Co-reporter:Cheng Wang;Jiao Liu;Huanhuan Liu
Journal of Industrial Microbiology & Biotechnology 2017 Volume 44( Issue 11) pp:1527-1540
Publication Date(Web):03 August 2017
DOI:10.1007/s10295-017-1974-4
Tacrolimus is widely used as an immunosuppressant in the treatment of various autoimmune diseases. However, the low fermentation yield of tacrolimus has thus far restricted its industrial applications. To solve this problem, the time-series response mechanisms of the intracellular metabolism that were highly correlated with tacrolimus biosynthesis were investigated using different exogenous feeding strategies in S. tsukubaensis. The metabolomic datasets, which contained 93 metabolites, were subjected to weighted correlation network analysis (WGCNA), and eight distinct metabolic modules and seven hub metabolites were identified to be specifically associated with tacrolimus biosynthesis. The analysis of metabolites within each metabolic module suggested that the pentose phosphate pathway (PPP), shikimate and aspartate pathway might be the main limiting factors in the rapid synthesis phase of tacrolimus accumulation. Subsequently, all possible key-limiting steps in the above metabolic pathways were further screened using a genome-scale metabolic network model (GSMM) of S. tsukubaensis. Based on the prediction results, two newly identified targets (aroC and dapA) were overexpressed experimentally, and both of the engineered strains showed higher tacrolimus production. Moreover, the best strain, HT-aroC/dapA, that was engineered to simultaneously enhanced chorismate and lysine biosynthesis was able to produce 128.19 mg/L tacrolimus, 1.64-fold higher than control (78.26 mg/L). These findings represent a valuable addition to our understanding of tacrolimus accumulation in S. tsukubaensis, and pave the way to further production improvements.
Co-reporter:Huiyan Geng;Huanhuan Liu;Jiao Liu
World Journal of Microbiology and Biotechnology 2017 Volume 33( Issue 6) pp:101
Publication Date(Web):02 May 2017
DOI:10.1007/s11274-017-2266-x
Rapamycin is a polyketide with a 31-membered macrolide ring that possesses powerful immunosuppressant activity. In this study, we firstly obtained a mutant, shikimate-resistant Streptomyces hygroscopicus strain UV-II, which displayed about 3.20-fold higher rapamycin production (305.9 mg/L) than the wild-type S. hygroscopicus ATCC29253 (95.5 mg/L). Under optimal conditions, with the addition of 2 g/L shikimic acid, the strain’s rapamycin production was further increased by approximately 34.9%, to 412.6 mg/L. To gain deeper insights into the effects of shikimic acid resistance and supplementation, the fermentation properties, metabolite concentrations, and transcriptional levels of relevant genes were analyzed and evaluated for differences between this improved mutant and its parental strain. The results showed that most of the metabolic modules involved in rapamycin biosynthesis were upregulated in the mutant strain. Analysis of metabolic pathways and gene expression levels further revealed that shikimic acid metabolism plays a crucial role in the synthesis of rapamycin, and identified the rapK gene as a potential target for genetic manipulation to obtain rapamycin-producing strains with improved product yield. Consequently, the rapK gene was overexpressed in the UV-II strain, which to our delight further improved rapamycin production to 457.3 mg/L. These findings thus provide a theoretical basis for further improvements in the production of not only rapamycin, but also of other, analogous macrolide compounds.
Co-reporter:Lanqing Dang;Jiao Liu;Cheng Wang
Journal of Industrial Microbiology & Biotechnology 2017 Volume 44( Issue 2) pp:259-270
Publication Date(Web):2017 February
DOI:10.1007/s10295-016-1880-1
Rapamycin, as a macrocyclic polyketide with immunosuppressive, antifungal, and anti-tumor activity produced by Streptomyces hygroscopicus, is receiving considerable attention for its significant contribution in medical field. However, the production capacity of the wild strain is very low. Hereby, a computational guided engineering approach was proposed to improve the capability of rapamycin production. First, a genome-scale metabolic model of Streptomyces hygroscopicus ATCC 29253 was constructed based on its annotated genome and biochemical information. The model consists of 1003 reactions, 711 metabolites after manual refinement. Subsequently, several potential genetic targets that likely guaranteed an improved yield of rapamycin were identified by flux balance analysis and minimization of metabolic adjustment algorithm. Furthermore, according to the results of model prediction, target gene pfk (encoding 6-phosphofructokinase) was knocked out, and target genes dahP (encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) and rapK (encoding chorismatase) were overexpressed in the parent strain ATCC 29253. The yield of rapamycin increased by 30.8% by knocking out gene pfk and increased by 36.2 and 44.8% by overexpression of rapK and dahP, respectively, compared with parent strain. Finally, the combined effect of the genetic modifications was evaluated. The titer of rapamycin reached 250.8 mg/l by knockout of pfk and co-expression of genes dahP and rapK, corresponding to a 142.3% increase relative to that of the parent strain. The relationship between model prediction and experimental results demonstrates the validity and rationality of this approach for target identification and rapamycin production improvement.
Co-reporter:Jun Wang;Huanhuan Liu;Di Huang;Lina Jin
Applied Microbiology and Biotechnology 2017 Volume 101( Issue 6) pp:2447-2465
Publication Date(Web):2017 March
DOI:10.1007/s00253-017-8136-5
FK506 (tacrolimus) is a 23-membered polyketide macrolide that possesses powerful immunosuppressant activity. In this study, feeding soybean oil into the fermentation culture of Streptomyces tsukubaensis improved FK506 production by 88.8%. To decipher the overproduction mechanism, comparative proteomic and metabolomic analysis was carried out. A total of 72 protein spots with differential expression in the two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), and 66 intracellular metabolites were measured by gas chromatography-mass spectrometer (GC-MS). The analysis of proteome and metabolome indicated that feeding soybean oil as a supplementary carbon source could not only strengthen the FK506 precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation, translation, and stress response, suggesting a better intracellular metabolic environment for the synthesis of FK506. Based on these analyses, 20 key metabolites and precursors of FK506 were supplemented into the soybean oil medium. Among them, lysine, citric acid, shikimic acid, and malonic acid performed excellently for promoting the FK506 production and biomass. Especially, the addition of malonic acid achieved the highest FK506 production, which was 1.56-fold of that in soybean oil medium and 3.05-fold of that in initial medium. This report represented the first comprehensive study on the comparative proteomics and metabolomics applied in S. tsukubaensis, and it would be a rational guidance to further strengthen the FK506 production.
Co-reporter:Qing Xu;Ying Liu;Shuang Li;Ling Jiang;He Huang
Bioprocess and Biosystems Engineering 2016 Volume 39( Issue 8) pp:1267-1280
Publication Date(Web):2016 August
DOI:10.1007/s00449-016-1605-x
Xylose is one of the most abundant lignocellulosic components, but it cannot be used by R. oryzae for fumaric acid production. Here, we applied high-throughput RNA sequencing to generate two transcriptional maps of R. oryzae following fermentation in glucose or xylose. The differential expression analysis showed that, genes involved in amino acid metabolism, fatty acid metabolism, and gluconeogenesis, were up-regulated in response to xylose. Moreover, we discovered the potential presence of oxidative stress in R. oryzae during xylose fermentation. To adapt to this unfavorable condition, R. oryzae displayed reduced growth and induce of a number of antioxidant enzymes, including genes involved in glutathione, trehalose synthesis, and the proteasomal pathway. These responses might divert the flow of carbon required for the accumulation of fumaric acid. Furthermore, using high-throughput RNA sequencing, we identified a large number of novel transcripts and a substantial number of genes that underwent alternative splicing. Our analysis provides remarkable insight into the mechanisms underlying xylose fermentation by R. oryzae. These results may reveal potential target genes or strategies to improve xylose fermentation.
Co-reporter:Baohua Wang;Jiao Liu;Huanhuan Liu
Journal of Industrial Microbiology & Biotechnology 2015 Volume 42( Issue 6) pp:949-963
Publication Date(Web):2015 June
DOI:10.1007/s10295-015-1611-z
Rapamycin is an important natural macrolide antibiotic with antifungal, immunosuppressive and anticancer activity produced by Streptomyces hygroscopicus. In this study, a mutant strain obtained by ultraviolet mutagenesis displayed higher rapamycin production capacity compared to the wild-type S. hygroscopicus ATCC 29253. To gain insights into the mechanism of rapamycin overproduction, comparative metabolic profiling between the wild-type and mutant strain was performed. A total of 86 metabolites were identified by gas chromatography–mass spectrometry. Pattern recognition methods, including principal component analysis, partial least squares and partial least squares discriminant analysis, were employed to determine the key biomarkers. The results showed that 22 potential biomarkers were closely associated with the increase of rapamycin production and the tremendous metabolic difference was observed between the two strains. Furthermore, metabolic pathway analysis revealed that amino acids metabolism played an important role in the synthesis of rapamycin, especially lysine, valine, tryptophan, isoleucine, glutamate, arginine and ornithine. The inadequate supply of amino acids, or namely “nitrogen starvation” occurred in the mutant strain. Subsequently, the exogenous addition of amino acids into the fermentation medium of the mutant strain confirmed the above conclusion, and rapamycin production of the mutant strain increased to 426.7 mg/L after adding lysine, approximately 5.8-fold of that in the wild-type strain. Finally, the results of real-time PCR and enzyme activity assays demonstrated that dihydrodipicolinate synthase involved with lysine metabolism played vital role in the biosynthesis of rapamycin. These findings will provide a theoretical basis for further improving production of rapamycin.
Co-reporter:Xinrong Pan, Haishan Qi, Li Mu, Jianping Wen, and Xiaoqiang Jia
Journal of Agricultural and Food Chemistry 2014 Volume 62(Issue 40) pp:9927-9935
Publication Date(Web):September 8, 2014
DOI:10.1021/jf502239d
The mixed microbes coculture method in cane molasses wastewater (CMW) was adopted to produce 2-phenylethanol (2-PE). Comparative metabolomics combined with multivariate statistical analysis was performed to profile the differences of overall intracellular metabolites concentration for the mixed microbes cocultured under two different fermentation conditions with low and high 2-PE production. In total 102 intracellular metabolites were identified, and 17 of them involved in six pathways were responsible for 2-PE biosynthesis. After further analysis of metabolites and verification by feeding experiment, an overall metabolic mechanism hypothesis for the microbial mixed cultures (MMC) utilizing CMW for higher 2-PE production was presented. The results demonstrated that the branches of intracellular pyruvate metabolic flux, as well as the flux of phenylalanine, tyrosine, tryptophan, glutamate, proline, leucine, threonine, and oleic acid, were closely related to 2-PE production and cell growth, which provided theoretical guidance for domestication and selection of species as well as medium optimization for MMC metabolizing CMW to enhance 2-PE yield.
Co-reporter:Haishan Qi;Sumin Zhao;Hong Fu
Journal of Industrial Microbiology & Biotechnology 2014 Volume 41( Issue 9) pp:1365-1374
Publication Date(Web):2014 September
DOI:10.1007/s10295-014-1473-9
Combinatorial approach of adsorbent resin HP20 addition and metabolic profiling analysis were carried out to enhance ascomycin production. Under the optimal condition of 5 % m/v HP20 added at 24 h, ascomycin production was increased to 380 from 300 mg/L. To further rationally guide the improvement of ascomycin production, metabolic profiling analysis was employed to investigate the intracellular metabolite changes of Streptomyces hygroscopicus var. ascomyceticus FS35 in response to HP20 addition. A correlation between the metabolic profiles and ascomycin accumulation was revealed by partial least-squares to latent structures discriminant analysis, and 11 key metabolites that most contributed to metabolism differences and ascomycin biosynthesis were identified. Based on the analysis of metabolite changes together with their pathways, the potential key factors associated with ascomycin overproduction were determined. Finally, rationally designed fermentation strategies based on HP20 addition were performed as follows: 2 % v/v n-hexadecane was added at 24 h; 1.0 g/L valine was supplemented at 48 h; 1.0 g/L lysine was added at 72 h. The ascomycin production was ultimately improved to 460 mg/L, a 53.3 % enhancement compared with that obtained in initial condition. These results demonstrated that the combination of HP20 addition and metabolic profiling analysis could be successfully applied to the rational guidance of production improvement of ascomycin, as well as other clinically important compounds.
Co-reporter:Wenjie Du;Di Huang;Menglei Xia
Journal of Industrial Microbiology & Biotechnology 2014 Volume 41( Issue 7) pp:1131-1143
Publication Date(Web):2014 July
DOI:10.1007/s10295-014-1450-3
FK506, a secondary metabolite produced by Streptomyces tsukubaensis, is well known for its immunosuppressant properties to prevent rejection of transplanted organs and treat autoimmune diseases. However, the low titer of FK506 in the original producer strain limits the further industrialization efforts and restricts its clinical applications. To address this issue, a highly efficient method combined genome shuffling and dynamic fed-batch strategies was systematically performed in this work. Firstly, after five rounds of genome shuffling based on precursors and product resistances, a higher yielding strain TJ-P325 was successfully acquired, whose production reached 365.6 mg/L, 11-fold increase compared with the original strain. Then, the possible mechanism of different production capabilities between TJ-P325 and the wild type was explored through comparative gene expression analysis of key genes. Results showed that the transcription level of key genes was altered significantly in the mutant. Moreover, precursors addition enhanced the FK506 production by 28 %, as well as reduced the by-products biosynthesis. Finally, the disodium malonate and disodium methylmalonate dynamic fed-batch strategies dramatically led to the production of 514.5 mg/L in a 7.5-L bioreactor. These results demonstrated that genome shuffling and dynamic fed-batch strategies could be successfully applied to generate high-yield strains with value-added natural products during industrial microbial fermentation.
Co-reporter:Di Huang;Menglei Xia;Shanshan Li
Journal of Industrial Microbiology & Biotechnology 2013 Volume 40( Issue 9) pp:1023-1037
Publication Date(Web):2013 September
DOI:10.1007/s10295-013-1301-7
FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis. However, the low titer at which it is produced is a bottleneck to its application and use in industrial processes. We have overexpressed five potential targets associated with FK506 production (fkbO, fkbL, fkbP, fkbM, fkbD) which were identified in our previous study, with the aim to improve FK506 production. The results of the analysis showed that the constructed strains with an additional copy of each gene increased FK506 production by approximately 10–40 % compared with the wild-type strain D852. The results of the gene expression analysis indicated that each gene was upregulated. Combinatorial overexpression of the five genes resulted in a 146 % increase in the FK506 titer to 353.2 mg/L, in comparison with the titer produced by D852. To further improve the production of FK506 by the engineered strain HT-FKBOPLMD, we supplemented the medium with various nutrients, including soybean oil, lactate, succinate, shikimate, chorismate, lysine, pipecolate, isoleucine and valine. Optimization of feeding concentrations and times resulted in HT-FKBOPLMD being able to produce approximately 70 % more FK506, thereby reaching the maximal titer of 457.5 mg/L, with lower amounts of by-products (FK520 and 37,38-dihydro-FK506). These results demonstrate that the combination of the metabolically engineered secondary pathways and the exogenous feeding strategies developed here was able to be successfully applied to improve the production of industrially and clinically important compounds.
Co-reporter:Peng Liu;Yunlin Chen
World Journal of Microbiology and Biotechnology 2013 Volume 29( Issue 6) pp:1121-1127
Publication Date(Web):2013 June
DOI:10.1007/s11274-013-1261-0
In this work, a mutant MX3004 with improved micronomicin (MCR) production was derived from Micromonosporasagamiensis ATCC21826, which was treated with femtosecond laser under the optimized irradiation conditions of 75 mW and 180 s, with a maximum of positive mutation rate of 17.8 % and the mortality rate of 69.2 %. A novel high-throughput method was established using microplate reader by quantifying the concentration of MCR for efficient screening of positive mutant from large numbers of mutants. Consequently, MX3004 displayed the highest MCR production capacity of 126 U/ml and a stable heredity (ten generations). Moreover, under the optimal fermentation conditions in a 7.5 l fermenter, the MCR production of MX3004 reached the maximum of 263 U/ml, which was increased by 484 % compared with the parent strain. The results suggest that femtosecond laser is a suitable method for the MCR production improvement and the screening method has a great potential application for aminoglycoside antibiotic production.
Co-reporter:Sumin Zhao;Di Huang;Haishan Qi
Applied Microbiology and Biotechnology 2013 Volume 97( Issue 12) pp:5329-5341
Publication Date(Web):2013/06/01
DOI:10.1007/s00253-013-4852-7
Rapamycin is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces hygroscopicus. To rationally guide the improvement of rapamycin production, comparative metabolic profiling analysis was performed in this work to investigate the intracellular metabolic changes in S. hygroscopicus U1-6E7 fermentation in medium M1 and derived medium M2. A correlation between the metabolic profiles and rapamycin accumulation was revealed by partial least-squares to latent structures analysis, and 16 key metabolites that most contributed to the metabolism differences and rapamycin production were identified. Most of these metabolites were involved in tricarboxylic acid cycle, fatty acids, and shikimic acid and amino acids metabolism. Based on the analysis of key metabolites changes in the above pathways, corresponding exogenous addition strategies were proposed as follows: 1.0 g/L methyl oleate was added at 0 h; 1.0 g/L lysine was added at 12 h; 0.5 g/L shikimic acid was added at 24 h; 0.5 g/L sodium succinate, 0.1 g/L phenylalanine, 0.1 g/L tryptophan, and 0.1 g/L tyrosine were added at 36 h, successively, and a redesigned fermentation medium (M3) was obtained finally on the basis of M2. The production of rapamycin in M3 was increased by 56.6 % compared with it in M2, reaching 307 mg/L at the end of fermentation (120 h). These results demonstrated that metabolic profiling analysis was a successful method applied in the rational guidance of the production improvement of rapamycin, as well as other industrially or clinically important compounds.
Co-reporter:Xiaoqiang Jia;Shanshan Li;Sha Xie
Applied Biochemistry and Biotechnology 2012 Volume 168( Issue 1) pp:1-9
Publication Date(Web):2012 September
DOI:10.1007/s12010-011-9268-1
Isobutanol can be biosynthesized via α-ketoisovalerate catalyzed by heterologous keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH). In this work, isobutanol biosynthesis pathway was designed in Bacillus subtilis, a notable solvent-tolerant host. In order to do that, a plasmid pPKA expressing KDC and ADH under the control of a B. subtilis strong promoter P43 was constructed. Isobutanol was detected in the products of the recombinant B. subtilis harboring pPKA plasmid, whereas none was detected by the wild-type strain. Effects of the medium ingredients such as glucose concentration and valine addition, and operating parameters such as initial pH, inoculation volume, and medium work volume on isobutanol production were also investigated. Isobutanol production reached to the maximum of 0.607 g/L after 35-h cultivation under the conditions: glucose concentration of 3%, valine addition of 2%, initial pH of 7.0, inoculum of 1%, and work volume of 50 mL/250 mL. Though the isobutanol production by the recombinant was low, it was the first successful attempt to produce isobutanol in engineered B. subtilis, and the results showed its great potential as an isobutanol-producing cell factory.
Co-reporter:Shanshan Li;Xiaoqiang Jia
Biotechnology Letters 2012 Volume 34( Issue 12) pp:2253-2258
Publication Date(Web):2012 December
DOI:10.1007/s10529-012-1041-1
High-level constitutive gene expression can result in cellular metabolic imbalance and limit production. To circumvent these problems, a PalsSD-controlled auto-inducible 2-ketoisovalerate biosynthetic pathway and a Pspac-controlled IPTG-inducible Ehrlich pathway were constructed in Bacillus subtilis to modulate gene expression. Based on the precise gene expression characteristics of the two inducible pathways, the optimal IPTG induction time point and dose for 2-methyl-1-propanol biosynthesis were determined as 9.5 h and 300 μM, respectively. Under the optimized conditions, strain BSUΔL-03 with inducible pathways produced up to 3.83 ± 0.46 g 2-methyl-1-propanol/l, which was about 60 % higher than BSUL04 with constitutive pathways.
Co-reporter:Di Huang;Guoying Wang;Guanghai Yu
Applied Microbiology and Biotechnology 2012 Volume 94( Issue 3) pp:637-649
Publication Date(Web):2012 May
DOI:10.1007/s00253-011-3773-6
In silico metabolic network models are valuable tools for strain improvement with desired properties. In this work, based on the comparisons of each pathway flux under two different objective functions for the reconstructed metabolic network of Streptomyces roseosporus, three potential targets of zwf2 (code for glucose-6-phosphate hydrogenase), dptI (code for α-ketoglutarate methyltransferase), and dptJ (code for tryptophan oxygenase) were identified and selected for the genetic modifications. Overexpression of zwf2, dptI, and dptJ genes increased the daptomycin concentration up to 473.2, 452.5, and 489.1 mg/L, respectively. Furthermore, co-overexpression of three genes in series resulted in a 34.4% higher daptomycin concentration compared with the parental strain, which ascribed to the synergistic effect of the enzymes responsible for daptomycin biosynthesis. Finally, the engineered strain enhanced the yield of daptomycin up to 581.5 mg/L in the fed-batch culture, which was approximately 43.2% higher than that of the parental strain. These results demonstrated that the metabolic network based on in silico prediction would be accurate, reasonable, and practical for target gene identification and strain improvement.
Co-reporter:Hai-shan Qi;Xing Xin;Shan-shan Li
Biotechnology and Bioprocess Engineering 2012 Volume 17( Issue 4) pp:770-779
Publication Date(Web):2012 August
DOI:10.1007/s12257-012-0114-2
Femtosecond laser irradiation technology was employed for the first time to improve the ascomycin (FK520) yield of Streptomyces hygroscopicus var. ascomyceticus NT2-11, which is an N-methyl-N-nitro-N-nitrosoguanidine (NTG)-induced strain derived from S. hygroscopicus (ATCC14891). The mutant FS35 with high and stable FK520 production capacity was then obtained in the optimal irradiation conditions (25 mW for 6 min) by the Titanium sapphire laser system (810 nm, 76 MHz, 150 fs). The FK520 production capacity of FS35 was 45% higher than that of the parental strain NT2-11. Moreover, under the optimal fermentation conditions, FK520 fermentation titer of FS35 reached 300 mg/L and the intrinsic kinetics of FS35 and NT2-11 were investigated comparatively in 3 phases. The mathematical models provided a good description of FK520 fermentation process for both strains and valuable information for optimizing operation and pilotplant enlargement research. The comparative studies on parameters of the models confirmed the advantages in production and the decrease of substrate inhibition through femtosecond laser irradiation. Therefore, femtosecond laser irradiation provides a promising way to enhance the production of FK520 in S. hygroscopicus.
Co-reporter:Chunsheng Qiu, Jianping Wen, Xiaoqiang Jia
International Journal of Hydrogen Energy 2011 Volume 36(Issue 14) pp:8243-8251
Publication Date(Web):July 2011
DOI:10.1016/j.ijhydene.2011.04.089
Extreme-thermophilic biohydrogen production from distillery wastewater was investigated in batch and continuous-mode operation. Hydrogen-producing mixed culture was enriched by repeated batch cultivations. Effect of temperature and pH on biohydrogen yield was investigated in batch experiments. The highest hydrogen yield of 196. mL/g-volatile solidsaddded (VSadded) was obtained at 70 °C and pH 7.0 in batch culture. Continuous biohydrogen production was performed in CSTR reactor with yield of 172.0 mL/g-VSadded at HRT (hydraulic retention time) of 4 days. The main metabolic products were acetate, lactate, and ethanol. Community structure of hydrogen-producing microflora was investigated by 16S rRNA gene sequence analysis. The microorganisms involved in both batch and continuous-mode operation were similar and hydrogen production was carried out by a group of extreme-thermophilic bacterial species related to Thermotoga, Coprothermobacter, Caldanaerobacter, Thermobrachium, and Caldicellulosiruptor.Highlights► Biohydrogen production from distillery wastewater was investigated and hydrogen yield of 172.0 mL/g-VSadded was obtained in CSTR at 70 °C. ► Hydrogen-producing mixed culture was enriched by repeated batch cultivations. ► Bacterial species related to Thermotoga and Coprothermobacter species were found to be the dominant bacteria in the mixed cunture.
Co-reporter:Shanshan Li;Xiaoqiang Jia
Applied Microbiology and Biotechnology 2011 Volume 91( Issue 3) pp:577-589
Publication Date(Web):2011 August
DOI:10.1007/s00253-011-3280-9
In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P43 was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.
Co-reporter:Guanghai Yu;Xiaoqiang Jia
Applied Biochemistry and Biotechnology 2011 Volume 163( Issue 6) pp:729-743
Publication Date(Web):2011 March
DOI:10.1007/s12010-010-9078-x
To improve the yield of daptomycin by Streptomyces roseosporus, a method of rational screening of He–Ne Laser and N-methyl-N-nitro-N-nitrosoguanidine (NTG)-induced mutants was employed in this work. Under the optimal mutagenesis conditions (NTG of 0.3 mg/L, 40 min; irradiation at 15 mW, 15 min), two steps of combined mutations were conducted. Screening of mutants was done according to the individual resistance to n-decanoic acid and daptomycin. A mutant strain LC-54-16, with the highest daptomycin production ability of 616 mg/L was obtained, which was over 5 times higher than the wild strain LC-52-6. The transcription levels of the main dpt genes in the mutant were approximately five times higher than those in the wild. The superiority of the mutant to the wild strain was further proved by the comparative studies on the kinetics of the mutant and the wild. The decrease of the inhibition of substrate and product was further confirmed, as well. It was concluded that the method of rational screening of He–Ne Laser and NTG-induced mutants could efficiently improve the daptomycin production ability of S. roseosporus.
Co-reporter:Xiaoqiang Jia;Peng Liu;Shuang Li
World Journal of Microbiology and Biotechnology 2011 Volume 27( Issue 9) pp:2117-2124
Publication Date(Web):2011 September
DOI:10.1007/s11274-011-0675-9
Based on its ability to produce lactic acid from glucose in mineral salt medium under anaerobic conditions, genetic modifications on Corynebacterium glutamicum Res 167 were carried out with the aim of producing optical pure D-lactic acid, involving the knockout of L-lactate dehydrogenase gene from C. glutamicum and the heterologous expression of D-lactate dehydrogenase gene from Lactobacillus bulgaricus into C. glutamicum. D-lactic acid production of the genetically engineered strain C. glutamicum Res 167Δldh/ldhA was 17.92 g/l (optical purity higher than 99.9%) after 16 h fermentation, which was 32.25% higher than the lactic acid production of the parental strain.
Co-reporter:Guanghai Yu;Xiaoqiang Jia
World Journal of Microbiology and Biotechnology 2011 Volume 27( Issue 8) pp:1859-1868
Publication Date(Web):2011 August
DOI:10.1007/s11274-010-0644-8
The effects of eight cofactors of enzymes on daptomycin production were investigated in this work, which included nicotinic acid (VPP), riboflavin (VB2), heme, thiamine (VB1), biotin (VH), cyanocobalamin (VB12), tetrahydrofolic acid (THF) and pyridoxal 5-phosphate (VB6). The dry cell weight (DCW), consumption of glucose, and daptomycin production were obviously improved when proper amount of exogenous cofactors were supplemented in the medium. The effects of heme, THF, VB12 and VB6 on daptomycin production were especially notable. The daptomycin yield enhanced 363, 104, 53 and 46%, respectively, when optimized amount of these four cofactors were supplemented in the broth. Moreover, the daptomycin yield further increased to 632 mg/l, which was over 4.5-fold higher than that of the control (without cofactors), at 132 h in a 7.5-l fermenter, by supplementation all of the eight cofactors at optimized concentrations (VPP 4 mg/l, VB2 0.5 mg/l, heme 9 mg/l, VB1 0.4 mg/l, VH 0.1 mg/l, VB12 0.04 mg/l, THF 6 mg/l and VB6 0.4 mg/l). Further, the effects of cofactors on the corresponding key enzymes and important intracellular metabolites were studied in order to elucidate the mechanism of enhancement of daptomycin production by manipulation of cofactors concentration in the fermentation culture. It is suggested that this strategy for increasing the daptomycin production in Streptomyces roseosporus LC-51 by manipulation of cofactors concentration in the fermentation culture may provide an alternative approach to enhance the production of metabolites in other Streptomyces.
Co-reporter:Xiaoqiang Jia, Jianping Wen, Xue Wang, Wei Feng and Yan Jiang
Industrial & Engineering Chemistry Research 2009 Volume 48(Issue 9) pp:4514-4529
Publication Date(Web):March 25, 2009
DOI:10.1021/ie800816d
A 3D transient CFD model was developed to simulate the dynamic behaviors of batch phenol biodegradation by immobilized Candida tropicalis in a gas−liquid−solid three-phase airlift loop reactor (ALR), coupling of three-phase fluid flow, species interphase mass transfer, and intrinsic bioreaction, with the bubble size distribution (BSD) determined by the multiple size group (MUSIG) model. Simulation of the time courses of the volume-averaged species mass concentrations in the riser and downcomer of the ALR was performed, whereas the phenol and oxygen concentrations in the liquid phase were validated by corresponding experiments. Source-term comparisons between species interphase mass transfer and bioreaction were done to distinguish the rate-limiting step in the process. Moreover, local transient phenol biodegradation characteristics such as oxygen concentration profiles in the gas, liquid, and solid phases; phenol concentration profiles in the liquid and solid phases; and cell concentration profiles in the solid phase were all predicted reasonably well. Furthermore, the performances of the ALR and a bubble column reactor (BCR) were compared, and the advantage of the former reactor was confirmed.
Co-reporter:Yan Liang;Wenyu Lu
Applied Biochemistry and Biotechnology 2009 Volume 152( Issue 3) pp:440-448
Publication Date(Web):2009 March
DOI:10.1007/s12010-008-8281-5
In this paper, a new spinosad-producing mutant UV-42-13 was obtained by employing rhamnose and sodium propionate resistant selection strategies in series with UV irradiation. Spinosad production of the mutant was 125.3 mg/L, improved 285.5% compared with that of the wild strain (32.5 mg/L).The results of experiment on tolerance of propyl alcohol addition showed that the tolerant ability to precursor was higher. The precursor-resistant ability of the mutant improved through tolerance experiment by adding propyl alcohol, and the spinosad production was greatly increased. The kinetic models for biomass, substrate consumption, and spinosad production of mutant strain and wild strain were studied by conducting batch fermentation in the shaking flask. The result showed that the kinetic models could describe the fermentation process of spinosad producing well.
Co-reporter:Guoying Wang;Hongmei Li
Journal of Industrial Microbiology & Biotechnology 2009 Volume 36( Issue 6) pp:809-814
Publication Date(Web):2009 June
DOI:10.1007/s10295-009-0555-6
Strain Candida albicans PDY-07 was used to study the anaerobic biodegradation of phenol and m-cresol as single and dual substrates in batch cultures. The strain had a higher potential to degrade phenol than m-cresol. The cell growth kinetics of batch cultures with various initial m-cresol concentrations was investigated, and the Haldane kinetic model adequately described the dynamic behavior of cell growth on m-cresol. When cells grew on the mixture of phenol and m-cresol, substrate interactions were observed. Phenol inhibited the utilization of m-cresol; on the other hand, m-cresol also inhibited the degradation of phenol. However, the presence of low-concentration phenol enhanced m-cresol biodegradation; 100 mg/l m-cresol could be completely degraded within a shorter period of time than m-cresol alone in the presence of 150–300 mg/l phenol. The maximum m-cresol biodegradation rate was obtained at the existence of 200 mg/l phenol. Phenol was preferably utilized by the strain as a carbon and energy source. In addition, a sum kinetics model was used to describe the cell growth behavior in binary mixture of phenol and m-cresol, and the interaction parameters were determined. The model adequately predicted the growth kinetics and the interaction between the substrates.
Co-reporter:Wenyu Lu, Jianping Wen, Xiaoqiang Jia, Bing Sun, Yu Chen, Minhui Liu
International Journal of Hydrogen Energy 2008 Volume 33(Issue 1) pp:34-42
Publication Date(Web):January 2008
DOI:10.1016/j.ijhydene.2007.09.010
Enterobacter aerogenes W-23 bacteria exhibiting hydrogen production (HP) ability were exposed to He–Ne laser irradiation (632.8 nm) to improve the HP ability. Upon the optimum irradiation dosage (18 mW for 22.2 min), a stable positive mutant strain E. aerogenes HB-5M was obtained. The maximum specific rate of HP of the mutant strain was 0.042h-1, which was about twice that of the wild strain. It was suggested that the higher HP ability of the mutant strain might be presumably attributed to the enhancement of hydrogenase activity. In addition, the kinetic parameters of cell growth, substrate consumption and HP of the wild strain and its mutant strain were regressed to simulate the fermentation process of E. aerogenes. The simulated results agreed well with the experimental data.
Co-reporter:Wenyu Lu, Jianping Wen, Yu Chen, Bing Sun, Xiaoqiang Jia, Minghui Liu, Qinggele Caiyin
International Journal of Hydrogen Energy 2007 Volume 32(Issue 8) pp:1059-1066
Publication Date(Web):June 2007
DOI:10.1016/j.ijhydene.2006.07.010
An aciduric high-yielding hydrogen yeast named Candida maltosa HY-35 was screened, which can grow and produce hydrogen at pH 1.3. Further research was carried out batchwise to measure the hydrogen-producing ability of a mixed culture of C. maltosa HY-35 and a facultative anaerobe Enterobacter aerogenes W-23. In this method, with the mixed culture of these two strains at 35∘C for 28 h, the hydrogen yield was 1735 ml/l, which was 17.15% and 119.90% higher than those of the monoculture of E. aerogenes W-23 and C. maltosa HY-35, respectively. Meanwhile, the average hydrogen production rate with the mixed culture was 261.1 ml/h/l, which was 7.85% and 146.23% higher than those of the monoculture of E. aerogenes W-23 and C. maltosa HY-35, respectively. The results suggested that mixed culture of these two strains had the synergistic effect on hydrogen production. The optimum fermentation medium and operation conditions for hydrogen production with mixed culture were also investigated.
Co-reporter:Yan Jiang;Li Lan;Zongding Hu
Biodegradation 2007 Volume 18( Issue 6) pp:719-729
Publication Date(Web):2007 December
DOI:10.1007/s10532-007-9100-3
Biodegradation of phenol and 4-chlorophenol (4-cp) using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2,000 mg l−1 phenol alone and 350 mg l−1 4-cp alone within 66 and 55 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Phenol beyond 800 mg l−1 could not be degraded in the presence of 350 mg l−1 4-cp. Comparatively, low-concentration phenol from 100 to 600 mg l−1 supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in the fact that 4-cp biodegradation velocity was higher than that without phenol. And the capacity of C. tropicalis to degrade 4-cp was increased up to 420 mg l−1 with the presence of 100–160 mg l−1 phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.
Co-reporter:Jianping Wen;Qing Yuan;Xueming Zhao;Xianling Liu
Journal of Chemical Technology and Biotechnology 2006 Volume 81(Issue 5) pp:817-822
Publication Date(Web):23 MAR 2006
DOI:10.1002/jctb.1528
An 80 m3 pilot-scale gas–liquid–solid three-phase flow airlift loop bioreactor (ALR) with a low ratio of height to diameter, in which a biological membrane replaced the activated sludge, was used in the nitrifying treatment of a real effluent from a fertilizer plant. The influences of pH value, air influx (Qg) and hydraulic residence time (HRT) on the reductions in chemical oxygen demand (COD) and ammonia nitrogen (NH4-N) were investigated and are discussed herein. The optimum operating conditions were obtained at a pH value of 7.0–8.0, air influx of 500 m3 h−1 and an HRT of 10 h. Under these optimum conditions, the effluent COD and NH4-N were less than 50 mg L−1 and 10 mg L−1 for a 40-day run, which were far below the primary discharge standard for the chemical fertilizer industry in the People's Republic of China (COD ≤ 100 mg L−1 and NH4-N ≤ 40 mg L−1). Furthermore, this pilot-scale airlift loop bioreactor generated only one-quarter of the sludge waste compared with the traditional activated sludge process. Copyright © 2006 Society of Chemical Industry
Co-reporter:Wen Jianping;Yuan Qing;Pan Lei;Mao Guozhu
Journal of Chemical Technology and Biotechnology 2005 Volume 80(Issue 7) pp:767-771
Publication Date(Web):14 MAR 2005
DOI:10.1002/jctb.1231
Aerobic treatment of refinery wastewater was carried out in a 200 dm3 gas–liquid–solid three-phase flow airlift loop bioreactor, in which a biological membrane replaced the activated sludge. The influences of temperature, pH, gas–liquid ratio and hydraulic residence time on the reductions in chemical oxygen demand (COD) and NH4-N were investigated and discussed. The optimum operation conditions were obtained as temperature of 25–35 °C, pH value of 7.0–8.0, gas–liquid ratio of 50 and hydraulic residence time of 4 h. The radial and axial positions had little influence on the local profiles of COD and NH4-N. Under the optimum operating conditions, the effluent COD and NH4-N were less than 100 mg dm−3 and 15 mg dm−3 respectively for more than 40 days, satisfying the national primary discharge standard of China (GB 8978-1996). Copyright © 2005 Society of Chemical Industry
Co-reporter:J. P. Wen;X. Q. Jia;W. Feng
Chemical Engineering & Technology 2005 Volume 28(Issue 1) pp:
Publication Date(Web):10 JAN 2005
DOI:10.1002/ceat.200407034
Hydrodynamic and mass transfer characteristics such as liquid velocity, gas holdup and gas-liquid volumetric mass transfer coefficient in the riser and downcomer of the gas-liquid-solid three-phase internal loop airlift reactors with nanometer solid particles were investigated. A mathematical model for the description of flow behavior and gas-liquid mass transfer of those reactors was also developed. The predicted results of this model agreed well with the experimental data.
Co-reporter:Chang-Lin Wang;Yan-Xin Liu;Jian-Ping Wen
Journal of Chemical Technology and Biotechnology 2004 Volume 79(Issue 4) pp:403-406
Publication Date(Web):17 MAR 2004
DOI:10.1002/jctb.1001
A new process for D-glucose hydrogenation in 50 wt% aqueous solution, into sorbitol in a 1.5 m3 gas–liquid–solid three-phase flow airlift loop reactor (ALR) over Raney Nickel catalysts has been developed. Five main factors affecting the reaction time and molar yield to sorbitol, including reaction temperature (TR), reaction pressure (PR), pH, hydrogen gas flowrate (Qg) and content of active hydrogen, were investigated and optimized. The average reaction time and molar yield were 70 min and 98.6% under the optimum operating conditions, respectively. The efficiencies of preparation of sorbitol between the gas–liquid–solid three-phase flow ALR and stirred tank reactor (STR) under the same operating conditions were compared. Copyright © 2004 Society of Chemical Industry
Co-reporter:Haoli Zhou, Wenyu Lu, Jianping Wen, Lijuan Ma
Journal of Molecular Catalysis B: Enzymatic (February 2009) Volume 56(Issues 2–3) pp:136-141
Publication Date(Web):1 February 2009
DOI:10.1016/j.molcatb.2008.02.005
Optimization of bioconversion of 16,17α-epoxyprogesterone by Rhizopus nigricans TJ 108 was investigated by means of uniform design. Batch cell growth and bioconversion kinetics were simulated under optimal conditions. Contois equation was used in the kinetics study of fungal growth on glucose. Simulation of bioconversion process was done at a constant value of substrate concentration according to the reaction mechanism. It was demonstrated that the above model satisfactorily described the kinetic behaviors of cell growth and bioconversion of the filamentous fungi.
Co-reporter:Lijuan Ma, Wenyu Lu, Jianping Wen
Journal of Molecular Catalysis B: Enzymatic (February 2009) Volume 56(Issues 2–3) pp:102-107
Publication Date(Web):1 February 2009
DOI:10.1016/j.molcatb.2008.04.011
Alginate–chitosan shell–core (AC) capsules doped with carbon nanotubes (CNTs) were prepared for lactate dehydrogenase (LDH, EC 1.1.1.27) encapsulation to convert pyruvic acid to lactic acid coupling with the oxidation of NADH to NAD+. LDH was entrapped within the liquid core of the capsules and the CNTs were incorporated in the alginate or chitosan matrices or both. The physical properties of the capsules and the immobilized LDH activity were investigated. The AC capsules doped with CNTs showed better mechanical strength than that without CNTs. The LDH loading efficiency of the AC capsules with CNTs (10 mg/mL) doped in both the shell and the core was 30.7% higher than that without CNTs. The optimal pH value for the bioconversion catalyzed by immobilized LDH was 7.0, lower than that by free LDH (7.5). The optimal temperature was 35 °C for both immobilized and free LDH. Operational stability of the immobilized LDH was greatly improved by doping CNTs in AC capsules. The results showed that this method was efficient for enzyme encapsulation in the biotechnology applications.
Co-reporter:Xinrong Pan, Huanhuan Liu, Jiao Liu, Cheng Wang, Jianping Wen
Bioresource Technology (December 2016) Volume 222() pp:
Publication Date(Web):1 December 2016
DOI:10.1016/j.biortech.2016.09.101
•Furfural seriously inhibited R. oryzae (ATCC 20344) for fumaric acid fermentation.•The metabolic response of R. oryzae to furfural was analyzed by metabolomics.•Potential key phospholipids to resist furfural were identified by lipidomics.•A great increase of fumaric acid titer was achieved by rational feeding.In order to relieve the toxicity of furfural on Rhizopus oryzae fermentation, the molecular mechanism of R. oryzae responding to furfural stress for fumaric acid-production was investigated by omics-based approaches. In metabolomics analysis, 29 metabolites including amino acid, sugars, polyols and fatty acids showed significant changes for maintaining the basic cell metabolism at the cost of lowering fumaric acid production. To further uncover the survival mechanism, lipidomics was carried out, revealing that phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and polyunsaturated acyl chains might be closely correlated with R. oryzae’s adapting to furfural stress. Based on the above omics analysis, lecithin, inositol and soybean oil were exogenously supplemented separately with an optimized concentration in the presence of furfural, which increased fumaric acid titer from 5.78 g/L to 10.03 g/L, 10.05 g/L and 12.13 g/L (increased by 73.5%, 73.8% and 110%, respectively). These findings provide a methodological guidance for hemicellulose-fumaric acid development.
Co-reporter:Lijuan Ma, Jianping Wen, Wenyu Lu, Qinggele Caiyin, Yan Liang
Enzyme and Microbial Technology (4 February 2008) Volume 42(Issue 3) pp:235-241
Publication Date(Web):4 February 2008
DOI:10.1016/j.enzmictec.2007.09.014
An efficient carrier, double-walled carbon nanotube-doped alginate gel (DWCNT-ALG), was researched for lactate dehydrogenase (LDH, EC 1.1.1.27) immobilization in this study. LDH was pre-adsorbed on DWCNTs and then they were encapsulated in the ALG matrices followed by Ca2+ cross-linking to form LDH-DWCNT-ALG biocomposites. Scanning and transmission electron microscope images were used to characterize the LDH-DWCNT-ALG biocomposites and the DWCNTs before and after being adsorbed by LDH. LDH leakage from LDH-DWCNT-ALG biocomposites was remarkably reduced about 61.7% compared with LDH-ALG biocomposites. The results of the optimum pH and temperature of the reaction catalyzed by immobilized LDH showed that higher activity was obtained by using LDH-DWCNT-ALG biocomposites. Operational and storage stabilities of immobilized LDH had been greatly improved after being immobilized in LDH-DWCNT-ALG hybrid biocomposites. This work showed both the biocompatibility of the organic component and the higher surface energy of nanotubes in enzyme immobilization.
Co-reporter:Lijuan Ma, Wenyu Lu, Zhendong Xia, Jianping Wen
Biochemical Engineering Journal (15 March 2010) Volume 49(Issue 1) pp:61-67
Publication Date(Web):15 March 2010
DOI:10.1016/j.bej.2009.11.011
Co-reporter:Cheng Wang, Jiao Liu, Huanhuan Liu, Junhua Wang, Jianping Wen
Biochemical Engineering Journal (15 July 2017) Volume 123() pp:45-56
Publication Date(Web):15 July 2017
DOI:10.1016/j.bej.2017.03.017
•FK506 fermentation could be predicted accurately by GS-DFBA in different cultural conditions.•Lots of uncover targets hidden in complex interconnectedness of central metabolism was predicted successfully.•FK506 production was improved effectively in the engineered strain.•This strategy has a huge potential to titer improvement of other products.FK506 is a 23-membered polyketide macrolide with a good immunosuppressant activity for the treatment of autoimmune diseases. However, the low fermentation yield is a bottleneck to limit its further industrialization and clinical applications. To address this issue, a genome-scale dynamic flux balance analysis (GS-DFBA) model was developed to seek for the bottlenecks of FK506 biosynthesis. After validating under different cultural conditions, lots of targets were predicted successfully with help of Minimization of Metabolic Adjustment (MOMA). Specifically, many uncover targets that heretofore had remained hidden in the complex interconnectedness of central metabolism had also shown significant effects on FK506 accumulation. To validate the model predictions, four identified targets (gcdh, tktB, msdh and ask gene) were engineered in Streptomyces tsukubaensis NRRL18488. All of the engineered strains showed a higher FK506 production, compared with the parent strain. Finally, the best strain HT-△gcdh-tktB/msdh/ask with gcdh-deletion and tktB-, msdh- and ask-overexpression could produce 126.61 ± 4.66 mg/L FK506, 129.8% higher than that of the wild-type strain (55.1 ± 3.83 mg/L). Results demonstrated that the GS-DFBA model could provide an effective approach in engineering S. tsukubaensis NRRL18488 for FK506 overproduction. This strategy developed here could also be extended to titer improvement of other products.
Co-reporter:Xue Wang, Jianping Wen, Xiaoqiang Jia
Biochemical Engineering Journal (15 December 2009) Volume 48(Issue 1) pp:42-50
Publication Date(Web):15 December 2009
DOI:10.1016/j.bej.2009.08.004
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