A simple unstructured model-based control for efficient expression of recombinant porcine insulin precursor by Pichia pastoris

Siliang Zhang,Hai-feng Hang,Wen Chen,Mei-jin Guo,Ju Chu,Ying-ping Zhuang 한국화학공학회 2008 Korean Journal of Chemical Engineering Vol.25 No.5

Based on the fact that Pichia cell growth follows a Monod equation under the condition of methanol concentration limitation, a kinetics model of recombinant methylotrophic yeast Pichia pastoris expressing porcine insulin precursor (PIP) was developed in the quasi-steady state in the induction phase. The model revealed that the relationship between specific growth rate (μ) and substrate methanol concentration was in accord with the Monod equation. The fermentation kinetic parameters maximum specific growth rate (μmax), saturation constant (Ks) and maintenance coefficient (M) were estimated to be 0.101 h−1, 0.252 g l−1, and 0.011 g MeOH g−1 DCW h−1, respectively. The unstructured model was validated in methanol induction phase with different initial cell densities. Results showed that the maximum specific protein production rate (qp.max) of 0.098 mg g−1 DCW h−1 was achieved when μ was kept at 0.016 h−1, and the maximum yield of PIP reached 0.97 g l−1, which was 1.5-fold as that of the control. Therefore, the simple Monod model proposed has proven to be a robust control system for recombinant porcine insulin precursor production by P. pastoris on pilot scale, which would be further applied on production scale.

Oxygen Uptake Rate Controlling Strategy Balanced with Oxygen Supply for Improving Coenzyme Q10 Production by Rhodobacter sphaeroides

Ze-Jian Wang,Xingzi Zhang,Ping Wang,Zhiwei Sui,Meijin Guo,Siliang Zhang,Yingping Zhuang 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.3

The effects of different oxygen uptake rates (OUR) on the physiological metabolism of Rhodobacter sphaeroides were investigated systematically in 50 L fermenters due to the significant influence on industrial coenzyme Q10 production under oxygen supply limitation. Meanwhile, the seriously decreased oxygen transfer rate caused by the increased broth viscosity was successfully prevented with OUR-directed continuous ammonium sulfate feeding in the late fermentation phase. The statistical analysis results showed that controlling OUR constantly at 45 ± 2.2 mmol/L/h by the oxygen supply level adjustment and the continuous ammonium sulfate feeding could greatly enhance Q10 production. This OUR-Stat controlling strategy successfully achieved the maximal coenzyme Q10 production (2584 ± 82 mg/L), which was 15.4% higher than that of the control. The highest specific CoQ10 content was 25.9 mg/(g DCW)), and the yield of CoQ10 to glucose consumption was up to 19.37 mg/g. These results demonstrated that the optimal OUR-Stat controlling strategy would be effective and economical for improving the industrial CoQ10 production.

In silico Analysis and Experimental Improvement of Taxadiene Heterologous Biosynthesis in Escherichia coli

Hailin Meng,Yong Wang,Qiang Hua,Siliang Zhang,Xiaoning Wang 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.2

The biosynthesis of terpenoids in heterologous hosts has become increasingly popular. Isopentenyl diphosphate (IPP) is the central precursor of all isoprenoids, and the synthesis can proceed via two separate pathways in different organisms: The 1-deoxylulose 5-phosphate (DXP)pathway and the mevalonate (MVA) pathway. In this study,an in silico comparison was made between the maximum theoretical IPP yields and the thermodynamic properties of the DXP and MVA pathways using different hosts and carbon sources. We found that Escherichia coli and its DXP pathway have the most potential for IPP production. Consequently, codon usage redesign, and combinations of chromosomal engineering and various strains were considered for optimizing taxadiene biosynthesis through the endogenic DXP pathway. A high production strain yielding 876 ± 60 mg/L taxadiene, with an overall volumetric productivity of 8.9 mg/(L × h), was successfully obtained by combining the chromosomal engineered upstream DXP pathway and the downstream taxadiene biosynthesis pathway. This is the highest yield thus far reported for taxadiene production in a heterologous host. These results indicate that genetic manipulation of the DXP pathway has great potential to be used for production of terpenoids, and that chromosomal engineering is a powerful tool for heterologous biosynthesis of natural products.

In silico Improvement of Heterologous Biosynthesis of Erythromycin Precursor 6-Deoxyerythronolide B in Escherichia coli

Hailin Meng,Zhiguo Lu,Yong Wang,Xiaoning Wang,Siliang Zhang 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.3

The heterologous biosynthesis of 6-deoxyerythronolide B (6dEB), a key intermediate in the biosynthesis of erythromycin, has recently been achieved in Escherichia coli, but the experimental product yield remains low. In this study, in silico strategies were adopted to evaluate and improve the biosynthesis of 6dEB in this strain. The theoretical capability of E. coli to produce 6dEB was first evaluated by analyzing the maximum theoretical molar yield (MTMY) of 6dEB utilizing three carbon sources,glucose, propionate and glycerol. Although propionate is presently most often used experimentally, our results indicated that glucose would be the most feasible substrate for 6dEB production from economic and long-term standpoints. Compared with Saccharomyces cerevisiae and Bacillus subtilis, E. coli was found to be a better heterologous host for the biosynthesis of 6dEB due to the higher MTMY value under the same conditions. Two strategies,including a flux distribution comparison analysis (FDCA)and linear minimization of metabolic adjustment based (LMOMA-based) methods, were proposed and employed for in silico strain improvement of 6dEB production, which yielded several potential gene targets for future experimental validation. In a further analysis, increasing the specific growth rate (SGR) or the non-growth associated maintenance (NGAM) was found to decrease the MTMY; while increasing the specific oxygen uptake rate (SOUR) or the specific carbon source uptake rate (SCUR) increased the MTMY. Taken together, our findings identified key factors directly affecting the MTMY of 6dEB production, which will guide future experimental research or even the industrial production of 6dEB.

On-line Specific Growth Rate Control for Improving Reduced Glutathione Production in Saccharomyces cerevisiae

Zhi-Qiang Xiong,Mei-jin Guo,Ju Chu,Ying-ping Zhuang,Siliang Zhang 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.5

Reduced glutathione (GSH), the abundant bioactive tripeptide in most living cells, is widely used in pharmaceutical, food, and cosmetic industries. Specific growth rate (μ) is a key physiological parameter for GSH high-cell-density cultivation using microbial cell factories. Here, based on a biomass probe, an on-line μ feedback control was developed to regulate glucose feeding rate during the fed-batch phase for overproducing GSH in Saccharomyces cerevisiae. Compared with real-time μ controlled at 0.15/h, μ controlled at 0.2/h achieved yeast dry weight (120 g/L), GSH concentration (1.5 g/L), and intracellular GSH content (1.25%), which improved by 9, 150, and 129.1%, respectively. To our knowledge, this is the first report about on-line μ feedback control for GSH production. On-line μ control led to 59.38 mg/L/h of GSH productivity and 3.52 mg/g of GSH yield on glucose, which improved by 107.6 and 7.2%, respectively, in comparison with those of traditional ethanol feedback control (maintaining ethanol concentration at 1%). Taken together, the on-line μ feedback control is a promising method as an efficient alternative to conventional feed control techniques presently practiced in the GSH industry, and has the potential for the production of other valuable chemicals.

High-throughput Screening Strategy Used for Enhanced Production of Pigment by Monascus purpureus D39-4

Jun Tan,Ju Chu,Wenjuan Shi,Cheng Lin,Yuanxin Guo,Ying-ping Zhuang,Siliang Zhang,Tadayuki Imanaka 한국식품과학회 2012 Food Science and Biotechnology Vol.21 No.6

Most of the fermentation experiment designs were limited by the low-throughput of shake flask, especially for the medium optimization. A simple high-throughput screening system was developed for the determination of pigment in Monascus purpureus fermentation samples. This downscaled system was designed to optimize medium composition combined with statistical methods. The total 29 experiments designed by the Box–Behnken were used to study the 4 most important operating variables on pigment production. The analysis revealed that the optimum concentrations of glucose, peptone, NaNO3, and KH2PO4were 51.42, 4.91, 1.00, and 1.00 g/L, respectively. A production of 69.5 U/mL was achieved in agreement with the prediction (68.9 U/mL) fermented in 24-deep-well microtiterplates. Furthermore, the fermentation medium optimized in the high-throughput system was verified in shake flasks, and the pigment production could be enhanced from 206.5 U/mL in un-optimized medium to 265.8 U/mL,giving nearly 1.30-fold increase in production.