高表达重组蛋白培养中葡萄糖能够用作生长相基质,并能实现重组蛋白的高效表达。     

Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Direct utilization of palm oil for the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids was demonstrated using Pseudomonas aeruginosa IFO3924. By secreted lipase, palm oil was hydrolyzed into glycerol and fatty acids. Fatty acids became favorable carbon sources for cell growth and PHA production via β-oxidation and glycerol for rhamnolipid production via de novo fatty acid synthesis. Both PHA and rhamnolipid syntheses started after the nitrogen source was exhausted and cell growth ceased. PHA synthesis continued until all fatty acids were exhausted, and at that time, PHA content in the cells reached a maximum, but stopped despite the remaining glycerol (<2g/l). In contrast, rhamnolipid synthesis continued until glycerol was exhausted.     

Influence of carbon source on α-amylase production by Aspergillus oryzae

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.     

Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, from Candida antarctica

Candida antarctica (sp. SY16) required avegetable oil as the carbon source to produce a biosurfactant, mannosylerythritol lipid (MEL-SY16). Biosurfactant production was 31 g l^–1 after 7 days in a batch culture and was not growth associated. In a two-stage culture, glycerol and oleic acid were used as an initial and a feeding carbon source, respectively, and 41 g l^–1 biosurfactant was produced after 8 days.     

Optimization of culture on the overproduction of TRAIL in high-cell-density culture by recombinant Escherichia coli

Different nutrient-feeding cultures were carried out in producing recombinant protein of truncated tumor necrosis factor related apoptosis-inducing ligand (TRAIL) (114–281 amino acids of TRAIL) in Escherichia coli strain C600/pBV-TRAIL. The effects of preinduction specific growth rate, postinduction carbon source (glucose and glycerol), and feeding strategies were investigated. The higher preinduction specific growth rate (μ=0.22 h⁻¹) contributed to the increase in the TRAIL production, at which TRAIL was accumulated in bacterial cells as 7.2% of total cellular protein, corresponding to 1.99 g l⁻¹ in contrast with 5.1% (1.29 g l⁻¹) at preinduction specific growth rate (μ=0.1 h⁻¹) during high-cell-density culture. Glycerol was superior to glucose as the postinduction carbon source for TRAIL production. Under similar culture conditions, the final concentration of TRAIL was produced 1.59-fold more when glycerol was used as postinduction carbon source than when glucose was used. At the same time, the results showed that it is efficient to adopt the pH-stat feeding strategy at postinduction for the overproduction of TRAIL. The TRAIL production was increased up to 4.51 g l⁻¹, approximately 16.1% of total cellular protein. The mechanisms behind the preinduction specific growth rate effect on the expression level may be ascribed to the leakage secretion of acetate.     

Anaerobic glycerol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains under hyperosmotic stress

Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80–90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

Comparison of citric acid production from glycerol and glucose by different strains of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

A wild type strain A-101 of Y. lipolytica and its three acetate-negative mutants (Wratislavia 1.31, Wratislavia AWG7, and Wratislavia K1) were compared for the production of citric acid from glucose and glycerol (pure and crude) in batch cultures. The substrates were used either as single carbon sources or as mixtures of glucose and pure or crude glycerol. The kinetic parameters, i.e., the volumetric citric acid production rate and yield obtained in the study show that the Wratislavia 1.31 and Wratislavia AWG7 strains produced the highest amount of citric acid from glycerol, with a yield from 0.40 to 0.53 g g⁻¹. This substrate was found to be a better carbon source for the biosynthesis of citric acid than glucose. The results obtained with the same strains have shown low content of isocitric acid and polyols, such as erythritol and mannitol. Y. lipolytica A-101 strain produced the highest amount of isocitric acid, from 13.8 to 21% isocitric acid in the sum of citric acids. However, the highest concentrations of erythritol were found in cultures with Y. lipolytica Wratislavia K1, from 18.1 to 30 g l⁻¹, for glucose and pure glycerol, respectively.     

The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In previous studies, we showed that cofactor manipulations can potentially be used as a tool in metabolic engineering. In this study, sugars similar to glucose, that can feed into glycolysis and pyruvate production, but with different oxidation states, were used as substrates. This provided a simple way of testing the effect of manipulating the NADH/NAD+ ratio or the availability of NADH on the metabolic patterns of Escherichia coli under anaerobic conditions and on the production of 1,2-propanediol (1,2-PD), which requires NADH for its synthesis. Production of 1,2-PD was achieved by overexpressing the two enzymes methylglyoxal synthase from Clostridium acetobutylicum and glycerol dehydrogenase from E. coli. In addition, the effect of eliminating a pathway competing for NADH by using a ldh ^– strain (without lactate dehydrogenase activity) on the production of 1,2-PD was investigated. The oxidation state of the carbon source significantly affected the yield of metabolites, such as ethanol, acetate and lactate. However, feeding a more reduced carbon source did not increase the yield of 1,2-PD. The production of 1,2-PD with glucose as the carbon source was improved by the incorporation of a ldh ^– mutation. The results of these experiments indicate that our current 1,2-PD production system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal. Electronic Publication     

Improving intracellular production of recombinant protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using an optimized preinduction glycerol-feeding scheme

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l⁻¹, short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l⁻¹. Thus, a novel strategy of induction at a cell density of 320 g l⁻¹ was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h⁻¹. The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g⁻¹ h⁻¹ from 0.043 mg g⁻¹ h⁻¹. Consequently, both high cell density (428 g l⁻¹) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h⁻¹ to the cell density of 320 g l⁻¹ and inducing the expression by mixed feeding.     

Modification and Evolution of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> for Enhanced Growth and Biotransformation Capabilities at Low Glucose Concentration

Gluconobacter oxydans is widely used in several biotechnological applications, where sorbitol or mannitol is commonly used as carbon source at high concentration. In this study, a membrane-bound glucose dehydrogenase-deficient strain (GDHK) was constructed to eliminate growth problems on glucose caused by direct oxidation of glucose in the medium. To achieve improved growth properties for the GDHK strain on glucose, a laboratory adaptive evolution experiment was performed with glucose as the sole carbon source. Results indicated evident, albeit modest, improvements in cell growth after a 50-day (about 430 generations) experimental evolution on glucose. The maximum specific growth rate and biomass yield of the resulting GDHE50 strain were increased around 1.35- to 1.4-fold compared with those of the GDHK strain. Meanwhile, two types of biotransformation reactions using resting cells of G. oxydans were investigated. Significant elevations in biotransformation performance of the GHDE50 strain were observed in comparison with that of the wild-type strain. In addition, resting cells of the GDHE50 strain grown on a relatively low concentration of glucose (10 g/l) could catalyze the biotransformation of glycerol to dihydroxyacetone and ethylene glycol to glycolic acid as efficient as the wild-type G. oxydans cultured on higher concentration of sorbitol or other carbon sources. These results suggest very favorable prospects of using glucose to lower production cost in many important industrial biocatalysis and biotransformation processes.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.