Phosphate limitation increases coenzyme Q10 production in industrial Rhodobacter sphaeroides HY01.

Coenzyme Q10 (CoQ10) is an important component of the respiratory chain in humans and some bacteria. As a high-value-added nutraceutical antioxidant, CoQ10 has excellent capacity to prevent cardiovascular disease. The content of CoQ10 in the industrial Rhodobacter sphaeroides HY01 is hundreds of folds higher than normal physiological levels. In this study, we found that overexpression or optimization of the synthetic pathway failed CoQ10 overproduction in the HY01 strain. Moreover, under phosphate- limited conditions (decreased phosphate or in the absence of inorganic phosphate addition), CoQ10 production increased significantly by 12% to220 mg/L, biomass decreased by 12%, and the CoQ10 productivity of unit cells increased by 27%. In subsequent fed-batch fermentation, CoQ10 production reached 272 mg/L in the shake-flask fermentation and 1.95 g/L in a 100-L bioreactor under phosphate limitation. Furthermore, to understand the mechanism associated with CoQ10 overproduction under phosphate- limited conditions, the comparatve transcriptome analysis was performed. These results indicated that phosphate limitation combined with glucose fed-batch fermentation represented an effective strategy for CoQ10 production in the HY01. Phosphate limitation induced a pleiotropic effect on cell metabolism, and that improved CoQ10 biosynthesis efficiency was possibly related to the disturbance of energy metabolism and redox potential.

Identification of novel knockout and up-regulated targets for improving isoprenoid production in E. coli.

Discovery of novel potential genetic targets to increase the supply of isoprenoid precursors, isopentyl/dimethylallyl diphosphate, is of importance for microbial production of isoprenoids. Here, to improve isoprenoid precursor supply, a flux distribution comparison analysis, based on the genome-scale model, was utilized to simultaneously predict the knockout, down- and up-regulated targets in Escherichia coli. 51 targets were in silico discovered. All knockout and up-regulated targets were experimentally tested to enhance lycopene production. Five knockout targets (deoB, yhfw, yahI, pta and eutD) and four up-regulated targets (ompN, ompE, ndk and cmk) led to 10-45% increases of lycopene yield, respectively, which had not been uncovered in previous studies. When engineering of the five most significant targets gdhA, eutD, tpiA, ompE and ompN, were combined the lycopene titer improved by 174% in shake-flask and 81% in bioreactor fermentations with a maximum yield of 454 mg l(-1).

Optimization of polyhydroxyalkanoates fermentations with on-line capacitance measurement.

The aim of this work was to provide an effective methodology for optimization of the polyhydroxyalkanoates (PHAs) fermentation with Ralstonia eutropha by the on-line capacitance measurement. The present study found the capacitance values could reflect variations of microbial morphology and viability. Furthermore, oxygen uptake rate, specific oxygen uptake rate and specific growth rate were measured in real-time and compared with the capacitance value. In addition, a fed-batch control strategy based on the on-line capacitance measurement was proposed to improve the PHAs production by 22%.

Systems perspectives on erythromycin biosynthesis by comparative genomic and transcriptomic analyses of S. erythraea E3 and NRRL23338 strains.

BACKGROUND: S. erythraea is a Gram-positive filamentous bacterium used for the industrial-scale production of erythromycin A which is of high clinical importance. In this work, we sequenced the whole genome of a high-producing strain (E3) obtained by random mutagenesis and screening from the wild-type strain NRRL23338, and examined time-series expression profiles of both E3 and NRRL23338. Based on the genomic data and transcriptpmic data of these two strains, we carried out comparative analysis of high-producing strain and wild-type strain at both the genomic level and the transcriptomic level. RESULTS: We observed a large number of genetic variants including 60 insertions, 46 deletions and 584 single nucleotide variations (SNV) in E3 in comparison with NRRL23338, and the analysis of time series transcriptomic data indicated that the genes involved in erythromycin biosynthesis and feeder pathways were significantly up-regulated during the 60 hours time-course. According to our data, BldD, a previously identified ery cluster regulator, did not show any positive correlations with the expression of ery cluster, suggesting the existence of alternative regulation mechanisms of erythromycin synthesis in S. erythraea. Several potential regulators were then proposed by integration analysis of genomic and transcriptomic data. CONCLUSION: This is a demonstration of the functional comparative genomics between an industrial S. erythraea strain and the wild-type strain. These findings help to understand the global regulation mechanisms of erythromycin biosynthesis in S. erythraea, providing useful clues for genetic and metabolic engineering in the future.

Direct proteomic mapping of Streptomyces avermitilis wild and industrial strain and insights into avermectin production.

An industrial mutant of Streptomyces avermitilis produced avermectin at a high level in industrial complex culture medium. However, almost no avermectin was detected in the cultures of tryptone soya broth (TSB). Its wild-type strain could not synthesize avermectin. To elucidate the regulatory mechanism about avermectin production, proteomic analysis of S. avermitilis was carried out. Results showed that during avermectin biosynthesis, fatty acid metabolism and TCA cycle were repressed. Partial enrichment of glycolytic pathway indicated the critical role of glucose catabolism during avermectin biosynthesis. Some enriched enzymes in amino acid metabolic pathways (glnA, leuC) confirmed the leucine as the possible precursor of avermectin. Highly expressed stress or stress-related proteins indicated a global regulation mechanism at the onset of avermectin production. And highly expressed morphology control proteins revealed an association between hyphal morphology and avermectin production. Further, this study proofed strengthened capability to utilize carbon and nitrogen source in the industrial strain. Some stress or stress-related proteins (eshA, clpC, dnaK and grpE) expressed at low level in the industrial strain cultivated in non-production medium (lower than that in the wild-type strain), but these highly expressed at the onset of avermectin production. More sensible response to environmental stress may be responsible for it.

Real-time fluid dynamics investigation and physiological response for erythromycin fermentation scale-up from 50 L to 132 m3 fermenter.

The physiological response of erythromycin fermentation scale-up from 50 L to 132 m(3) scale was investigated. A relatively high oxygen uptake rate (OUR) in early phase of fermentation was beneficial for erythromycin biosynthesis. Correspondingly, the maximal consistency coefficient (K) reflecting non-Newtonian fluid characteristics in 50 L and 132 m(3) fermenter also appeared in same phase. Fluid dynamics in different scale bioreactor was further investigated by real-time computational fluid dynamics modeling. The results of simulation showed that the impeller combination in 50 L fermenter could provide more modest flow field environment compared with that in 132 m(3) fermenter. The decrease of oxygen transfer rate (OTR) in 132 m(3) fermenter was the main cause for impairing cell physiological metabolism and erythromycin biosynthesis. These results were helpful for understanding the relationship between hydrodynamic environment and physiological response of cells in bioreactor during the scale-up of fermentation process.

Incomplete protein disulphide bond conformation and decreased protein expression result from high cell growth during heterologous protein expression in Pichia pastoris.

Previous report has shown that the expression of recombinant human consensus interferon-α mutant (cIFN) in Pichia pastoris in bioreactor is limited with respect to the incorrectly folded cIFN with incomplete disulfide bond, which lead to the degradation and aggregation of cIFN. In this study, the origin of incorrectly folded cIFN is firstly studied. Fed-batch fermentation in bioreactor shows that the incorrectly folded cIFN is formed intramolecularly and secreted to the extracellular environment. Further chemostat cultures indicate that the specific growth rate is the critical factor for the production of incorrect cIFN. In addition, cell shows reduced expression level of cIFN at high specific growth rate. We also demonstrate that the incorrectly folded cIFN could form aggregates intracellularly and these aggregates are non-covalent forms. Taken together, these results suggest that the efficient heterologous expression of cIFN is limited by high cell growth that is unique from expression limitations seen for soluble proteins. A balance has to be found between the increase for high efficient expression of heterologous proteins and requirement of the high cell growth during the expression of recombinant proteins in P. pastoris.

An assessment of seed quality on erythromycin production by recombinant Saccharopolyspora erythraea strain.

An assessment of seed quality on erythromycin production by recombinant strain Saccharopolyspora erythraea ZL1004 was investigated in 15 l fermenter. Adding 10 g/l corn steep liquor and 30 g/l soybean flour in seed medium were beneficial to improve cell growth, and the maximal biomass reached 36% at 40 h. Enzyme activity in cell showed that the maximal protease and minimum amylase were appeared in this stage. Compared with the control in 50 l fermenter, the cell metabolism with inoculation of the optimized seed cultivation was obviously quicker, and physiological response such as oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) were also improved. The maximal erythromycin A production was 9160 U/ml at 215 h, which was increased by 21.63% with respect to the control. It was the first report to integrate cell growth characteristics and physiological response method to assess the seed quality for erythromycin production.

Incomplete formation of intramolecular disulfide bond triggers degradation and aggregation of human consensus interferon-alpha mutant by Pichia pastoris.

Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-alpha mutant (cIFN) were serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro conversion assay based on incubation with different concentrations of beta-mercaptoethanol. The results showed that only a wound such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers. Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which result in sharp decrease of bioactivity of cIFN in bioreactor.

Regulation of branched-chain amino acid catabolism: glucose limitation enhances the component of isovalerylspiramycin for the bitespiramycin production.

4''-O-isovalerylspiramycins are the major components of bitespiramycin complex consisting of a group of 4''-O-acylated spiramycins. The availability of isovaleryl group, usually in vivo derived from leucine, one of the branched-chain amino acids, affects the content of isovaleryispiramycin significantly. In this study, the effect of glucose on the activity of branched-chain alpha-keto acid dehydrogenase (BCKDH), which catalyzed the rate-limiting as well as the first irreversible reaction oxidative decarboxylation for branched-chain amino acids degradation, and isovaleryispiramycin biosynthesis was investigated. In the initial glucose concentration experiment, when the residual glucose concentration in the medium declined to 2-4 g/L, the BCKDH activity rose rapidly, and glucose deprivation and the summit of BCKDH activity appeared nearly at the same time. After a delay of about 6 h, the maximal isovalerylspiramycin content was observed. However, the shortage of glucose at the later production phase resulted in the marked decrease in BCKDH activity and isovaleryispiramycin content. In the fermentation in a 50 L fermentor, glucose feeding at the late production phase helped to maintain the residual glucose concentration between 0 and 1 g/L, leading to the high level of BCKDH activity and thus isovalerylspiramycin content. These suggested that glucose concentration could be used as a key parameter to regulate BCKDH activity and isovaleryispiramycin biosynthesis in the bitespiramycin production.

Improved vitamin B(12) production by step-wise reduction of oxygen uptake rate under dissolved oxygen limiting level during fermentation process.

Effects of different oxygen transfer rates (OTR) on the cell growth and vitamin B(12) biosynthesis of Pseudomonas denitrificans were first investigated under dissolved oxygen limiting conditions. The results demonstrated that high OTR accelerated cell growth and initial vitamin B(12) biosynthesis rate, while lower OTR was critical for higher productivity in the late fermentation process. The oxygen uptake rates (OUR) corresponded well with OTR. Based on the metabolic intermediate analysis, a step-wise OUR control strategy was proposed. The strategy was successfully implemented in scale-up to an industrial fermenter (120,000 l). A stable maximum vitamin B(12) production of 208 + or - 2.5 mg/l was achieved, which was increased by 17.3% compared with the control. Furthermore, the glucose consumption coefficient to vitamin B(12) was 34.4% lower than that of the control. An efficient and economical fermentation process based on OUR criterion was established for industrial vitamin B(12) fermentation by P. denitrificans.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinantspiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l-1 Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3 percent higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5 percent. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85 percent and 72.1 percent of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41 percent. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9 percent while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l-1 leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l-1 leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1 percent to 46.9 percent.

Time-resolved transcriptome analysis of Bacillus subtilis responding to valine, glutamate, and glutamine.

Microorganisms can restructure their transcriptional output to adapt to environmental conditions by sensing endogenous metabolite pools. In this paper, an Agilent customized microarray representing 4,106 genes was used to study temporal transcript profiles of Bacillus subtilis in response to valine, glutamate and glutamine pulses over 24 h. A total of 673, 835, and 1135 amino-acid-regulated genes were identified having significantly changed expression at one or more time points in response to valine, glutamate, and glutamine, respectively, including genes involved in cell wall, cellular import, metabolism of amino-acids and nucleotides, transcriptional regulation, flagellar motility, chemotaxis, phage proteins, sporulation, and many genes of unknown function. Different amino acid treatments were compared in terms of both the global temporal profiles and the 5-minute quick regulations, and between-experiment differential genes were identified. The highlighted genes were analyzed based on diverse sources of gene functions using a variety of computational tools, including T-profiler analysis, and hierarchical clustering. The results revealed the common and distinct modes of action of these three amino acids, and should help to elucidate the specific signaling mechanism of each amino acid as an effector.

Production and distribution of beta-glucosidase in a mutant strain Trichoderma viride T 100-14.

The characterization of beta-glucosidase's production and distribution in a mutant strain Trichoderma viride T 100-14 at extracellular and intracellular levels were studied in this paper. Three experiment groups were done automatically with pH controlled at 4.8 during fermentation process, with 1mg/ml 2-deoxy-d-glucose addition or without pH control and 2-deoxy-d-glucose addition (control). Activity assay and electron microscopic immunogold labeling experiments were performed at different culture periods (24, 48, 72, 96 and 120 hours). Under constant pH 4.8, high density of immunogold labeling particles, highest intracellular enzyme activity, total enzyme activity and specific activity were observed at 24 hours of fermentation. After 72 hours, the extracellular and total activities fluctuated little and the maximal activity in extracellular fraction was 2.7 times higher than control. By contrast, with 2-deoxy-d-glucose addition, the secreted and total beta-glucosidase activities achieved their maximum at 96 hours of fermentation, and the maximal secreted activity increased 2.05-fold than the control. Additionally, the secretion ratio (maximal secreted beta-glucosidase activity/maximal total activity) with pH control or 2-deoxy-d-glucose addition was elevated profoundly near to a level as the cellulase in fungi.

Enhancement of erythromycin A production with feeding available nitrogen sources in erythromycin biosynthesis phase.

Effects of feeding different available nitrogen sources from 80 h in erythromycin biosynthesis phase on the erythromycin A (Er-A) production were investigated in 50 l fermenter. Feeding corn steep liquor and yeast extract, the Er-A production was enhanced, while the biotransformation from erythromycin C (Er-C) to Er-A had no increase. When ammonium sulphate was fed at high feeding rate, the maximal Er-A production and ratio of Er-A to Er-C were 7953 U/ml and 98.18:1 at 184 h, respectively, which were higher than that of the control (6742 U/ml and 5.47:1). The feeding ammonium sulphate process was successfully scaled up from 50 l to 25 m(3) fermenter. The maximal Er-A production reached 7938 U/ml at 203 h, which was enhanced by 22.1% compared with the control (6501 U/ml at 192 h). The ratio of Er-A to Er-C was 24.05:1, which was higher than that of the control (4.77:1).

Efficient extraction of intracellular reduced glutathione from fermentation broth of Saccharomyces cerevisiae by ethanol.

Reduced glutathione (GSH) from fermentation broth of Saccharomyces cerevisiae was extracted with ethanol without disruption of the cells. The effects of ethanol concentration, extraction temperature and extraction time were assessed by using 2(3) full factorial designs (FFD). Preliminary studies showed that ethanol concentration had the most influence on GSH yield by ethanol extraction, based on the first order regression coefficients derived using MINITAB software, and an optimal ethanol concentration (25%, v/v) was obtained. However, compared to the conventional extraction technique (hot water extraction), there was no significant advantage in yield of GSH from yeast cells using ethanol extraction under these optimized conditions. But ethanol extraction has several advantages, such as lower energy consumption and lower protein concentration of extraction broth, which may reduce the complexity and cost of the purification process. Hence, ethanol extraction which does not disrupt yeast cells could be an inexpensive, simple and efficient alternative to conventional extraction techniques in the GSH industry.

Optimization of cellulase mixture for efficient hydrolysis of steam-exploded corn stover by statistically designed experiments.

To improve the enzymatic hydrolytic efficiency and reduce production cost, a statistically designed experimental approach was used to optimize the composition of cellulase mixture so as to maximize the amount of glucose produced from steam-exploded corn stover (SECS). Using seven purified enzymes (cellobiohydrolases, Cel7A, Cel6A, Cel6B; endoglucanases, Cel7B, Cel12A, Cel61A; and beta-glucosidase) from Trichoderma viride T 100-14 mutant strain, a multi-enzyme mixture was constituted after screening and optimization. The final optimal composition (mol%) of the multi-enzyme mixture was Cel7A (19.8%), Cel6A (37.5%), Cel6B (4.7%), Cel7B (17.7%), Cel12A (15.2%), Cel61A (2.3%) and beta-glucosidase (2.8%). The subsequent verification experiments followed by glucose assay together with scanning electron microscopy (SEM) observation confirmed the validity of the models. The multi-enzyme mixture displayed a high performance in converting the cellulosic substrate (SECS). The amount of glucose produced (15.5mg/ml) was 2.1 times as that of the crude cellulase preparation. The results indicated that the optimized cellulase mixture is an available and efficient paradigm for the hydrolysis of lignocellulosic substrate. The enhanced cellulolytic activity displayed by the constructed cellulase mixture could be used as an effective tool for producing bioethanol efficiently from cellulose.

Leucine improves the component of isovalerylspiramycins for the production of bitespiramycin.

Bitespiramycin, a group of 4''-O-acylated spiramycins with 4''-O-isovalerylspiramycins as the major components, is produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4''-O-acyltransferase gene from a carbomycin-producing strain S. mycarofaciens 1748. The effects of leucine feeding on the bitespiramycin fermentation, especially the synthesis of isovalerylspiramycin components, were investigated. The experiment was initially performed in flask culture under the condition of feeding 15.4 mmol/l of leucine at 72 h fermentation, and the culture without leucine feeding was used as control. When 15.4 mmol/l leucine was fed at 72 h, 51.3 +/- 0.33% total isovalerylspiramycins was recorded compared to 40.9 +/- 0.26% under the control condition after 96 h of fermentation. The improvement of total isovalerylspiramycin content was further achieved in 15 l fermentation when 15.4 mmol/l of leucine was supplemented from 65 to 72 h. These results indicated that isovaleryl group derived from leucine catabolism could act as the precursor of the 4'' side chain of bitespiramycin, which profoundly enhanced the synthesis of isovalerylspiramycins in the bitespiramycin complex.

Oxygen uptake rate optimization with nitrogen regulation for erythromycin production and scale-up from 50 L to 372 m3 scale.

Effects of different nitrogen sources on the erythromycin production were investigated in 50 l fermenter with multi-parameter monitoring system firstly. With the increase of soybean flour concentration from 27 g/l to 37 g/l to the culture medium, the erythromycin production had no obvious increase. Whereas adding corn steep liquor 15 g/l in the medium was beneficial for the production of erythromycin, the maximum erythromycin production was 22.2% higher than that of the control. It was found that corn steep liquor can regulate and enhance the oxygen uptake rate (OUR) which characterizes the activity of the microbial metabolism by inter-scale observation and data association. Both Intracellular and extracellular organic acids of central metabolism were analyzed, and it was found that the whole levels of lactic acid, pyruvic acid, citric acid, and propionic acid were higher than those of control before 64th h. The consumption amount of amino acids, which could be transformed into the precursors for erythromycin synthesis (i.e. threonine, serine, alanine, glycine and phenylalanine), were elevated compared with the control in erythromycin biosynthesis phase. The results indicated that corn steep liquor can regulate OUR to certain level in the early phase of fermentation, and enhance the metabolic flux of erythromycin biosynthesis. Erythromycin production was successfully scaled up from a laboratory scale (50 l fermenter) to an industrial scale (132 m(3) and 372 m(3)) using OUR as the scale-up parameter. Erythromycin production on industrial scale was similar to that at laboratory scale.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins.

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l(-1) Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3% higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5%. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85% and 72.1% of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41%. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9% while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l(-1) leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l(-1) leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1% to 46.9%.