Expression and Characterization of a New L-amino Acid Oxidase AAO Producing α-ketoglutaric Acid from L-glutamic Acid

Rao Ben,Liao Xianqing,Liu Fang,Chen Wei,Zhou Ronghua,Ma Lixin,Wang YaPing 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.6

L-amino acid oxidase (AAO) was reported to be capable of converting L-glutamic acid to α-aketoglutaric acid (α-KG). The sequence of AAO from Kitasatospora cheerisanensis was synthesized based on Pichia pastoris codon-usage preferences. AAO gene was cloned into plasmid pPICZα which was transformed into P. pastoris. Next, multi-copy expression plasmids were constructed by using plasmid pHBM905BDM. High-density fermentation was performed and the recombinant enzyme was characterized. The conversion conditions were optimized. By using Escherichia coli expression system, no soluble or active AAO was obtained from two strains after fermentation and induction. We can’t obtain high-level expression of recombinant strains by using plasmid pPICZα. Therefore, we constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PAAO1, PAAO2, PAAO3, PAAO4, and PAAO5, respectively. The following results showed that expression of AAO in multicopy strains increased as designed and strain PAAO5 was chosen for high-density fermentation and enzyme activity experiments. After high-density fermentation, we achieved an AAO-expression yield of 120.8 U/mL. After temperature and pH optimization, the highest AAO activity was observed at a temperature and pH of 20°C and 6, respectively. After optimization of the conversion conditions, the average production rate of L-glutamic acid to α-KG was 3.46 g/L/h and the highest α-KG titer (103 g/L) was converted from 120 g/L L-glutamic acid. In this study, AAO was abundantly expressed by using P. pastoris expression system. The following experiments indicated that AAO is suitable for use in industrial applications.

Efficient Surface Display of L-glutamate Oxidase and L-amino Acid Oxidase on Pichia pastoris Using Multi-copy Expression Strains

Rao Ben,Zhou Ronghua,Dong Qing,Liao Xianqing,Liu Fang,Chen Wei,Liu Xiaoyan,Min Yong,Wang YaPing 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.4

L-glutamate oxidase (GLOD) and L-amino acid oxidase (AAO) were reported to be capable of convert L-glutamic acid to α-aketoglutaric acid (α-KG). These two enzymes gene have been successfully expressed by using pHBM905BDM in Pichia pastoris to produce α-aketoglutaric acid from L-glutamic acid in our previous studies. Here these two enzymes were displayed on P. pastoris to achieve the conversion. We constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PGLOD(1-3)-AGα1 and PAAO(1-3)-AGα1, respectively. The following results showed that expression of GLOD(1-3)- AGα1 and AAO(1-3)-AGα1 in multi-copy strains increased as designed and strain PGLOD3-AGα1 and PAAO3-AGα1 was chosen for high-density fermentation and enzyme activity experiments. By using a multi-copy expression approach and high-density fermentation, we achieved a GLOD expression yield of 688.5 U/g dry cell weight and AAO expression yield of 626.7 U/g dry cell weight. By using displayed GLOD, the average production rate of L-glutamic acid to α-KG was 6.22 g/L/h and the highest α-KG titer (124.5 g/L) was converted from 135 g/L L-glutamic acid. By using displayed AAO, the average production rate of L-glutamic acid to α-KG was 5.78 g/L/h and the highest α-KG titer (115.6 g/L) was converted from 135 g/L L-glutamic acid. It showed that displaying enzymes on P. pastoris are suitable for use in industrial applications.

An Auto-inducible Expression System Based on the RhlI-RhlR Quorum-sensing Regulon for Recombinant Protein Production in E. coli

Rao Ben,Fan Jiying,Sun Jian’an,Truong Ngoc Tu,Sun Jing,Zhou Jingsong,Qiuyi,Shen Yaling 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.1

An artificial network which can accomplish recombinant protein synthesis guided by cell population in E. coli was constructed. The successful functioning of this network requires two plasmids, pWNB and pET. pWNB is responsible for production of T7 RNA polymerase, which controls pET; pET, in turn, regulates the production of target proteins. Several model proteins were tested and the results show that this E. coli system can be used to efficiently express various recombinant proteins. Since system contains T7 RNA polymerase production elements, it is transferable and applicable to well-characterized E. coli strains. Compared to the IPTG-induced system, an equal or greater amount of target protein can be obtained using this auto-inducible expression system in flasks and bioreactors. Our results suggest that it is a competitive alternative to other expression systems used in labs or for industrial applications.

Enhanced Acetoin Production by Serratia marcescens H32 Using Statistical Optimization and a Two-stage Agitation Speed Control Strategy

Jianan Sun,Liaoyuan Zhang,Ben Rao,Yunbin Han,Ju Chu,Jiawen Zhu,Yaling Shen,Dong-Zhi Wei 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.3

Enhanced acetoin production was carried out by Serratia marcescens H32. First, medium compositions were optimized statistically for shake flask fermentations to produce acetoin. Sucrose and corn steep liquor powder (CSLP) were identified as the most significant factors by Plackett–Burman design. The path of steepest ascent and response surface methodology were then employed to determine the optimal concentrations of the two factors. Acetoin yield was up to 41.5 g/L in flask fermentations using the optimized medium. Furthermore, the optimal medium was used to conduct fermentation experiments in a 3.7-L bioreactor. The influences of different agitation speeds on acetoin production were investigated. Based on a process analysis, a two-stage agitation speed control strategy was proposed, in which the agitation speed was controlled at 700 rpm during the first 8 h and then switched to 600 rpm. A relatively high acetoin concentration (44.9 g/L)and high acetoin productivity (1.73 g/L/h) were achieved by applying this strategy. Fed-batch fermentation based on the two-stage agitation speed control strategy was performed,and a maximum acetoin concentration of 60.5 g/L with productivity of 1.44 g/L/h was achieved. Enhanced acetoin production was carried out by Serratia marcescens H32. First, medium compositions were optimized statistically for shake flask fermentations to produce acetoin. Sucrose and corn steep liquor powder (CSLP) were identified as the most significant factors by Plackett–Burman design. The path of steepest ascent and response surface methodology were then employed to determine the optimal concentrations of the two factors. Acetoin yield was up to 41.5 g/L in flask fermentations using the optimized medium. Furthermore, the optimal medium was used to conduct fermentation experiments in a 3.7-L bioreactor. The influences of different agitation speeds on acetoin production were investigated. Based on a process analysis, a two-stage agitation speed control strategy was proposed, in which the agitation speed was controlled at 700 rpm during the first 8 h and then switched to 600 rpm. A relatively high acetoin concentration (44.9 g/L)and high acetoin productivity (1.73 g/L/h) were achieved by applying this strategy. Fed-batch fermentation based on the two-stage agitation speed control strategy was performed,and a maximum acetoin concentration of 60.5 g/L with productivity of 1.44 g/L/h was achieved.

Calculation of Torque and Loss of Coaxial Magnetic Gear with Halbach Array in 3D Model

Jing Libing,Liu Wei,Tang Weizhao,Rao Yingying,Tan Can,Ben Tong 대한전기학회 2022 Journal of Electrical Engineering & Technology Vol.17 No.3

In two-dimensional fi eld, the end eff ect exists in the calculation of the electromagnetic characteristics of the magnetic gear, which leads to the inaccurate calculation of the air-gap magnetic fi eld and torque. In order to improve the calculation accuracy of the electromagnetic characteristics of the magnetic gear, a 3D coaxial magnetic gear (CMG) with Halbach arrays distribution is proposed in this paper. Using the unilateral eff ect of the Halbach arrays, the magnetic fi eld on the air gap side is strengthened, and the magnetic fi eld on the iron yoke side is weakened. The topology of CMG with 4 inner and 17 outer poles is established and compared with the 2D model with the same parameters. The results show that the 3D CMG with Halbach array distribution can eff ectively reduce the torque ripple, and the accuracy of torque and loss calculation is improved compared with 2D model. The analysis results can provide a basis for the design and manufacture of magnetic gear.

High-level Expression of an Acidic and Thermostable Chitosanase in Pichia pastoris Using Multi-copy Expression Strains and High-celldensity Cultivation

Zhou Ronghua,Liao Xianqing,Liu Fang,Dong Qing,Chen Wei,Wang YaPing,Rao Ben 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.4

Chitin is a linear homopolymer of acetylated β- (1,4)-linked glucosamine residues and among the most abundant polysaccharides in the world. Here, we identified and purified a novel chitosanase (CCHA) from Aspergillus oryzae NKY2017 obtained from Hu’bei province in China. Construction of a cDNA library from this strain revealed the gene sequence subsequently expressed in Pichia pastoris and subsequent construction of multi-copy expression plasmids (CCHA1/2/3/4). The results demonstrated elevated levels of CCHA expression in multi-copy strains, with strain CCHA4 chosen for high-density fermentation and enzyme-activity experiments. High-density fermentation achieved a CCHA yield of 22,500 U/mL, and temperature and pH optimization resulted in the highest CCHA activity at 40°C and 4.0, respectively. We used this enzyme for a large-scale preparation of oligosaccharides: 4 g enzyme could convert 150 kg chitosan into oligosaccharides in 24 h at 40°C. These results demonstrated abundant CCHA expression in P. pastoris and suggested the efficacy of CCHA for use in industrial applications.

Generating iPSCs: Translating Cell Reprogramming Science into Scalable and Robust Biomanufacturing Strategies

Silva, M.,Daheron, L.,Hurley, H.,Bure, K.,Barker, R.,Carr, Andrew J.,Williams, D.,Kim, H.W.,French, A.,Coffey, Pete J.,Cooper-White, Justin J.,Reeve, B.,Rao, M.,Snyder, Evan Y.,Ng, Kelvin S.,Mead, Ben Cell Press 2015 Cell stem cell Vol.16 No.1

Induced pluripotent stem cells (iPSCs) have the potential to transform drug discovery and healthcare in the 21<SUP>st</SUP> century. However, successful commercialization will require standardized manufacturing platforms. Here we highlight the need to define standardized practices for iPSC generation and processing and discuss current challenges to the robust manufacture of iPSC products.