Haifeng Hang

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.

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).

Transcriptional investigation of the effect of mixed feeding to identify the main cellular stresses on recombinant Pichia pastoris

Heterologous protein expression using Pichia pastoris causes metabolic stress on the physiology of host cells, which may compromise the yields of secreted foreign proteins. Thus, understanding these metabolic stresses during secretory expression allows us to circumvent these undesirable effects. We investigated the effect of co-feeding two alternative carbon resources, sorbitol and yeast extract (YE), on the physiology of A3, a P. pastoris strain carrying 18 copies of the porcine insulin precursor (PIP) gene. Comparative transcriptional analysis was performed on 13 selected genes involved in important cellular processes. Results showed that co-feeding of either sorbitol or YE along with methanol improved the performance of A3. The co-feeding of YE enhanced the specific growth rate of A3 and the specific PIP productivity. However, the oxidative stress in the yeast cells increased. The co-feeding of methanol and sorbitol increased the specific growth rate of A3 but did not affect the specific PIP productivity. The transcriptional results suggested that sorbitol may have repressed the expression of foreign proteins. These observations will not only guide the application of mixed feeding strategies but also give a deeper understanding of the metabolic burden in the secretory expression of foreign proteins.

Biochemical parameters of Saccharopolyspora Erythraea during feeding ammonium sulphate in erythromycin biosynthesis phase

The physiology of feeding ammonium sulphate in erythromycin biosynthesis phase of Saccharopolyspora erythraea on the regulation of erythromycin A (Er-A) biosynthesis was investigated in 50 L fermenter. At an optimal feeding ammonium sulphate rate of 0.03 g/L per h, the maximal Er-A production was 8281 U/mL at 174 h of growth, which was increased by 26.3% in comparison with the control (6557 U/mL at 173 h). Changes in cell metabolic response of actinomycete were observed, i.e. there was a drastic increase in the level of carbon dioxide evolution rate and oxygen consumption. Assays of the key enzyme activities and organic acids of S. erythraea and amino acids in culture broth revealed that cell metabolism was enhanced by ammonium assimilation, which might depend on the glutamate transamination pathway. The enhancement of cell metabolism induced an increase of the pool of TCA cycle and the metabolic flux of erythromycin biosynthesis. In general, ammonium assimilation in the erythromycin biosynthesis phase of S. erythraea exerted a significant impact on the carbon metabolism and formation of precursors of the process for dramatic regulation of secondary metabolites biosynthesis.

Kinetic analysis of curdlan production by Alcaligenes faecalis with maltose, sucrose, glucose and fructose as carbon sources

Curdlan has wide-ranging benefits in food and pharmaceutical industries for its unique rheological and thermal gelling properties. To analyze the cell growth and curdlan biosynthesis kinetics of Alcaligenes faecalis, the kinetic properties of the curdlan fermentation under different carbon sources conditions (maltose, sucrose, glucose and fructose) were investigated using Logistic and Luedeking-Piret equations. The results demonstrated that curdlan fermentation is partial growth-associated process. With maltose as the sole carbon source, the highest curdlan production (Pm = 39.3 g/L), the maximum specific growth rate (μm = 0.44/h) and the growth-associated rate constant (α = 2.05 g curdlan/g cell) were achieved. In contrast, the fructose was the less desired carbon source in both the cell growth and curdlan production. Further, the results demonstrated that slow-releasing glucose from maltose boosted cell growth and curdlan production.

Sustainable biosynthesis of curdlan from orange waste by using Alcaligenes faecalis: A systematically modeled approach

This study presents an engineered approach for sustainable biosynthesis of curdlan by Alcaligenes faecalis using orange peels. To confirm the substrate suitability a four step study was organized. Firstly, drying of substrate was carried within temperature range of 60-120 °C, along with the application of moisture diffusion control model. Secondly, fermentation medium was obtained via saccharification and detoxification, releasing highest sugar at 72.34 g/L with phenolics removal of 95-98%. Thirdly, curdlan fermentation was conducted in detoxified orange peel hydrolysate followed by optimization of batch culture fermentation via kinetic modeling using Logistic and Luedeking-Piret equations. In 5 L bioreactor, highest specific growth rate (μm = 0.233/h), highest curdlan production (Pm = 23.24 g/L) and growth associated rate constant (α = 3.403) were achieved. Moreover, the total sugar consumption and conversion rates were 83.27% and 53.20%. Lastly, characterization techniques such as FTIR, NMR, XRD, TGA, HPGPC and EDS were applied to biosynthesized curdlan for qualitative validation.

Kinetic analysis of sodium gluconate production by Aspergillus niger with different inlet oxygen concentrations

To further understand fermentation kinetics of sodium gluconate (SG) production by Aspergillus niger with different inlet oxygen concentrations, logistic model for cell growth and two-step models for SG production and glucose consumption were established. The results demonstrated that the maximum specific growth rate (µm) presented exponential relationship with inlet oxygen concentration and the maximum biomass (Xm) exhibited linear increase. In terms of SG production, two-step model with Luedeking–Piret equation during growth phase and oxygen-dependent equation during stationary phase could well fit the experimental data. Notably, high inlet oxygen concentration exponentially improved SG yield (YP/S), whereas biomass yield to glucose (YX/S) and cell maintenance coefficient (m) were almost independent on inlet oxygen concentration, indicating that high oxygen supply enhancing SG synthesis not only functioning as a substrate directly, but also regulating glucose metabolism towards SG formation. Finally, the applicability and predictability of the proposed models were further validated by additional experiments.

Mixomics analysis of Bacillus subtilis: effect of oxygen availability on riboflavin production

BackgroundRiboflavin, an intermediate of primary metabolism, is one kind of important food additive with high economic value. The microbial cell factory Bacillus subtilis has already been proven to possess significant importance for the food industry and have become one of the most widely used riboflavin-producing strains. In the practical fermentation processes, a sharp decrease in riboflavin production is encountered along with a decrease in the dissolved oxygen (DO) tension. Influence of this oxygen availability on riboflavin biosynthesis through carbon central metabolic pathways in B. subtilis is unknown so far. Therefore the unveiled effective metabolic pathways were still an unaccomplished task till present research work.ResultsIn this paper, the microscopic regulation mechanisms of B. subtilis grown under different dissolved oxygen tensions were studied by integrating 13C metabolic flux analysis, metabolomics and transcriptomics. It was revealed that the glucose metabolic flux through pentose phosphate (PP) pathway was lower as being confirmed by smaller pool sizes of metabolites in PP pathway and lower expression amount of ykgB at transcriptional level. The latter encodes 6-phosphogluconolactonase (6-PGL) under low DO tension. In response to low DO tension in broth, the glucose metabolic flux through Embden–Meyerhof–Parnas (EMP) pathway was higher and the gene, alsS, encoding for acetolactate synthase was significantly activated that may result due to lower ATP concentration and higher NADH/NAD+ ratio. Moreover, ResE, a membrane-anchored protein that is capable of oxygen regulated phosphorylase activity, and ResD, a regulatory protein that can be phosphorylated and dephosphorylated by ResE, were considered as DO tension sensor and transcriptional regulator respectively.ConclusionsThis study shows that integration of transcriptomics, 13C metabolic flux analysis and metabolomics analysis provides a comprehensive understanding of biosynthesized riboflavin’s regulatory mechanisms in B. subtilis grown under different dissolved oxygen tension conditions. The two-component system, ResD–ResE, was considered as the signal receiver of DO tension and gene regulator that led to differences between biomass and riboflavin production after triggering the shifts in gene expression, metabolic flux distributions and metabolite pool sizes.

Human Papillomavirus 16 L1 Protein Expression and Self-Assembly in Recombinant Pichia pastoris

Human papillomavirus virus-like particles (HPV VLPs) have been shown to be effective in preventing cervical cancer when used as a prophylactic vaccine. However, little attention has been paid to the process of L1 expression and self-assembly of VLPs in vivo. In this study, the methanol concentration is optimized during fermentation to optimize L1 expression and self-assembly into VLPs. HPV 16 L1 was expressed in Pichia pastoris GS115, and successfully self-assembled into HPV 16 VLPs. The results demonstrate that there is a strong correlation between methanol concentration and the expression of HPV L1 as well as VLP formation. During the methanol induction phase, low concentrations of residual methanol (0.1-0.3%) promoted the expression of free L1 and increased the total content of L1, whereas a higher residual methanol concentration (0.32-0.6%) was conducive for the self-assembly of LI into VLPs. To conclude, if the residual methanol concentration is too low or too high, the levels of L1 can easily decrease during the fermentation process.