Regulation of meilingmycin in Streptomyces nanchangensis: Effect of ammonium ion

Ying-ping Zhuang,Yong Wang,Ping Wang,Ju Chu,Si-liang Zhang 한국화학공학회 2010 Korean Journal of Chemical Engineering Vol.27 No.3

Effects of different ammonium sulfate concentrations on meilingmycin biosynthesis were studied in this research. The results show that a lower concentration of ammonium ions stimulates the biosynthesis of meilingmycin,while a concentration higher than 5mmol/L inhibits the mycelial growth and the biosynthesis of the products. However,increased sugar consumption rate with the elevated concentration of ammonium sulfate was observed during the fermentation process. On this basis, six enzymes, which are responsible for the meilingmycin biosynthesis and the glucose metabolism, were measured and analyzed during the bioprocess. The results suggest that glucose-6-phosphate dehydrogenase, citrate synthase, succinate dehydrogenase and fatty acid synthase are stimulated by higher concentration of ammonium ions, while valine dehydrogenase and methylmalonyl-CoA carboxyltransferase are inhibited. From the results it follows that the precursor supply was restricted with the higher concentration of ammonium ions, which results in the lower production of meilingmycin. Thus, the strategy of maintaining a low level of FAS activity is a critical factor for high yield of meilingmycin.

Ex vivo Expansion of Bone Marrow Mesenchymal Stem Cells Using Microcarrier Beads in a Stirred Bioreactor

Lang Zhou,Jiantao Kong,Ying-ping Zhuang,Ju Chu,Si-Liang Zhang,Mei-Jin Guo 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.1

Bone marrow mesenchymal stem cells (bmMSCs)have recently gained attention as a useful resource in the fields of regenerative medicine and tissue engineering. However, the number of bmMSCs obtained from available donors is very low. Here we developed a culture strategy for in vitro expansion of bmMSCs in a 1.5 L stirred bioreactor with microcarrier beads. First, the microcarriers (Cytodex 3) were equilibrated in culture medium containing 3% fetal bovine serum (FBS) for at least 30 min prior to cell addition. After inoculation, the FBS concentration of the medium was maintained at 3% (v/v) in the first 24 h and thereafter maintained at 1% (v/v) and a developed feeding regimen was applied over 5 days. The maximum cell density of 2.6 × 106 cells/mL was achieved at day 5,corresponding to a 10.4 ± 0.8 fold increases in total cell number. Among the harvested cells, 98.95% expressed CD29and 84.48% expressed CD90, suggesting that the majority of expanded bmMSCs still retained their differentiation potential. Therefore, the developed microcarrier-based stirred bioreactor culture system is an effective method to generate significant numbers of bmMSCs for potential applications and research studies.

An Alkaline pH Control Strategy for Methionine Adenosyltransferase Production in Pichia pastoris Fermentation

Xiaoqing Hu,Ju Chu,Si-Liang Zhang,Ying-ping Zhuang,Xin Wu,Huaxin Chen,Zhongyuan Lv,Zhongyi Yuan 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.5

Pichia pastoris is a successful system forexpressing heterologous proteins and its fermentation pH isalways maintained below 7.0. However, particular proteinsare unstable under acidic conditions, such as methionineadenosyltransferase (MAT), and thus fermentation underacidic pH conditions is unsuitable because protein activityis lost owing to denaturation. Here, a strategy employingalkaline pH in the late fermentation period was developedto improve MAT production. Initially, P. pastoris KM71was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, theexpressed MAT displayed highest stability at pH 8.0;however, pH 8.0 inhibited cell growth and induced cellrupture, thus affecting protein production. To balance MATstability and Pichia cell viability, different pH controlstrategies were compared. In strategy A (reference), theinduction pH was maintained at 6.0, whereas in strategy B,it was gradually elevated to 8.0 through a 25 h transitionperiod (80 ~ 105 h). MAT activity was 0.86 U/mg (twofoldhigher than the control). However, MAT content wasreduced by 50% when compared with strategy A, becauseof proteases released upon cell lysis. To improve cellviability under alkaline conditions, glycerol was added inaddition to methanol (strategy C). When compared withstrategy B, the MAT-specific activity remained nearlyconstant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MATproduction represents an excellent alternative for expressingproteins that are stable only under alkaline conditions.

Enhanced Protein Production by Sorbitol Co-feeding with Methanol in Recombinant Pichia pastoris Strains

Li Chen,Ali Mohsin,Ju Chu,Ying-ping Zhuang,Yamei Liu,Mei-Jin Guo 한국생물공학회 2017 Biotechnology and Bioprocess Engineering Vol.22 No.6

Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.

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.

Mutation Breeding of High Avermectin B1a-producing Strain by the Combination of High Energy Carbon Heavy Ion Irradiation and Sodium Nitrite Mutagenesis Based on High Throughput Screening

Xiaoqing Song,Yun Zhang,Xudong Zhu,Yonghong Wang,Ju Chu,Ying-ping Zhuang 한국생물공학회 2017 Biotechnology and Bioprocess Engineering Vol.22 No.5

Microbial mutation breeding has been widely used because it is one of the most efficient and practical breeding strategies in the fermentation industry. However, different mutagenesis methods cause various degrees of DNA damage to individual microorganisms, which lead to diverse characteristics of the mutants. In this study, the effects of four different mutagenesis methods on the mutation breeding of Streptomyces avermitilis for improving avermectin B1a production were investigated with an optimized liquid microtiter plate (MTP) culture system. First, an effective and feasible MTP system for mutant strain screening was evaluated through the optimization of the oxygen transfer rate and rapid titer determination. Then, high energy carbon heavy ion irradiation, diethyl sulfate, ultraviolet- (UV) irradiation combined with lithium chloride, and sodium nitrite were used as the mutagens for mutation breeding, respectively. Results showed that carbon heavy ion irradiation had the advantages of possessing the highest positive mutation rate and mean-production of positive mutant strains in the first generation. Sodium nitrite treatment resulted in mutant strains with better inherited stability than the other three methods. Through the combined treatment of carbon heavy ion irradiation and sodium nitrite treatment, an inheritstable mutant S. avermitilis S-233 with high avermectin B1a production was successfully obtained. The fermentation verification in a 500-liter (L) bioreactor demonstrated that the avermectin B1a produced by mutant S. avermitilis S-233 reached 6818 μg/mL, which was 23.8% higher than that of parent strains.

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.

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.

Gas-liquid mass transfer studies: The influence of single- and double-impeller configurations in stirred tanks

Ao Pan,Minghui Xie,Jianye Xia,Ju Chu,Ying-ping Zhuang 한국화학공학회 2018 Korean Journal of Chemical Engineering Vol.35 No.1

The influence of impeller structure on the mass transfer characteristics was studied with the steady-state method for gas-liquid volumetric mass transfer coefficient (kLa). The single-impeller configurations included eight impeller types (three radial flow impellers, four axial flow impellers and one mixed flow impeller), and the doubleimpeller included three configurations (RT+RT, RT+WHD, WHD+WHD). For single-impeller, the gas-liquid mass transfer rates of radial flow impellers were better than those of axial flow impellers under the same rotation speed and gas flow rate. The mass transfer performance (defined as the volumetric mass transfer coefficient per unit power input) of radial flow impellers were also better than that of axial flow impellers. With the same kLa value under a certain gas flow rate, the local bubble size distribution between radial flow impeller and axial flow impeller was similar. As for double impellers, RT+RT provided the highest mass transfer rate under certain rotation speed and gas flow rate, while WHD+WHD gave the highest values of gas-liquid mass transfer coefficient with the same power consumption.

Fermentation Optimization and Industrialization of Recombinant Saccharopolyspora erythraea Strains for Improved Erythromycin A Production

Xiang Zou,Chang-fa Chen,Xia-chang Qi,Jiang-chao Qian,Ju Chu,Ying-ping Zhuang,Si-liang Zhang,Wei Zeng,Wan-jun Li 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.6

Improvement of Erythromycin A (Er-A) production and purity by metabolic engineering of the industrial erythromycin-producing strains Saccharopolyspora erythraea strians ZL1004 and ZL1007, in which the amounts of tailoring enzymes EryK (a P450 hydroxylase)and EryG (an S-adenosylmethionine-dependent O-methyltransferase)for biotransformation of Erythromycin D to Er-A were modulated, was performed in a 50 L fermentor. Addition of 15 g/L of corn steep liquor to the medium increased Er-A production; maximum Er-A production was 8,196 U/mL at 191 h, which was 81.8% higher than that of control (4,507 U/mL at 184 h). Er-B impurities were completely eliminated, whereas Er-C impurities were only 153 U/mL at 191 h. Analysis of intra- and extracellular metabolites and key enzyme activities in central carbon metabolism revealed that the pool of TCA cycle intermediates was enhanced by the addition of corn steep liquor and induced an increase in erythromycin biosynthesis. There were no significant differences between strains ZL1004 and ZL1007 regarding Er-A production and impurity accumulation. Compared to wild type strain,Er-A production was improved by 23.9% while Er-C was reduced by 83.9% and Er-B was completely eliminated. Furthermore, fermentation of recombinant strain ZL1004was successfully scaled up from laboratory scale (50 L fermentor) to industrial scale (25 and 132 m3), with similar levels of Er-A production and purity obtained.