Co-reporter:Peilian Wei, Qinhu Wang, Baojian Hang, Feng Shi, Jin Cai, Lei Huang, Zhinan Xu
Process Biochemistry 2017 Volume 59, Part B(Volume 59, Part B) pp:
Publication Date(Web):1 August 2017
DOI:10.1016/j.procbio.2017.05.014
•The bioactivity of aquaporin in E.coli was demonstrated by in-vivo expression of AqpSS9 from P. profundum SS9.•A high productivity of soluble AqpSS9 (571 mg/l) was achieved by using in-vitro E. coli cell-free expression system (D-CF mode).•High water permeability (Pf 310.7 ± 3.2 μm/s) was determined with AqpSS9 incorporated proteoliposome.•The construction of AqpSS9 embedded layer-by-layer (LbL) membrane with improved water permeability and salt rejection.AqpSS9 is a novel aquaporin derived from the deep sea bacterium Photobactetrium profundum SS9 and has attracted many attentions in developing water filtration biomimetic membranes. Functional characterization of AqpSS9 was carried out first by its expression in E. coli MM1211 (aqpZ−). Results showed that it was similar to bacterial aquaporin Z (AqpZ) and functioned as a real aquaporin. In-vitro expression of AqpSS9 were systematically investigated using three different modes: precipitate-based cell-free (P-CF) mode, the detergent-based cell-free (D-CF) mode and lipid-based cell-free expression mode (L-CF). D-CF mode showed more superiority than P-CF and L-CF mode, and the highest expression level of 571 mg/l was achieved by adding 0.7% Brij-78. Then AqpSS9 was purified by affinity chromatograph and incorporated into DOPC liposomes. Osmotic water permeability values (Pf) of reconstituted AqpSS9 proteoliposomes was measured as 310.7 ± 3.2 μm/s, which was about 3.5 times of empty control liposomes and comparable to reported Aqps. The AqpSS9 embedded layer-by-layer (LbL) membrane was fabricated and tested, which showed enhanced water permeability and salt rejection in comparison with the control membrane. This work demonstrated the good performance of AqpSS9 as a water channel protein, which may become an alternative candidate for biomimetic membrane construction for water filtration.Download high-res image (141KB)Download full-size image
Co-reporter:Yunping Tang;Xiuliang Yang;Baojian Hang
Applied Biochemistry and Biotechnology 2016 Volume 178( Issue 7) pp:1458-1470
Publication Date(Web):2016 April
DOI:10.1007/s12010-015-1959-6
Mature collagen is abundant in human bodies and very valuable for a range of industrial and medical applications. The biosynthesis of mature collagen requires post-translational modifications to increase the stability of collagen triple helix structure. By co-expressing the human-like collagen (HLC) gene with human prolyl 4-hydroxylase (P4H) and d-arabinono-1, 4-lactone oxidase (ALO) in Escherichia coli, we have constructed a prokaryotic expression system to produce the hydroxylated HLC. Then, five different media, as well as the induction conditions were investigated with regard to the soluble expression of such protein. The results indicated that the highest soluble expression level of target HLC obtained in shaking flasks was 49.55 ± 0.36 mg/L, when recombinant cells were grew in MBL medium and induced by 0.1 mM IPTG at the middle stage of exponential growth phase. By adopting the glucose feeding strategy, the expression level of target HLC can be improved up to 260 mg/L in a 10 L bench-top fermentor. Further, HPLC analyses revealed that more than 10 % of proline residues in purified HLC were successfully hydroxylated. The present work has provided a solid base for the large-scale production of hydroxylated HLC in E. coli.
Co-reporter:Weijun Zhao;Feng Shi;Baojian Hang;Lei Huang
Applied Biochemistry and Biotechnology 2016 Volume 178( Issue 6) pp:1263-1272
Publication Date(Web):2016 March
DOI:10.1007/s12010-015-1943-1
S-Adenosyl-l-methionine (SAM) plays important roles in trans-methylation, trans-sulfuration, and polyamine synthesis in all living cells, and it is also an effective cure for liver disease, depressive syndromes, and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, a multiple-copy integrative plasmid pYMIKP-SAM2 was introduced into the chromosome of wild-type Saccharomyces cerevisiae strain ZJU001 to construct the recombined strain R1-ZJU001. Further studies showed that the recombinant yeast exhibited higher enzymatic activity of methionine adenosyltransferase and improved its SAM biosynthesis. With a three-phase fed-batch strategy in 15-liter bench-top fermentor, 8.81 g/L SAM was achieved after 52 h cultivation of R1-ZJU001, about 27.1 % increase over its parent strain ZJU001, whereas the SAM content was also improved from 64.6 mg/g DCW to 91.0 mg/g DCW. Our results shall provide insights into the metabolic engineering of SAM pathway in yeast for improved productivity of SAM and subsequent industrial applications.
Co-reporter:Zhenjun Si;Jianzhong Zhu;Wenguang Wang
Applied Microbiology and Biotechnology 2016 Volume 100( Issue 24) pp:10321-10330
Publication Date(Web):2016 December
DOI:10.1007/s00253-016-7739-6
A novel and efficient screening method for pyrroloquinoline quinone (PQQ) high-yielding methylotrophic strains was developed by using glucose dehydrogenase apoenzyme (GDHA) which depended on PQQ as the cofactor. Using this high-throughput method, PQQ high-yielding strains were rapidly screened out from thousands of methylotrophic colonies at a time. The comprehensive phylogenetic analysis revealed that the highest PQQ-producing strain zju323 (CCTCC M 2016079) could be assigned to a novel species in the genus Methylobacillus of the Betaproteobacteria. After systematic optimization of different medium components and cultivation conditions, about 33.4 mg/L of PQQ was obtained after 48 h of cultivation with Methylobacillus sp. zju323 at the shake flask scale. Further cultivations of Methylobacillus sp. zju323 were carried out to investigate the biosynthesis of PQQ in 10-L bench-top fermenters. In the batch operation, the PQQ accumulation reached 78 mg/L in the broth after 53 h of cultivation. By adopting methanol feeding strategy, the highest PQQ concentration was improved up to 162.2 mg/L after 75 h of cultivation. This work developed a high-throughput strategy of screening PQQ-producing strains from soil samples and also demonstrated one potential bioprocess for large-scale PQQ production with the isolated PQQ strain.
Co-reporter:Zhouming Shi;Lei Huang;Xiangting Wu;Lanping Luo;Kaijun Xiao;Jin Cai
Journal of Chemical Technology and Biotechnology 2014 Volume 89( Issue 12) pp:1883-1889
Publication Date(Web):
DOI:10.1002/jctb.4271
Abstract
BACKGROUND
Butyric acid is an important chemical with wide industrial applications, but large-scale bio-production still requires an efficient bioprocess. For this purpose, one novel moving fibrous-bed bioreactor (MFBB) was developed in this work for economical production of butyric acid.
RESULTS
The cells of Clostridium tyrobutyricum were immobilized on a fibrous matrix packed in porous levitated sphere carriers. Each carrier was packed with a fiber quantity 2 cm × 6 cm (0.42 g) and was rotated at 100 rpm in a stirred-tank fermentor having pH and temperature controls. Batch and repeated-batch fermentations were carried out to produce butyric acid with a high yield (0.48 ± 0.015 g g-1). Subsequent fed-batch fermentation using the MFBB resulted in a high concentration of butyric acid (65.4 ± 1.4 g L-1). This novel MFBB could be employed to produce high concentration butyric acid (64.5 ± 1.2 g L-1) by a simple repeated-batch addition of a high concentration of glucose (>100 g L-1).
CONCLUSIONS
The MFBB system developed could practically and economically produce butyric acid from glucose for long-term operation. © 2013 Society of Chemical Industry
Co-reporter:Fan Zhu;Jin Cai;Qiang Zheng;Xiangcheng Zhu;Peilin Cen
Journal of Chemical Technology and Biotechnology 2014 Volume 89( Issue 4) pp:616-622
Publication Date(Web):
DOI:10.1002/jctb.4169
Abstract
BACKGROUND
Poly-γ-glutamic acid (γ-PGA) is an extracellular anionic polymer with various potential applications. Renewable and economical carbon sources as the major fermentation feedstock are in need for γ-PGA production.
RESULTS
Xylose has been demonstrated to support cell growth and γ-PGA biosynthesis in Bacillus subtilis HB-1. Batch fermentation using xylose could produce about 23.63 g L−1 γ-PGA in the broth with a high production rate of 0.74 g L−1 h−1. Subsequent fed-batch fermentation could further improve the concentration of γ-PGA up to 28.15 g L−1 with a high conversion rate of 0.97 g g−1 (l-glutamate). Based on these preliminary results, the multiple-sugar containing substrate (corncob fibres hydrolysate) was chosen as the alternative complex carbon source to produce γ-PGA, and one high concentration (24.92 g L−1) of γ-PGA was attained.
CONCLUSIONS
Our pilot trial results suggested that the low-cost and renewable lignocellulosic biomass can be used for the environmental friendly and efficient production of γ-PGA. © 2013 Society of Chemical Industry
Co-reporter:Shuxiang Zheng;Peilian Wei;Lei Huang;Jin Cai
Food Science and Biotechnology 2014 Volume 23( Issue 2) pp:445-450
Publication Date(Web):2014/04/01
DOI:10.1007/s10068-014-0061-0
Glucuronic acid is an important biochemical with wide applications in the food and medical industries. The myo-inositol oxygenase (MIOX) gene was synthesized and expressed in Escherichia coli BL 21(DE3). After optimization of induction conditions and the culture temperature, the highest MIOX activity (45.46 kU/L) was achieved when 0.1 mM isopropyl-thio-β-d-galactoside (IPTG) was added to cell cultures with an OD600 value 1.0 followed by induction at 26°C for 8 hours. The purified MIOX enzyme exerted characteristics similar to the native form. Conversion of myo-inositol to glucuronic acid was performed using whole cells in a pH 8.5 buffer system. Whole cells harboring myo-inositol oxygenase were used as a biocatalyst to produce 2.13 g/L of glucuronic acid with a conversion rate of 99%. A promising novel process for glucuronic acid production from abundant agricultural byproducts is presented.
Co-reporter:Xu Zhang;Jiayuan Sheng;Lei Huang
World Journal of Microbiology and Biotechnology 2014 Volume 30( Issue 3) pp:893-901
Publication Date(Web):2014 March
DOI:10.1007/s11274-013-1502-2
High-level production of G protein-coupled receptors (GPCRs) is usually difficult to achieve in heterologous cell systems. The inherent hydrophobicity of these receptors could cause aggregation and possible cytotoxicity. Cell-free (CF) expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. Here we reported the CF production of an olfactory receptor from Caenorhabditis elegans, odorant response abnormal protein 10 (ODR-10), a member of GPCRs, using the Escherichia coli extracts. Different expression vectors were investigated and 175 μg/ml total ODR-10 was achieved with pIVEX2.4c. To obtain soluble ODR-10, different detergents and liposome with varied concentrations were respectively added into the CF system. High-level expression of soluble ODR-10 (150 μg/ml) was attained with the addition of 1.5 % polyoxyethylene-(20)-cetyl-ether (Brij58) into the CF system. Furthermore, the yield of total ODR-10 was improved to 350 μg/ml by supplementing liposomes into the CF system, and the maximal concentration of the soluble receptor (102 μg/ml) was achieved in this liposome-assisted CF system. Both strategies produced ODR-10 efficiently by using CF system, and the direct reconstitution of the in vitro expressed receptor into liposomes will be preferred for its potential applications in many areas.
Co-reporter:Yunping Liu;Jianfeng Pan;Peilian Wei
Biotechnology and Bioprocess Engineering 2012 Volume 17( Issue 4) pp:693-702
Publication Date(Web):2012 August
DOI:10.1007/s12257-011-0660-z
In order to improve the production of alcohol oxidase (AOX), a recombinant Pichia pastoris (P. pastoris) system was constructed by transformation of the plasmid pPIC9K-AOX into P. pastoris GS115. The effects of different expression conditions on alcohol oxidase activity in the culture supernatant were investigated in the shake flask scale. The results showed that the highest extracellular activity (562 U/L) of alcohol oxidase was obtained after 56 h induction with 4% methanol at OD600 1.0 in the medium containing 50 g/L maltose, which is about 4.2 folds higher than previously reported. High-purity functional recombinant AOX (>90%) was purified from the culture with the Ni-NTA affinity column and Sephadex G-100 chromatographical methods, with a total recovery rate of 68.9%. Further studies showed that the purified rAOX had similar enzymatic characteristics as the native enzyme, except that the thermal stability and resistance to H2O2 inhibition of rAOX were significantly greater compared to the previous report. The purified rAOX was well tolerant to various water-miscible organic solvents. This efficient expression and purification process will be promising for large-scale production of rAOX as an important diagnostic enzyme for alcohol detection in many areas.
Co-reporter:Lei Huang;Yijuan Xiang;Jin Cai;Ling Jiang
Korean Journal of Chemical Engineering 2011 Volume 28( Issue 12) pp:2312-2315
Publication Date(Web):2011 December
DOI:10.1007/s11814-011-0110-9
The effects of three main sugars in cane molasses were investigated systematically to prepare a cost-effective medium for butyric acid bioproduction. Additionally, 30 g/L corn steep liquor was screened out as the suitable nitrogen source. In the batch fermentation of free cells, when 60 g/L glucose was the only carbon source, 21.28 g/L butyric acid was achieved after 30 h cultivation. Similar product concentration, productivity and yield were obtained when 60 g/L fructose was applied. The utilization of sucrose would bring about lower productivity (0.29 g/L·h) and product concentration (18.15 g/L), but the yield of butyric acid/sucrose (0.34 g/g) is almost the same as that from glucose or fructose (0.35 g/g). Finally, the sugar mixture (15 g/L glucose, 20 g/L fructose and 35 g/L sucrose) was employed to produce butyric acid in a fibrous-bed bioreactor (FBB), and 40.11 g/L butyric acid was produced with one simple fed-batch strategy.
Co-reporter:Ling Jiang;Jufang Wang;Shizhong Liang;Jin Cai
Applied Biochemistry and Biotechnology 2011 Volume 165( Issue 1) pp:98-108
Publication Date(Web):2011 September
DOI:10.1007/s12010-011-9236-9
The great performance of a fibrous bed bioreactor (FBB) is mainly dependent on the cell adhesion and immobilization into the fibrous matrix. Therefore, understanding the mechanism and factors controling cell adhesion in the fibrous matrix is necessary to optimize the FBB setup and further improve the fermentability. The adhesion behavior of a strain of Clostridium tyrobutyricum isolated from an FBB was studied, which was proven to be affected by the different environmental conditions, such as growth phase of cells, pH, ionic strength, ionic species, and composition of media. Our results also suggested that electrostatic interactions played an important role on bacteria adhesion into the fibrous matrix. This study demonstrated that the compositions of fermentation broth would have a significant effect on cell adhesion. Consequently, a two-stage glucose supply control strategy was developed to improve the performance of FBB with higher viable cell density in the operation of the FBB setup.
Co-reporter:Huili Zhang;Jin Cai;Jiaqi Dong;Danping Zhang
Applied Microbiology and Biotechnology 2011 Volume 92( Issue 2) pp:295-303
Publication Date(Web):2011 October
DOI:10.1007/s00253-011-3358-4
Poly (β-l-malic acid) (PMLA) is a water-soluble polyester with many attractive properties in chemical industry and medicine development. However, the low titer of PMLA in the available producer strains limits further industrialization efforts and restricts its many potential applications. In order to solve this problem, a new strain with the distinguished high productivity of PMLA was isolated from fresh plants samples. It was characterized as the candidate of Aureobasidium pullulans based on the morphology and phylogenetic analyses of the internal transcribed spacer sequences. After the optimization of culture conditions, the highest PMLA concentration (62.27 g l−1) could be achieved in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO3 in the medium. This high-level fermentation process was further scaled up in the 10 l benchtop fermentor with a high PMLA concentration (57.2 g l−1) and productivity (0.35 g l−1 h−1), which are the highest level in all the literature. Finally, the suitable acid hydrolysis conditions of PMLA were also investigated with regard to the production of l-malic acid, and the kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. The present work paved the road to produce this multifunctional biomaterial (PMLA) at industrial scale and promised one alternative method to produce l-malic acid in the future.
Co-reporter:Qiang Zheng, Jiangang Lu, Hao Chen, Lei Huang, Jin Cai, Zhinan Xu
Analytical Biochemistry 2011 410(2) pp: 171-176
Publication Date(Web):
DOI:10.1016/j.ab.2010.10.024
Co-reporter:Dong Yang;Xiangcheng Zhu;Xueyun Wu
Applied Microbiology and Biotechnology 2011 Volume 89( Issue 6) pp:1709-1719
Publication Date(Web):2011 March
DOI:10.1007/s00253-010-3025-1
iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3–18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO3 to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT–PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies.
Co-reporter:Xueyun Wu;Dong Yang;Xiangcheng Zhu
Biotechnology and Bioprocess Engineering 2010 Volume 15( Issue 4) pp:664-669
Publication Date(Web):2010 August
DOI:10.1007/s12257-009-3129-6
The heterologous production of iso-migrastatin (iso-MGS) was successfully demonstrated in an engineered S. lividans SB11002 strain, which was derived from S. lividans K4-114, following introduction of pBS11001, which harbored the entire mgs biosynthetic gene cluster. However, under similar fermentation conditions, the iso-MGS titer in the engineered strain was significantly lower than that in the native producer — Streptomyces platensis NRRL 18993. To circumvent the problem of low iso-MGS titers and to expand the utility of this heterologous system for iso-MGS biosynthesis and engineering, systematic optimization of the fermentation medium was carried out. The effects of major components in the cultivation medium, including carbon, organic and inorganic nitrogen sources, were investigated using a single factor optimization method. As a result, sucrose and yeast extract were determined to be the best carbon and organic nitrogen sources, resulting in optimized iso-MGS production. Conversely, all other inorganic nitrogen sources evaluated produced various levels of inhibition of iso-MGS production. The final optimized R2YE production medium produced iso-MGS with a titer of 86.5 mg/L, about 3.6-fold higher than that in the original R2YE medium, and 1.5 fold higher than that found within the native S. platensis NRRL 18993 producer.
Co-reporter:Meihua Wang;Jin Cai;Lei Huang;Zhengbin Lv
Applied Biochemistry and Biotechnology 2010 Volume 162( Issue 7) pp:2027-2036
Publication Date(Web):2010 November
DOI:10.1007/s12010-010-8978-0
The bioactivity of swollenin is beneficial to cellulose decomposition by cellulase despite the lack of hydrolytic activity itself. In order to improve the productivity of swollenin, the effects of culture conditions on the expression level in recombinant Aspergillus oryzae were investigated systematically. With regard to the bioactivity of swollenin, glycerin and peanut meal were the optimal carbon or nitrogen source, respectively. The highest level production of swollenin (50 mg L−1) was attained after 88 h cultivation with the initial pH of 5.6 in the culture medium. Then the soluble swollenin was effectively purified from the cultural supernatant by ammonium sulfate precipitation and cationic exchange chromatography with recovery yield of 53.2%. The purified swollenin was fully bioactive due to its strong synergistic activity with cellulose.
Co-reporter:Jiewen Zhou;Lei Huang;Jiazhang Lian;Jiayuan Sheng;Jin Cai
Biotechnology Letters 2010 Volume 32( Issue 10) pp:1481-1486
Publication Date(Web):2010 October
DOI:10.1007/s10529-010-0315-8
A functional glucokinase (YqgR), N-acetylglucosamine-phosphate mutase (Agm1) and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) were synthesized simultaneously in an Escherichia coli cell-free system. These three enzymes were coupled to reconstruct a biosynthetic pathway which could produce UDP-N-acetylglucosamine with N-acetylglucosamine as the substrate.
Co-reporter:Lei Huang;Peilian Wei;Limei Fan;Dan Ye
World Journal of Microbiology and Biotechnology 2010 Volume 26( Issue 4) pp:649-655
Publication Date(Web):2010 April
DOI:10.1007/s11274-009-0218-9
Powdery mildew is one of the most destructive plant diseases and causes dramatic yield losses in agricultural production. Mildiomycin (MIL) is an effective nucleoside antibiotics used to against such a devastating disease. In order to further improve the bioactivity of MIL, a cytosine-substituted mildiomycin analogue (MIL-C) was biosynthesized by supplementing 0.9 g/L cytosine into the fermentation medium as the precursor. The bioactivity of MIL-C against powdery mildew disease in vivo and in vitro were examined systematically, and compared to MIL and triadimefon. The minimum inhibitory concentration (MIC) of MIL-C in vitro was 78.8 mg/L, which was only half of that of MIL. Furthermore, in the greenhouse trials, the EC90 value of MIL-C (52.89 mg/L) was much lower than that of either MIL (153.22 mg/L) or triadimefon (288.90 mg/L). In the field trials, the efficiency of MIL-C against powdery mildew of cucumber or pumpkin was better than that of MIL or triadimefon. Further acute toxicological and Ames tests proved that MIL-C was a potent bio-pesticide with good bio-compatibility and low toxicity, and its usage can be proposed to popularize for controlling the powdery mildew in the future.
Co-reporter:Jie Chen;Feng Shi;Bin Zhang;Fan Zhu
Applied Biochemistry and Biotechnology 2010 Volume 160( Issue 2) pp:370-377
Publication Date(Web):2010 January
DOI:10.1007/s12010-008-8307-z
Poly-γ-glutamic acid (γ-PGA) is a kind of water-soluble and biodegradable polymer made from d- and l-glutamic acid units, which are linked by amide bonds formed between α-amino and γ-carboxylic acid groups. As a potential targeted biopolymer that can be refined from biomass directly, γ-PGA has been increasingly applied to food, cosmetic, and pharmaceutical industries. In this work, a suitable nitrogen source was screened out for the high and cost-effective production of γ-PGA in Bacillus subtilis ZJU-7. The effects of inoculation time and initial glucose concentration on the γ-PGA production were investigated systematically in both shake flasks and a bench-top 15-l fermentor. Under the optimized culture conditions, a high γ-PGA productivity (46.4 g/l) was obtained after 48 h cultivation at 37 °C. Finally, the large-scale fermentation of γ-PGA production was successfully scaled up to a 100-l fermentor, with the highest γ-PGA productivity for over 54.0 g/l.
Co-reporter:Ling Jiang;Jufang Wang;Shizhong Liang
Applied Biochemistry and Biotechnology 2010 Volume 160( Issue 2) pp:350-359
Publication Date(Web):2010/01/01
DOI:10.1007/s12010-008-8305-1
Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g−1, respectively, in batch fermentation when 30 g L−1 glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L−1 and yield 0.40 g g−1 of butyric acid were obtained with 25 g L−1 xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L−1 with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).
Co-reporter:Ningning Zhang;Xiangcheng Zhu;Dong Yang
Applied Microbiology and Biotechnology 2010 Volume 86( Issue 5) pp:1345-1353
Publication Date(Web):2010 May
DOI:10.1007/s00253-009-2406-9
Tallysomycin (TLM) H-1, a novel TLM analog, is the major product isolated from Streptoalloteichus hindustanus SB8005, a genetically engineered strain from S. hindustanus E465-94 ATCC 31158. Based on the structural comparison and experimental assays, TLM H-1 represents a novel bleomycin (BLM) analog displaying DNA cleavage activity similar to its parent compounds TLM and BLM, both representatives of the glycopeptide anticancer antibiotics. The low titer of TLM H-1 in the engineered SB8005 strain has greatly limited its further study. In this paper, fermentation optimization for TLM H-1 production in the SB8005 strain is described; single-factor optimization and response surface methodology proved invaluable. The results indicated that three variables including distiller’s grains and solubles, copper sulfate, and maltose out of eight parameters could significantly influence the TLM H-1 production. With systematic comparison and evaluation, the final optimized fermentation medium was determined. The optimized yield of TLM H-1 in the bench-top fermentor was 249.9 mg/L, which is 26.8 times higher than reported using the original medium, and 12.9-fold higher than that of the parent compound TLM produced by the wild-type strain. This work provides important parameters for TLM H-1 production by fermentation and should facilitate further mechanistic studies and clinical developments of TLM H-1 as an anticancer agent.
Co-reporter:Lei Huang;Peilian Wei;Ru Zang;Peilin Cen
Annals of Microbiology 2010 Volume 60( Issue 2) pp:287-292
Publication Date(Web):2010 June
DOI:10.1007/s13213-010-0039-y
Fumaric acid is an important four-carbon dicarboxylic acid as a potential biorefinery target. A high-throughput screening method for fumaric acid overproduction strains was established. Nystatin (50 mg/L) was added into the production medium to restrict the spread of Rhizopus oryzae hyphae on agar plates. With bromocerol green as a pH indicator in the agar plates, the capability of fumaric acid biosynthesis by single colony was positively correlated with the diameter ratio of the colored ring and the colony. With this novel strategy, one high-yield mutant (Rhizopus oryzae ZJU11) was isolated from a large colony library of Rhizopus oryzae after UV irradiation. Starting with an optimized glucose concentration of 85 g/L, Rhizopus oryzae ZJU11 can produce 57.4 g/L fumaric acid in flask and 41.1 g/L in 5-L fermentor, which were 205% and 160% higher than that of the parent strain, respectively. Further studies showed that the production of fumaric acid by Rhizopus oryzae ZJU11 remained at the same level after three consecutive generations on the fermentation medium.
Co-reporter:Jiazhang Lian;Yi Ma;Jin Cai;Ming Wu
Applied Microbiology and Biotechnology 2009 Volume 85( Issue 2) pp:
Publication Date(Web):2009 November
DOI:10.1007/s00253-009-2055-z
The overexpression of subunit b of F1F0 adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level. With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 μg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F1F0 ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device.
Co-reporter:Jiazhang Lian;Shinghua Ding;Jin Cai
Applied Microbiology and Biotechnology 2009 Volume 82( Issue 3) pp:463-470
Publication Date(Web):2009 March
DOI:10.1007/s00253-008-1774-x
Aquaporin Z (AqpZ), a typical orthodox aquaporin with six transmembrane domains, was expressed as a fusion protein with TrxA in E. coli in our previous work. In the present study, three fusion partners (DsbA, GST and MBP) were employed to improve the expression level of this channel protein in E. coli. The result showed that, compared with the expression level of TrxA-AqpZ, five- to 40-fold increase in the productivity of AqpZ with fusion proteins was achieved by employing these different fusion partners, and MBP was the most efficient fusion partner to increase the expression level. By using E. coli C43 (DE3)/pMAL-AqpZ, the effects of different expression conditions were investigated systematically to improve the expression level of MBP-AqpZ in E. coli. The high productivity of MBP-AqpZ (200 mg/l) was achieved under optimized conditions. The present work provides a novel approach to improve the expression level of membrane proteins in E. coli.
Co-reporter:Jie Chen;Heming Chen;Xiangchen Zhu;Yinghua Lu
Applied Microbiology and Biotechnology 2009 Volume 82( Issue 2) pp:241-248
Publication Date(Web):2009 February
DOI:10.1007/s00253-008-1769-7
The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 108 cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l−1) was achieved with a high productivity (23 μg l−1 h−1). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l−1 h−1 in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.
Co-reporter:Xiyang Chen;Peilian Wei;Limei Fan;Dong Yang
Applied Microbiology and Biotechnology 2009 Volume 83( Issue 3) pp:507-512
Publication Date(Web):2009 June
DOI:10.1007/s00253-009-1918-7
Rapamycin is a 31-member ring macrolide produced by Streptomyces hygroscopicus and has many applications in clinical medicine. In the present work, several protoplasts-related techniques including protoplasts mutation, intraspecies and interspecies protoplasts fusion were tried to improve the rapamycin productivity in S. hygroscopicus. Although mutation and fusion of different protoplasts of S. hygroscopicus did not improve the productivity of rapamycin significantly, the interspecies fusion of protoplasts of S. hygroscopicus D7-804 and Streptomyces erythreus ZJU325 could have brought about one high-yield (345 mg/L) rapamycin producer with 23.6% higher than that of the parental strain. Then, with seven mutants of S. hygroscopicus with different features and rapamycin productivities as the parental strains, only one-round genome shuffling has generated a high-yield rapamycin-producing strain with an outstanding yield of 445 mg/L. The systematic research of protoplast-related techniques has established an applicable way to generate high-yield strains from original microorganisms which can only produce low amount of expected natural products, without information of target gene clusters and gene sequences.
Co-reporter:Dan YE;Zhi-nan Xu;Pei-lin CEN
Journal of Zhejiang University-SCIENCE B 2008 Volume 9( Issue 1) pp:77-84
Publication Date(Web):2008 January
DOI:10.1631/jzus.B071372
Cytosine-substituted mildiomycin analogue (MIL-C) was produced effectively by supplementing cytosine into the culture of Streptoverticillium rimofaciens. In order to improve the yield of MIL-C, statistically-based experimental designs were applied to optimize the fermentation medium for S. rimofaciens ZJU 5119. Fifteen culture conditions were examined for their significances on MIL-C production using Plackett-Burman design. The Plackett-Burman design and one-variable-at-a-time design indicated that glucose and rice meal as the complex carbon sources, and peanut cake meal and NH4NO3 as the complex nitrogen sources were beneficial for MIL-C production in S. rimofaciens ZJU 5119. The results of further central composition design (CCD) showed that the optimal concentration of glucose, rice meal and peanut cake meal were 18.7 g/L, 64.8 g/L and 65.1 g/L, respectively. By using this optimal fermentation medium, the MIL-C concentration was increased up to 1336.5 mg/L, an approximate 3.8-fold improvement over the previous concentration (350.0 mg/L) with un-optimized medium. This work will be very helpful to the large-scale production of MIL-C in the future.
Co-reporter:Haiqin Chen;Cheng Wang
Frontiers of Chemical Science and Engineering 2008 Volume 2( Issue 2) pp:224-229
Publication Date(Web):2008 June
DOI:10.1007/s11705-008-0029-9
In the new era of “Omics”, the traditional techniques of protein expression in vivo can not come up with the exponential increase of genetic information. The cellfree protein synthesis system provides a new strategy of protein expression with advantages of rapid, convenient and high-throughput expression. The preparation of cell extracts, the optimization of substrate concentrations and the energy regeneration system are the key factors for the successful construction of cell-free protein expression system. In this work, the cell extract was prepared from RNase I- defective strain E. coli A19. The cell growth phase, the pressure for cell disruption and the storage condition of cell extracts were optimized. Meanwhile, the optimal substrate concentrations and the energy regeneration system were selected. Under the optimized conditions, the green fluorescent protein (GFP) reporter gene was expressed in the E. coli cell-free system with high expression level (Ca. 154 μg/mL) which was 29 times higher than the expression level before optimization.
Co-reporter:Feng Shi;PeiLin Cen
Science China Chemistry 2007 Volume 50( Issue 3) pp:
Publication Date(Web):2007 June
DOI:10.1007/s11426-007-0061-5
Three kinds of poly amino acids, poly-γ-glutamic acid, poly(ɛ-L-lysine) and multi-L-arginyl-poly (L-aspartic acid) can be synthesized by enzymatic process independently from ribosomal protein biosynthesis pathways in microorganism. These biosynthesized polymers have attracted more and more attentions because of their unique properties and various applications. In this review, the current knowledge on the biosynthesis, biodegradations and applications of these three poly amino acids are summarized.
Co-reporter:Zhinan Xu;Keju Jing;Ying Liu;Peilin Cen
Journal of Industrial Microbiology & Biotechnology 2007 Volume 34( Issue 1) pp:83-90
Publication Date(Web):2007 January
DOI:10.1007/s10295-006-0168-2
Two glucose dehydrogenase (E.C. 1.1.1.47) genes, gdh223 and gdh151, were cloned from Bacillus megaterium AS1.223 and AS1.151, and were inserted into pQE30 to construct the expression vectors, pQE30-gdh223 and pQE30-gdh151, respectively. The transformant Escherichia coli M15 with pQE30-gdh223 gave a much higher glucose dehydrogenase activity than that with the plasmid pQE30-gdh151. Thus it was used to optimize the expression of glucose dehydrogenase. An proximately tenfold increase in GDH activity was achieved by the optimization of culture and induction conditions, and the highest productivity of glucose dehydrogenase (58.7 U/ml) was attained. The recombinant glucose dehydrogenase produced by E. coli M15 (pQE30-gdh223) was then used to regenerate NADPH. NADPH was efficiently regenerated in vivo and in vitro when 0.1 M glucose was supplemented concomitantly in the reaction system. Finally, this coenzyme-regenerating system was coupled with a NADPH-dependent bioreduction for efficient synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate from ethyl 4-chloro-3-oxobutanoate.
Co-reporter:Lei Huang;Zhixia Zhong;Li Peng
Applied Biochemistry and Biotechnology 2007 Volume 142( Issue 2) pp:139-147
Publication Date(Web):2007 August
DOI:10.1007/s12010-007-0009-4
Human β-defensin-3(HBD3) is a low molecular weight cationic peptide with a broad antimicrobial spectrum. A recombinant Escherichia coli (pET32-smHBD3) was constructed to produce HBD3 fusion protein (TrxA-HBD3) before, but the productivity is relatively low. In the present work, the effects of different expression conditions were systematically investigated to improve the expression level of the fusion protein. With regard to the volumetric productivity, the optimal conditions were determined as follows: cultivation at 34 °C in MBL medium, induction at middle stage of the exponential growth phase with 0.4 mM isopropylthio-d-galactoside, and postinduction expression for 8 h. Under these conditions, the volumetric productivity of the fusion protein reached 2.55 g/L, i.e., 0.55 g mature HBD3/L, which was about 2.6 times of that obtained under the unoptimized conditions. And the target protein still maintained high solubility (≥97.9%) and accounted for 66% of the total soluble protein. A cationic exchange purification step was employed to obtain high-purity target protein (90%) with a recovery ratio of 78%. This soluble expression level of HBD3 fusion protein was the highest among all the reported literature and facilitated the development of high efficient purification of HBD3.
Co-reporter:Haiqin Chen;Xiufei Yin;Peilin Cen
Applied Microbiology and Biotechnology 2007 Volume 77( Issue 2) pp:347-354
Publication Date(Web):2007 November
DOI:10.1007/s00253-007-1156-9
Sixteen kinds of human immunodeficiency virus (HIV) target genes were cloned by polymerase chain reaction (PCR) amplification, and specific plasmids were constructed as the templates for the expression of these genes in the cell-free system. Similarly, the linear PCR templates of these genes for cell-free protein expression were also constructed by using two PCR amplification process. These different templates can be employed to biosynthesize HIV proteins in the cell-free system simultaneously and can be adapted for some high-throughput processes. HIV protease (P10) was performed as a target protein, and two different templates (plasmid and PCR product) were prepared and used for P10 expression in the Escherichia coli cell-free system. The target protein P10 was detected in sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels either by using a plasmid template or by a PCR template. These results are promising and helpful to develop a high throughput process for drug discovery.
Co-reporter:Jie Chen;Yinghua Lu;Peilin Cen;Xiangming Fang
Biotechnology Letters 2007 Volume 29( Issue 6) pp:859-863
Publication Date(Web):2007 June
DOI:10.1007/s10529-007-9341-6
Human Fas ligand (hFasL) is a member of the tumor necrosis factor (TNF) family with many medical interests. To produce this protein efficiently, an improved vector which could express the recombinant hFasL protein with a 6-his tag at its C-terminal was constructed. The new vector was transformed into Dictyostelium discoideum AX3 which then produced 157 μg hFasL l−1. Using one-step Ni-affinity chromatography, it was purified with a recovery of 92% and purity of 91%.
Co-reporter:Chengliang Shu, Jin Cai, Lei Huang, Xiangcheng Zhu, Zhinan Xu
Journal of Molecular Catalysis B: Enzymatic (November 2011) Volume 72(Issues 3–4) pp:139-144
Publication Date(Web):1 November 2011
DOI:10.1016/j.molcatb.2011.05.011
A novel method involving polyethylenimine (PEI) coating and glutaraldehyde cross-linking processes was developed to immobilize Candida rugosa lipase onto cotton cloth. After the systematic investigation, the optimal lipase immobilization was achieved when 0.1 g lipase was loaded on 1.5 g support, which was pretreated with 10 ml of 1.0 mg/ml PEI solution at pH 8.0. Subsequent catalytic analysis of immobilized lipase for ethyl butyrate synthesis was also carried out in the Erlenmeyer flasks. The results indicated that when optimal 0.25 M ethanol and 0.6 M butyric acid were catalyzed by the immobilized lipase at 25 °C, the highest conversion yield of 91.2% and 1.27 mmol h−1 g−1 productivity of ethyl butyrate were obtained. Furthermore, a kinetic model of Ping Pong Bi-Bi mode with inhibition of both substrates was proposed and validated by experimental data. To explore the practical potential of immobilized lipase, its operational stability was evaluated and the residual activity was remained about 50% after 12 repeated recycles, with a half-life time of about 300 h for the immobilized lipase. Finally, a recycle batch reactor using immobilized lipase was developed for ethyl butyrate production. The achieved result of 0.85 M final ethyl butyrate concentration, with the conversion of 70.6% and the productivity of 1.45 mmol h−1 g−1, had revealed the promising potential of this immobilized lipase in practical applications.Graphical abstractDownload full-size imageHighlights► Candida rugosa lipase was immobilized onto cotton cloth. ► The effects of PEI, pH value and the amount of support were investigated to improve the immobilization efficiency. ► The half-life time of the immobilized lipase is about 300 h. ► Ping Pong Bi-Bi mode with inhibition of substrates was proposed and validated. ► A recycle batch reactor using immobilized lipase was established.
Co-reporter:Baojian Hang, Jianfeng Pan, Desheng Ni, Qiang Zheng, Xu Zhang, Jin Cai, Lei Huang, Peilian Wei, Zhinan Xu
Process Biochemistry (May 2016) Volume 51(Issue 5) pp:599-606
Publication Date(Web):1 May 2016
DOI:10.1016/j.procbio.2016.02.002
•Aquaporin Z (AqpZ) was expressed as the fusion protein maltose-binding protein (MBP)–AqpZ–polyhistidine (HIS).•The MBP tag of MBP–AqpZ–HIS was removed by factor Xa.•AqpZ–HIS was purified using affinity chromatography.•The yield of functional AqpZ–HIS was 15–20 mg/l.Aquaporin Z (AqpZ) is a water channel protein belonging to the major intrinsic protein (MIP) family and is found in the membrane of Escherichia coli. Due to its high water permeability and low activation energy, AqpZ is a potential candidate for developing biomimetic water filtration technologies. In the present study, membrane-associated AqpZ was expressed in E. coli using the maltose-binding protein (MBP)/polyhistidine (HIS) dual-affinity tag fusion system. The effects of different host strains and the expression conditions on the production of the fusion protein MBP–AqpZ–HIS were systematically studied. Furthermore, an efficient protocol was developed to purify AqpZ–HIS via affinity chromatography, and approximately 15–20 mg of functional AqpZ–HIS was obtained from 1 l of culture with a specific water transport activity of around 2 × 10−14 cm3 s−1 monomer−1. The related sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and tryptophan fluorescence emission analyses indicated that purified AqpZ–HIS assembled as a homotetramer with high thermostability, in turn indicating the correct folding of AqpZ. Our results showed that the MBP/HIS dual-affinity tag system was highly efficient for the overexpression, membrane targeting, and purification of MIP channel proteins in E. coli.Download full-size image
Co-reporter:Yufeng Yang, Lei Huang, Jufang Wang, Zhinan Xu
Enzyme and Microbial Technology (January 2015) Volume 68() pp:43-49
Publication Date(Web):1 January 2015
DOI:10.1016/j.enzmictec.2014.10.002
•FAD-GDH from A. terreus was successfully expressed in soluble form in E. coli.•Chaperone coexpression and osmotic stress induced by simple carbon sources both proved effective for improving the yield of soluble FAD-GDH.•Enzyme variants with improved substrate specificity were generated using site-directed mutagenesis.An FAD-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus NIH2624 was expressed in Escherichia coli with a yield of 228 ± 16 U/L of culture. Co-expression with chaperones DnaK/DnaJ/GrpE and osmotic stress induced by simple carbon sources enhanced productivity significantly, improving the yield to 23883 ± 563 U/L after optimization. FAD-GDH was purified in two steps with the specific activity of 604 U/mg. Using d-glucose as substrate, the optimal pH and temperature for FAD-GDH were determined to be 7.5 and 50 °C, respectively. Activity was stable across the pH range 3.5–9.0, and the half-life was 52 min at 42 °C. Km and Vmax were calculated as 86.7 ± 5.3 mM and 928 ± 35 U/mg, and the molecular weight was approximately 65.6 kDa based on size exclusion chromatography, indicating a monomeric structure. The 3D structure of FAD-GDH was simulated by homology modelling using the structure of A. niger glucose oxidase (GOD) as template. From the model, His551, His508, Asn506 and Arg504 were identified as key residues, and their importance was verified by site-directed mutagenesis. Furthermore, three additional mutants (Arg84Ala, Tyr340Phe and Tyr406Phe) were generated and all exhibited a higher degree of substrate specificity than the native enzyme. These results extend our understanding of the structure and function of FAD-GDH, and could assist potential commercial applications.
Co-reporter:Jingle Liang, Zhinan Xu, Tongfeng Liu, Jianping Lin, Peilin Cen
Enzyme and Microbial Technology (January 2008) Volume 42(Issue 2) pp:145-150
Publication Date(Web):1 January 2008
DOI:10.1016/j.enzmictec.2007.08.012
Natamycin is a very attractive antifungal agent with wide applications in medical and food industries. In order to improve the productivity of natamycin, the effects of cultivation conditions were investigated with Streptomyces gilvosporeus LK-196 in the shake flasks and 30-L fermentors. The results showed that dissolved oxygen and shear force would affluence the biosynthesis of natamycin significantly. The high concentration of natamycin (2.03 g/L) was achieved under the suitable culture conditions in the shake flask scale. Further investigations in 30-L fermentors showed that the optimal pH was controlled at 6.0 during the whole bioprocess, and the dissolved oxygen level should be more than 30% by adjusting the aeration and agitation rates for high production of natamycin. Under these optimal conditions the high concentration of natamycin (3.94 g/L) was achieved with Str. gilvosporeus LK-196 in the 30-L fermentor. Finally, the high-level fermentation process was successfully scaled up to 1000-L fermentors and 18,000-L fermentors in the pilot plant.
Co-reporter:Yue Pu, Chang Dong, Baojian Hang, Lei Huang, Jin Cai, Zhinan Xu
Process Biochemistry (December 2016) Volume 51(Issue 12) pp:2011-2016
Publication Date(Web):1 December 2016
DOI:10.1016/j.procbio.2016.08.003
•PQQGDH was evolved by multiplex sites in situ engineering for enhanced thermal stability and substrate specificity.•A color reaction-based high-throughput screen method was constructed to analyze mutants.•Two variant proteins showed enhanced substrate specificity and thermal stability than the parent PQQGDH enzyme.Pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) is a novel oxygen-independent oxidoreductase. In this work, the PQQGDH gene was integrated into the chromosome of Escherichia coli EcNR2 and then modulated by multiplex-site in situ engineering to achieve improved substrate specificity and thermal stability. After four mutagenesis cycles executed within a single day, six PQQGDH variants were selected and characterized. The variants Ser231Lys/Asn452Thr or Ser231Lys showed higher stability relative to the wild-type enzyme. Additionally, the wild-type PQQGDH could only utilize maltose and lactose at 60% efficiency relative to glucose, while four variants (Ser231Cys/Asn452Thr, Ser231Lys/Asn452Thr, Asn452His, and Asn452Thr) showed significant reductions in their ability to utilize both maltose (30%) and lactose (45%). In particular, Ser231Cys/Asn452Thr and Ser231Lys/Asn452Thr possessed both improved substrate specificity and thermal stability compared to the wild-type PQQGDH. Our study provided a novel and efficient approach to simultaneously introduce multiplex mutations in situ, which is a potential strategy for target enzyme evolution in vivo.Download full-size image
Co-reporter:Wei Luo, Yunping Liu, Xiangcheng Zhu, Weijun Zhao, Lei Huang, Jin Cai, Zhinan Xu, Peilin Cen
Enzyme and Microbial Technology (6 May 2011) Volume 48(Issues 6–7) pp:438-444
Publication Date(Web):6 May 2011
DOI:10.1016/j.enzmictec.2011.03.008
With improved enzymatic activity and easy accessibility, the recombinant purine nucleoside phosphorylase (PNPase) could be a very promising alternative for nucleoside biosynthesis. In our work, the deoD gene encoding PNPase was successfully cloned from Escherichia coli MG1665 and overexpressed in E. coli BL 21(DE3). After optimization of expression conditions including temperature, induction timing and isopropyl-thio-β-d-galactoside (IPTG) concentration, over 70% of expressed total protein was His-tagged PNPase, in the soluble and functional form. Followed assays indicated that the recombinant enzyme exhibited similar substrate specificity and pH preference as the wild type PNPase. Furthermore, the immobilization technology was applied to develop the possible application of recombinant enzyme. Agar from four different polymer carriers was selected as a suitable matrix for whole recombinant cell entrapment. Subsequent enzyme assays, kinetic analysis and stability evaluation of free and immobilized recombinant cells were compared. The results indicated that although the immobilization process reduced the substrate affinity and catalytic efficiency of recombinant cells, it could significantly enhance the stability and reusability of these cells. Finally, the immobilized whole cell biocatalyst was applied to produce ribavirin, as a model nucleoside synthesis reaction. The obtained relative high productivity of rabavirin and quick reaction time suggested the great potential and feasibility of immobilized PNPase in efficient and valuable industrial utilizations.
Co-reporter:Xu Zhang, Jiazhang Lian, Lei Kai, Lei Huang, Peilin Cen, Zhinan Xu
Enzyme and Microbial Technology (5 February 2014) Volume 55() pp:26-30
Publication Date(Web):5 February 2014
DOI:10.1016/j.enzmictec.2013.12.002
•Several leader peptides were fused to increase AqpZ expression in cell-free system.•Leader peptide sequences could be in situ removed by adding detergents or liposome.•The purified AqpZ from these cell-free systems was fully bioactive.Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10−14 cm3 s−1 monomer−1. Using this in situ removable leader peptide strategy, the transcription–translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve.
Co-reporter:Zhouming Shi, Peilian Wei, Xiangcheng Zhu, Jin Cai, Lei Huang, Zhinan Xu
Enzyme and Microbial Technology (10 October 2012) Volume 51(Issue 5) pp:263-268
Publication Date(Web):10 October 2012
DOI:10.1016/j.enzmictec.2012.07.007
Hydrolysate of Jerusalem artichoke was applied for the production of l-lactic acid by immobilized Lactococcus lactis cells in a fibrous bed bioreactor system. Preliminary experiments had indicated that the high quality hydrolysate, which was derived from the 40 min acid treatment at 95 °C and pH 1.8, was sufficient to support the cell growth and synthesis of l-lactic acid. With the addition of 5 g/l yeast extract, the fermentative performance of free cell system was evidently improved. After the basal settlement of hydrolysate based fermentation, the batch mode and the fed-batch mode fermentation were carried out in the free cell system and the fibrous bed bioreactor system, respectively. In all cases the immobilized cells presented the superior ability to produce l-lactic acid. The comparison of batch mode and fed-batch mode also indicated that the growth-limiting feeding strategy could reduce the lag phase of fermentation process and enhance the production of l-lactic acid. The achieved maximum concentration of l-lactic acid was 142 g/l in the fed-batch mode. Subsequent repeated-batch fermentation of the fibrous bed bioreactor system had further exhibited the persistence and stability of this system for the high production of l-lactic acid in a long term. Our work suggested the great potential of the fibrous bed bioreactor system and hydrolysate of J. artichoke in the economical production of l-lactic acid at industrial scale.Highlights► High yield of sugars from Jerusalem artichoke was obtained using mild acid treatment. ► JA hydrolysate could support the high level production of lactic acid in FBB. ► Immobilization of Lactococcus lactis greatly improved the lactic acid productivity.
Co-reporter:Fan Zhu, Jin Cai, Xiangting Wu, Jin Huang, Lei Huang, Jianzhong Zhu, Qiang Zheng, Peilin Cen, Zhinan Xu
Journal of Biotechnology (10 March 2013) Volume 164(Issue 1) pp:67-74
Publication Date(Web):10 March 2013
DOI:10.1016/j.jbiotec.2012.12.009
Poly-γ-glutamic acid (γ-PGA) is an extracellular anionic polymer with many potential applications. Although microbial fermentation is the common approach to produce γ-PGA, the broth at the latter stage usually becomes very viscous and severely influences the metabolites producing pattern of target microbe. In this study, acetoin and 2,3-butanediol were confirmed to be the main byproducts of γ-PGA-producing strain B. subtilis ZJU-7 (B. subtilis CGMCC1250), and their effects on the cell growth and γ-PGA biosynthesis were further investigated in shake flasks. The outcome indicated that both acetoin and 2,3-butanediol showed clear impairment on γ-PGA production of B. subtilis ZJU-7. Moreover, the extracellular metabolites profiles of fermentation under three different pH values were acquired and the metabolic flux redistribution of pathways related to γ-PGA biosynthesis was calculated based on the collected data. As a result, the metabolic flux favored to distribute toward glycolytic pathway at pH 6.5, in which the ingestion rate of extracellular glutamic acid was higher and the subsequent γ-PGA biosynthesis was enhanced. The present work provided us a deep insight into the metabolic flux control of γ-PGA biosynthesis, which will stimulate some novel metabolic engineering strategies to improve the productivity of γ-PGA in future.Highlights► Acetoin and 2,3-butanediol were confirmed as the main byproducts in B. subtilis ZJU-7. ► Extracellular metabolite profiles of PGA fermentation were acquired at three pH values. ► Pathways of byproducts were included in metabolic network modeling of PGA biosynthesis. ► Metabolic flux redistribution of PGA and byproducts formation was calculated.
Co-reporter:Lei Huang, Yue Pu, Xiuliang Yang, Xiangcheng Zhu, Jin Cai, Zhinan Xu
Journal of Biotechnology (10 April 2015) Volume 199() pp:55-61
Publication Date(Web):10 April 2015
DOI:10.1016/j.jbiotec.2015.02.006
•Improved metabolite overproduction in E. coli using a CRP engineering strategy.•More than a 25% improvement in lycopene productivity was achieved by CRP evolution.•The DNA microarray analyses provided a possible explanation for the varying lycopene production.Transcriptional engineering has received significant attention for improving strains by modulating the behavior of transcription factors, which could be used to reprogram a series of gene transcriptions and enable multiple simultaneous modifications at the genomic level. In this study, engineering of the cAMP receptor protein (CRP) was explored with the aim of subtly balancing entire pathway networks and potentially improving lycopene production without significant genetic intervention in other pathways. Amino acid mutations were introduced to CRP by error-prone PCR, and three variants (mcrp26, mcrp159 and mcrp424) with increased lycopene productivity were screened. Combinations of three point mutations were then created via site-directed mutagenesis. The best mutant gene (mcrp26) was integrated into the genome of E. coli BW25113-BIE to replace the wild-type crp gene (MT-1), which resulted in a higher lycopene production (18.49 mg/g DCW) compared to the original strain (WT). The mutant strain MT-1 was further investigated in a 10-L bench-top fermentor with a lycopene yield of 128 mg/l at 20 h, approximately 25% higher than WT. DNA microarray analyses showed that 396 genes (229 up-regulated and 167 down-regulated) were differentially expressed in the mutant MT-1 compared to WT. Finally, the introduction of the mutant crp gene (mcrp26) increased β-carotene production in E. coli. This is the first report of improving the phenotype for metabolite overproduction in E. coli using a CRP engineering strategy.
![4,5-Dioxo-4,5-dihydro-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid](/data/chemimg/654800/72909-34-3.png)
![4,5-Dioxo-4,5-dihydro-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid](/data/chemimg/654800/72909-34-3_b.png)
