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Advances in engineered microorganisms for improving metabolic conversion via microgravity effects

Advances in engineered microorganisms for improving metabolic conversion via microgravity effects ADDENDUM Bioengineered 6:4, 251--255; July/August 2015; © 2015 Taylor & Francis Group, LLC Advances in engineered microorganisms for improving metabolic conversion via microgravity effects Jie Huangfu, Genlin Zhang, Jun Li, and Chun Li* School of Life Science; Beijing Institute of Technology; Beijing, China s an extreme and unique environ- highly dependent upon chemical and Ament, microgravity has significant physical environmental parameters, the effects on microbial cellular processes, changes of environmental factors such as such as cell growth, gene expression, nat- temperature, osmolality and oxygen avail- ural pathways and biotechnological prod- ability can seriously affect the physiologi- ucts. Application of microgravity effects cal characteristics and gene expression of to identify the regulatory elements in engineered microorganisms. reengineering microbial hosts will draw To overcome the problems of high cost much more attention in further research. and long spaceflight duration, the special- In this commentary, we discuss the ized ground based bioreactors were microgravity effects in engineered micro- designed to simulate weightlessness organisms for improving metabolic con- aspects in the laboratory, which were version, including cell growth kinetics, described as low shear modeled micro- antimicrobial susceptibility, resistance to gravity. Simulated microgravity (SMG) is stresses, secondary metabolites produc- characterized by decreased gravitational tion, recombinant protein production force and significant reduction in fluid and enzyme activity, as well as gene shear due to absent of convection currents. expression changes. Application of The sedimentation, which leads to the dif- microgravity effects in engineered micro- ferent dispersion of nutrients and wastes organisms could provide valuable plat- within the vessel, is prevented under form for innovative approaches in microgravity. SMG condition can be bioprocessing technology to largely modeled by special bioreactors in ground- improve the metabolic conversion effi- based experiments. One such bioreactor is cacy of biopharmaceutical products Rotary Cell Culture System (RCCS, Synthecon Inc., NASA). Microgravity presents a novel condition to study meta- Keywords: bioprocessing technology, bolic conversion in engineered microor- Commentary cellular process, engineered microorgan- ganisms. Cells and other molecules are isms, microgravity effects, metabolic kept suspending in a fluid medium with- The use of engineered microorganisms conversion out imparting significant shear forces that to produce primary or secondary metabo- *Correspondence to: Chun Li; Email: lichun@bit.edu.cn accompany stirred terrestrial systems. Pre- lites is becoming more common in bio- vious studies showed that microgravity Submitted: 05/04/2015 processing technology. A large of conditions have significant effects on Revised: 05/20/2015 chemicals, pharmaceuticals, biofuels and numerous microbial cellular processes. agricultural compounds have been pro- Accepted: 05/26/2015 duced at high enough efficiencies through http://dx.doi.org/10.1080/21655979.2015.1056942 metabolically engineered microorganisms, Improvement of microbial of which Escherichia coli, S. cerevisiae and characteristics using microgravity efficacy Addendum to: Huangfu J, Qi F, Liu H, Zou H, P. pastoris are widely used for expressing Most studies suggested that micrograv- Ahmed MS, Li C. Novel helper factors influencing recombinant protein production in Pichia pasto- therapeutic proteins and industrial ity effects were advantageous for microbial 3-7 ris based on proteomic analysis under simulated enzymes. However, largely obtaining growth. Under microgravity, microor- microgravity. Appl Microbiol Biotechnol. 2015; amounts of products is still a major bottle- ganisms show a shortened lag phase, 99(2):653-65; PMID: 25359479; http://dx.doi.org/ neck for efficiently commercial promo- an increased exponential growth rate 10.1007/s00253-014-6175-8. tion. Modern bioprocessing technology is and a higher final cell count during the www.tandfonline.com Bioengineered 251 17 the space microgravity condition. The gramicidins (GS) produced by Bacillus brevis was higher when cultured in simu- lated microgravity bioreactor of RWV than in shaking flask. Zhang L et al. uti- lized the diamagnetic levitation to simu- late an altered gravity environment and found that the secondary metabolite pro- duced by Streptomyces avermitilis was increased at this culturing condition. The dramatic shift in the site of MccB17 accumulation from E. coli ZK 650 to extracellular fluid when the cell growth took place in the low shear stress charac- teristic of simulated microgravity bioreac- tors. Importantly, the question of whether microgravity effects will similarly enhance secondary metabolites produc- tion have been raised. The specific mecha- nisms responsible for antibiotic production under microgravity need to be elucidated. In recent years, synthetic biology pro- vides a significant driving force to develop complex natural products and drugs origi- Figure 1. (a). Changes of E. coli growth under SMG and NG conditions in RCCSs. (b). Resistance of E. nated from engineered microorganisms. coli to antibiotics under SMG and NG conditions in RCCSs. (c). Expression efficiency of the recombi- nant PGUS-E at 19 C under SMG and NG conditions in RCCSs. The natural biosynthetic pathways show surprising levels of built-in modularity at many levels, which can be exploited by stationary phase in comparison to normal greatly potential in conventional biopro- synthetic biology approaches. However, gravity (NG) that are generated by the cessing techniques, and ultimately can the high-value chemicals production from bioreactor. The growth of E. coli cultured lower the industrial production cost. microbial engineering is still much lower under SMG and NG conditions were Microorganisms exposed to microgravity than that from utilizing chemical meth- exhibited in Figure 1a. The increased environment are significantly more resis- ods. The application of engineered optical density of microorganisms cul- tant to the antibiotic agents due to the microorganisms is constrained for the rea- tured under microgravity might be caused enhanced biofilms. The explanations son of intracellular biotechnological prod- by the increased cell size and number. can be used to construct the antibiotic ucts. Microgravity effects can be Microgravity effects also disturb spatially producing microorganisms with improv- introduced to improve metabolic conver- programmed budding patterns, generating ing biomass or reducing drug resistance of sion efficacy in bioprocessing technology. strain dependent growth differences in superbugs. yeast colonies on semi-solid medium. Tuning recombinant protein Furthermore, microgravity effects were Regulation of secondary metabolites expression by microgravity reported to affect other distinct physiolog- production by microgravity It is widely used for expressing pro- ical changes including resistance to radia- Several researches revealed that micro- karyotic and eukaryotic origin proteins tion, susceptibility to osmotic, thermal gravity effects could inhibit microbial sec- using engineered microorganisms. How- and acidic stress, effective substrates utili- ondary metabolism or have no effects, i.e. ever, there is evidence of major bottle- 10 11 zation and antibiotics resistance. The the b-lactam antibiotics produced by necks to primarily obtain large amounts 13,14 antibiotics resistance of E. coli cultured Streptomyces clavuligerus, the microcin of functional proteins. Some studies dem- 13,17 under SMG and NG conditions were B17 produced by E. coli and the rapa- onstrated that the expression of recombi- exhibited in Figure 1b. The improvement mycin produced by Streptomyces hygrosco- nant proteins from engineered 13,15 of these performances is thought to be rel- picus. Only a few reports microorganisms was changed when the evant to reduced extracellular mass trans- demonstrated that microgravity effects cells were cultured under microgravity, 20-24 port that occurs in the absence of could enhance the accumulation of sec- compared with normal gravity. The sedimentation and buoyancy-driven con- ondary metabolites. The microbial anti- microgravity effects could promote pro- vection in suspended cultures. The biotic actinomycin D produced by tein expression, such as the expression of unique microgravity effects may have Streptomyces plicatus was enhanced under the recombinant b-galactosidase and the 252 Bioengineered Volume 6 Issue 4 Figure 2. Main cellular changes in protein and gene levels upon a shift from SMG to NG in RCCSs. Upward arrows indicated higher abundance under SMG conditions. glycodelin in human cells, the recombi- coli was 3.7 times higher under micro- transcriptional and translational levels. nant b-glucuronidase in E. coli (Fig. 1c) gravity than under which of normal grav- These identified elements were stress- 21,22 28-29 30 and P. pastoris and the human mono- ity (Table 1). responsive genes, miRNAs and clonal antibody in Sp2/0 myeloma mouse The further investigation of mecha- transcriptional factors. Mangala LS cell line. These results suggested that nisms through which microbial cells et al. explored the changes in global microgravity effects could be used for sense microgravity effects signals can expression of microRNA (miR-150, miR- efficient production of recombinant pro- give rise to advanced knowledge about 34a, miR-423-5p, miR-22, miR-141, teins from engineered microorganisms. microbial process. The effects of micro- miR-618, and miR-222) and related tran- Interestingly, the enzyme catalysis process gravity can potentially be used in cata- scription factors including EGR2, ETS1, 25-27 30 was also changed under microgravity. lytic bioprocess. Additionally, the and c-REL under SMG conditions. The activities of intracellular antioxidant research can also elevate the amount of Global microarray and proteomic analyses enzymes, superoxide dismutase and cata- recombinant proteins produced by engi- revealed that 167 transcripts and 73 pro- lase, were higher under space micrograv- neered microorganisms. teins changed expression with the con- ity condition. Qi et al. revealed the served RNA-binding protein Hfq significant alterations in catalytic proper- identified as a likely global regulator Further application of microgravity ties of the recombinant b-glucuronidase involved in the response of Salmonella effects in response to low-shear modeled micro- typhimurium to space microgravity and The physiological responses of cells to gravity effect. The catalytic efficiency ground-based microgravity culture external stress of microgravity effects are (kcat/Km) of this enzyme expressed by E. model. Qi et al. found that the up- related with significantly changed genes at Table 1. Kinetic constants of the recombinant PGUS expressed by E. coli under SMG and NG conditions ¡1 ¡1 ¡1 ¡1 ¡1 PGUS eexpression Vmax(umol min mg ) Km (mM) Kcat (s ) Kcat/Km (mM s ) SMG 147.20 104.61 415.4 3.97 NG 7.29 12.01 21.5 1.79 www.tandfonline.com Bioengineered 253 Figure 3. Approaches of using microgravity effects in bioprocessing technology to improve biopharmaceutical products yields in the engineered microorganisms. regulated genes of methanol metabolism environmental changes and help to Conclusion and carbohydrate metabolic process, cell investigate potential synergetic actions of Studies concerning the influence of the redox homeostasis and oxidative stress, trans- individual helper targets for cell engi- microgravity effects on microbial cells lation and protein folding-related, and pro- neering. Notably, microgravity effects may receive much attention. Utilizing tein transportation were contributed to can help to obtain more accurate data microgravity effects combined with sys- enhancing production and secretion of the needed for the improvement of certain tems biotechnology based strategy, and recombinant protein from P. pastoris under ground-based processes and products. synthetic biology techniques to find novel SMG condition. It was observed that Huangfu et al. examined the proteomic SMG had a significant effect on P. pastoris profiling of the recombinant P. pastoris target genes will boost metabolic conver- molecular progress (Fig. 2). Bradamante grew under simulated microgravity and sion in bioprocessing technology. et al. demonstrated the cells developed a identified some potential helper genes “multiple ways oxidative stress response” to which could be used for strain improve- Disclosure of Potential Conflicts of Interest prevent oxidative damage created by the ment. Overexpressing the gene encoding microgravity effects. The regulation of the detoxifying enzyme of thiol peroxidase No potential conflicts of interest was pathways appeared to be differentially con- was proved to efficiently counteract oxi- disclosed. trolled during spaceflight and SMG environ- dative stress arising from heterologous Funding ment,comparedtoconventional culture. protein production and improve the The present researches are only at the very productivity of recombinant proteins in This work was financially supported by beginning of clarifying the role of micro- methylotrophic P. pastoris. The utiliza- the National Natural Science Foundation gravity effects at the molecular levels. The tion of deeply “omics” analyzing plat- of China (21425624). important mechanisms have not been forms for engineered microorganism unraveled. under microgravity condition to References The analysis of omic-data can provide improve the metabolic conversion effi- 1. Baumann K, Carnicer M, Dragosits M, Graf AB, Stadl- important platforms to define the molec- cacy of biopharmaceutical products is an mann J, Jouhten P, Maaheimo H, Gasser B, Albiol J, ular mechanisms of response to attractive field (Fig. 3). Mattanovich D, et al. A multi-level study of 254 Bioengineered Volume 6 Issue 4 recombinant Pichia pastoris in different oxygen condi- 13. Demain AL, Fang A. Secondary metabolism in simu- sp. PCC 7120 (Cyanobacterium) under simulated tions. BMC Syst Biol 2010; 22(4):141; lated microgravity. Chem Rec 2001; 1:333-346. microgravity. Acta Astronaut 2004; 55(11):953-7; PMID:20969759; http://dx.doi.org/10.1186/1752- PMID:11893073 PMID:15806733; http://dx.doi.org/10.1016/j. 0509-4-141 14. Fang A, Pierson DL, Mishra SK, Koenig DW, Demain actaastro.2004.04.014 2. Klaus DM. Microgravity and its implication for fer- AL. Secondary metabolism in simulated microgravity: 26. Gaubin Y, Prevost MC, Cariven C, Pianezzi B, Planel mentation biotechnology. Trends Biotechnol 1998; 16 b-lactam production by Streptomyces clavuligerus. J Ind H, Soleilhavoup JP. Enzyme activities and membrane (9):369-73; PMID:9776612 Microbiol Biotechnol 1997; 18(1):22-5; lipids in Artemia cysts after a long duration space. Adv 3. Kim HW, Matin A, Rhee MS. Microgravity alters the PMID:9079284 Space Res 1996; 18(12):221-227; http://dx.doi.org/ physiological characteristics of Escherichia coli O157: 15. Fang A, Pierson DL, Mishra SK, Demain AL. Growth 10.1016/0273-1177(96)00043-9 H7 ATCC 35150, ATCC 43889, and ATCC 43895 of Streptomyces hygroscopicus in rotating-wall bioreactor 27. Qi F, Dai D, Liu Y, Kaleem I, Li C. Effects of low-shear under different nutrient conditions. Appl Environ under simulated microgravity inhibits rapamycin pro- modeled microgravity on the characterization of recom- Microbiol 2014; 80(7):2270-8; PMID:24487539; duction. Appl Microbiol Biotechnol 2000; 54(1):33-6; binant b-D-glucuronidase expressed in Pichia pastoris. http://dx.doi.org/10.1128/AEM.04037-13 PMID:10952002 Appl Biochem Biotechnol 2011; 163(1):162-72; 4. Arunasri K, Adil M, Venu Charan K, Suvro C, Hima- 16. Benoit MR, Li W, Stodieck LS, Lam KS, Winther CL, PMID:20607443; http://dx.doi.org/10.1007/s12010- bindu Reddy S, Shivaji S. Effect of simulated micro- Roane TM, Klaus DM. Microbial antibiotic produc- 010-9025-x gravity on E. coli K12 MG1655 growth and gene tion aboard the International Space Station. Appl 28. Sheehan KB, McInnerney K, Purevdorj-Gage B, Alten- expression. PLoS One 2013; 8(3):e57860; Microbiol Biotechnol 2006; 70(4):403-11; burg SD, Hyman LE. Yeast genomic expression pat- PMID:23472115; http://dx.doi.org/10.1371/journal. PMID:16091928 terns in response to low-shear modeled microgravity. pone.0057860 17. Fang A, Pierson DL, Mishra SK, Demain AL. Relief BMC Genomics 2007; 8:3; PMID:17201921; http:// 5. Raja V, Eric M, Laura L. Changes in gene expression of from glucose interference in microcin B17 biosynthesis dx.doi.org/10.1186/1471-2164-8-3 E. coli under conditions of modeled reduced gravity. by growth in a rotating-wall bioreactor. Lett Appl 29. Crabbe A, Schurr MJ, Monsieurs P, Morici L, Schurr J, Microgravity Sci Technol 2008; 20:41-57; Microbiol 2000; 31(1):39-41; PMID:10886612 Wilson JW, Ott CM, Tsaprailis G, Pierson DL, Stefa- PMID:17343762 18. Fang A, Pierson DL, Koenig DW, Mishra SK, Demain nyshyn-Piper H, et al. Transcriptional and proteomic 6. Johanson K, Allen PL, Lewis F, Cubano LA, Hyman AL. Effect of simulated microgravity and shear stress on responses of Pseudomonas aeruginosa PAO1 to space- LE, Hammond TG. Saccharomyces cerevisiae gene microcin B7 production by Escherichia coli and on its flight conditions involve Hfq regulation and reveal a expression changes during rotating wall vessel suspen- excretion into the medium. Appl Environ Microbiol role for oxygen. Appl Environ Microbiol 2011; 77 sion culture. J Appl Physiol (1985) 2002; 93(6):2171- 1997; 63(10):4090-2; PMID:9327574 (4):1221-30; PMID:21169425; http://dx.doi.org/ 80; PMID:12391061 19. Liu M, Gao H, Shang P, Zhou X, Ashforth E, Zhuo Y, 10.1128/AEM.01582-10 7. Van Mulders SE, Stassen C, Daenen L, Devreese B, Chen D, Ren B, Liu Z, Zhang L. Magnetic field is the 30. Mangala LS, Zhang Y, He Z, Emami K, Ramesh GT, Siewers V, van Eijsden RG, Nielsen J, Delvaux FR, dominant factor to induce the response of Streptomyces Story M, Rohde LH, Wu H. Effects of simulated Willaert R. The influence of microgravity on invasive avermitilis in altered gravity simulated by diamagnetic microgravity on expression profile of microRNA in growth in Saccharomyces cerevisiae. Astrobiology 2011; levitation. PLoS One 2011; 6(10):e24697; human lymphoblastoid cells. J Biol Chem 2011; 286 11(1):45-55; PMID:21345087; http://dx.doi.org/ PMID:22039402; http://dx.doi.org/10.1371/journal. (37):32483-90; PMID:21775437; http://dx.doi.org/ 10.1089/ast.2010.0518 pone 10.1074/jbc.M111.267765 8. Kobayashi Y, Narumi I, Satoh K, Funayama T, Kikuchi 20. Stephen N. Rotary bioreactor for recombinant protein 31. Wilson JW, Ott CM, H€oner zu Bentrup K, Ramamur- M, Kitayama S, Watanabe H. Radiation response production. Cell Technology for Cell Products 2007; thy R, Quick L, Porwollik S, Cheng P, McClelland M, mechanisms of the extremely radioresistant bacterium 3:567-569; http://dx.doi.org/10.1007/978-1-4020- Tsaprailis G, Radabaugh T, et al. Space flight alters Deinococcus radiodurans. Biol Sci Space 2004; 18 5476-1_98 bacterial gene expression and virulence and reveals a (3):134-5; PMID:15858357 21. Xiang L, Qi F, Dai D, Li C, Jiang Y. Simulated micro- role for global regulator Hfq. Proc Nat Acad Sci U S A 9. Rosenzweig JA, Ahmed S, Eunson J Jr, Chopra AK. gravity affects growth of E.coli and recombinant beta- 2007; 104(41):16299-304; PMID:17901201 Low-shear force associated with modeled microgravity D-glucuronidase production. Appl Biochem Biotechnol 32. Qi F, Wang C, Liu Y, Kaleem I, Li Q, Li C. Transcrip- and spaceflight does not similarly impact the virulence 2010; 162(3):654-61; PMID:19921492; http://dx.doi. tional profiling of protein expression related genes of P. of notable bacterial pathogens. Appl Microbiol Biotech- org/10.1007/s12010-009-8836-0 pastoris under simulated microgravity. PLoS One 2011; nol 2014; 98(21):8797-807; PMID:25149449; http:// 22. Qi F, Imdad K, Lv B, Guo X, Li C. Enhancement of 6(11):e26613; PMID:3206813; http://dx.doi.org/ dx.doi.org/10.1007/s00253-014-6025-8 recombinant b-D-glucuronidase production under 10.1371/journal.pone.0026613 10. Brown RB, Klaus D, Todd P. Effects of space flight, cli- low-shear modeled microgravity in P.pastoris. J Chem 33. Bradamante S, Villa A, Versari S, Barenghi L, Orlandi norotation, and centrifugation on the substrate utiliza- Technol Biot 2011; 86(4):505-511; http://dx.doi.org/ I, Vai M. Oxidative stress and alterations in actin cyto- tion efficiency of E. coli. Microgravity Sci Technol 10.1002/jctb.2541 skeleton trigger glutathione efflux in Saccharomyces cere- 2002; 13(4):24-9; PMID:12521048 23. Foster LJ, Catzel D, Atwa S, Zarka M, Mahler SM. visiae. Biochim Biophys Acta 2010; 1803(12):1376-85; 11. Searles SC, Woolley CM, Petersen RA, Hyman LE, Increase in synthesis of human monoclonal antibodies PMID:20708643; http://dx.doi.org/10.1016/j. Nielsen-Preiss SM. Modeled microgravity increases fila- by transfected Sp2/0 myeloma mouse cell line under bbamcr.2010.07.007 mentation, biofilm formation, phenotypic switching, conditions of microgravity. Biotechnol Lett 2003; 25 34. HuangfuJ,QiF,Liu H, ZouH,Ahmed MS,LiC. and antimicrobial resistance in Candida albicans. Astro- (15):1271-4. PMID:14514080 Novel helper factors influencing recombinant pro- biology 2011; 11(8):825-36; PMID:21936634; http:// 24. Boyle D, Montelone B, Cornejo A, Takemoto L. tein production in Pichia pastoris based on proteo- dx.doi.org/10.1089/ast.2011.0664 Effects of Microgravity upon Growth, Morphology, mic analysis under simulated microgravity. Appl 12. Breitling R, Takano E. Synthetic biology advances for and Expression of Recombinant Protein in E. coli. Cos- Microbiol Biotechnol 2015; 99(2):653-65; pharmaceutical production. Curr Opin Biotechnol mic Research 1995; 34:609 PMID:25359479; http://dx.doi.org/10.1007/ 2015; 35C:46-51; PMID:25744872; http://dx.doi.org/ 25. Li GB, Liu YD, Wang GH, Song LR. Reactive oxygen s00253-014-6175-8 10.1016/j.copbio species and antioxidant enzymes activity of Anabaena www.tandfonline.com Bioengineered 255 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioengineered Taylor & Francis

Advances in engineered microorganisms for improving metabolic conversion via microgravity effects

Bioengineered , Volume 6 (4): 5 – Jul 4, 2015

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Abstract

ADDENDUM Bioengineered 6:4, 251--255; July/August 2015; © 2015 Taylor & Francis Group, LLC Advances in engineered microorganisms for improving metabolic conversion via microgravity effects Jie Huangfu, Genlin Zhang, Jun Li, and Chun Li* School of Life Science; Beijing Institute of Technology; Beijing, China s an extreme and unique environ- highly dependent upon chemical and Ament, microgravity has significant physical environmental parameters, the effects on microbial cellular processes, changes of environmental factors such as such as cell growth, gene expression, nat- temperature, osmolality and oxygen avail- ural pathways and biotechnological prod- ability can seriously affect the physiologi- ucts. Application of microgravity effects cal characteristics and gene expression of to identify the regulatory elements in engineered microorganisms. reengineering microbial hosts will draw To overcome the problems of high cost much more attention in further research. and long spaceflight duration, the special- In this commentary, we discuss the ized ground based bioreactors were microgravity effects in engineered micro- designed to simulate weightlessness organisms for improving metabolic con- aspects in the laboratory, which were version, including cell growth kinetics, described as low shear modeled micro- antimicrobial susceptibility, resistance to gravity. Simulated microgravity (SMG) is stresses, secondary metabolites produc- characterized by decreased gravitational tion, recombinant protein production force and significant reduction in fluid and enzyme activity, as well as gene shear due to absent of convection currents. expression changes. Application of The sedimentation, which leads to the dif- microgravity effects in engineered micro- ferent dispersion of nutrients and wastes organisms could provide valuable plat- within the vessel, is prevented under form for innovative approaches in microgravity. SMG condition can be bioprocessing technology to largely modeled by special bioreactors in ground- improve the metabolic conversion effi- based experiments. One such bioreactor is cacy of biopharmaceutical products Rotary Cell Culture System (RCCS, Synthecon Inc., NASA). Microgravity presents a novel condition to study meta- Keywords: bioprocessing technology, bolic conversion in engineered microor- Commentary cellular process, engineered microorgan- ganisms. Cells and other molecules are isms, microgravity effects, metabolic kept suspending in a fluid medium with- The use of engineered microorganisms conversion out imparting significant shear forces that to produce primary or secondary metabo- *Correspondence to: Chun Li; Email: lichun@bit.edu.cn accompany stirred terrestrial systems. Pre- lites is becoming more common in bio- vious studies showed that microgravity Submitted: 05/04/2015 processing technology. A large of conditions have significant effects on Revised: 05/20/2015 chemicals, pharmaceuticals, biofuels and numerous microbial cellular processes. agricultural compounds have been pro- Accepted: 05/26/2015 duced at high enough efficiencies through http://dx.doi.org/10.1080/21655979.2015.1056942 metabolically engineered microorganisms, Improvement of microbial of which Escherichia coli, S. cerevisiae and characteristics using microgravity efficacy Addendum to: Huangfu J, Qi F, Liu H, Zou H, P. pastoris are widely used for expressing Most studies suggested that micrograv- Ahmed MS, Li C. Novel helper factors influencing recombinant protein production in Pichia pasto- therapeutic proteins and industrial ity effects were advantageous for microbial 3-7 ris based on proteomic analysis under simulated enzymes. However, largely obtaining growth. Under microgravity, microor- microgravity. Appl Microbiol Biotechnol. 2015; amounts of products is still a major bottle- ganisms show a shortened lag phase, 99(2):653-65; PMID: 25359479; http://dx.doi.org/ neck for efficiently commercial promo- an increased exponential growth rate 10.1007/s00253-014-6175-8. tion. Modern bioprocessing technology is and a higher final cell count during the www.tandfonline.com Bioengineered 251 17 the space microgravity condition. The gramicidins (GS) produced by Bacillus brevis was higher when cultured in simu- lated microgravity bioreactor of RWV than in shaking flask. Zhang L et al. uti- lized the diamagnetic levitation to simu- late an altered gravity environment and found that the secondary metabolite pro- duced by Streptomyces avermitilis was increased at this culturing condition. The dramatic shift in the site of MccB17 accumulation from E. coli ZK 650 to extracellular fluid when the cell growth took place in the low shear stress charac- teristic of simulated microgravity bioreac- tors. Importantly, the question of whether microgravity effects will similarly enhance secondary metabolites produc- tion have been raised. The specific mecha- nisms responsible for antibiotic production under microgravity need to be elucidated. In recent years, synthetic biology pro- vides a significant driving force to develop complex natural products and drugs origi- Figure 1. (a). Changes of E. coli growth under SMG and NG conditions in RCCSs. (b). Resistance of E. nated from engineered microorganisms. coli to antibiotics under SMG and NG conditions in RCCSs. (c). Expression efficiency of the recombi- nant PGUS-E at 19 C under SMG and NG conditions in RCCSs. The natural biosynthetic pathways show surprising levels of built-in modularity at many levels, which can be exploited by stationary phase in comparison to normal greatly potential in conventional biopro- synthetic biology approaches. However, gravity (NG) that are generated by the cessing techniques, and ultimately can the high-value chemicals production from bioreactor. The growth of E. coli cultured lower the industrial production cost. microbial engineering is still much lower under SMG and NG conditions were Microorganisms exposed to microgravity than that from utilizing chemical meth- exhibited in Figure 1a. The increased environment are significantly more resis- ods. The application of engineered optical density of microorganisms cul- tant to the antibiotic agents due to the microorganisms is constrained for the rea- tured under microgravity might be caused enhanced biofilms. The explanations son of intracellular biotechnological prod- by the increased cell size and number. can be used to construct the antibiotic ucts. Microgravity effects can be Microgravity effects also disturb spatially producing microorganisms with improv- introduced to improve metabolic conver- programmed budding patterns, generating ing biomass or reducing drug resistance of sion efficacy in bioprocessing technology. strain dependent growth differences in superbugs. yeast colonies on semi-solid medium. Tuning recombinant protein Furthermore, microgravity effects were Regulation of secondary metabolites expression by microgravity reported to affect other distinct physiolog- production by microgravity It is widely used for expressing pro- ical changes including resistance to radia- Several researches revealed that micro- karyotic and eukaryotic origin proteins tion, susceptibility to osmotic, thermal gravity effects could inhibit microbial sec- using engineered microorganisms. How- and acidic stress, effective substrates utili- ondary metabolism or have no effects, i.e. ever, there is evidence of major bottle- 10 11 zation and antibiotics resistance. The the b-lactam antibiotics produced by necks to primarily obtain large amounts 13,14 antibiotics resistance of E. coli cultured Streptomyces clavuligerus, the microcin of functional proteins. Some studies dem- 13,17 under SMG and NG conditions were B17 produced by E. coli and the rapa- onstrated that the expression of recombi- exhibited in Figure 1b. The improvement mycin produced by Streptomyces hygrosco- nant proteins from engineered 13,15 of these performances is thought to be rel- picus. Only a few reports microorganisms was changed when the evant to reduced extracellular mass trans- demonstrated that microgravity effects cells were cultured under microgravity, 20-24 port that occurs in the absence of could enhance the accumulation of sec- compared with normal gravity. The sedimentation and buoyancy-driven con- ondary metabolites. The microbial anti- microgravity effects could promote pro- vection in suspended cultures. The biotic actinomycin D produced by tein expression, such as the expression of unique microgravity effects may have Streptomyces plicatus was enhanced under the recombinant b-galactosidase and the 252 Bioengineered Volume 6 Issue 4 Figure 2. Main cellular changes in protein and gene levels upon a shift from SMG to NG in RCCSs. Upward arrows indicated higher abundance under SMG conditions. glycodelin in human cells, the recombi- coli was 3.7 times higher under micro- transcriptional and translational levels. nant b-glucuronidase in E. coli (Fig. 1c) gravity than under which of normal grav- These identified elements were stress- 21,22 28-29 30 and P. pastoris and the human mono- ity (Table 1). responsive genes, miRNAs and clonal antibody in Sp2/0 myeloma mouse The further investigation of mecha- transcriptional factors. Mangala LS cell line. These results suggested that nisms through which microbial cells et al. explored the changes in global microgravity effects could be used for sense microgravity effects signals can expression of microRNA (miR-150, miR- efficient production of recombinant pro- give rise to advanced knowledge about 34a, miR-423-5p, miR-22, miR-141, teins from engineered microorganisms. microbial process. The effects of micro- miR-618, and miR-222) and related tran- Interestingly, the enzyme catalysis process gravity can potentially be used in cata- scription factors including EGR2, ETS1, 25-27 30 was also changed under microgravity. lytic bioprocess. Additionally, the and c-REL under SMG conditions. The activities of intracellular antioxidant research can also elevate the amount of Global microarray and proteomic analyses enzymes, superoxide dismutase and cata- recombinant proteins produced by engi- revealed that 167 transcripts and 73 pro- lase, were higher under space micrograv- neered microorganisms. teins changed expression with the con- ity condition. Qi et al. revealed the served RNA-binding protein Hfq significant alterations in catalytic proper- identified as a likely global regulator Further application of microgravity ties of the recombinant b-glucuronidase involved in the response of Salmonella effects in response to low-shear modeled micro- typhimurium to space microgravity and The physiological responses of cells to gravity effect. The catalytic efficiency ground-based microgravity culture external stress of microgravity effects are (kcat/Km) of this enzyme expressed by E. model. Qi et al. found that the up- related with significantly changed genes at Table 1. Kinetic constants of the recombinant PGUS expressed by E. coli under SMG and NG conditions ¡1 ¡1 ¡1 ¡1 ¡1 PGUS eexpression Vmax(umol min mg ) Km (mM) Kcat (s ) Kcat/Km (mM s ) SMG 147.20 104.61 415.4 3.97 NG 7.29 12.01 21.5 1.79 www.tandfonline.com Bioengineered 253 Figure 3. Approaches of using microgravity effects in bioprocessing technology to improve biopharmaceutical products yields in the engineered microorganisms. regulated genes of methanol metabolism environmental changes and help to Conclusion and carbohydrate metabolic process, cell investigate potential synergetic actions of Studies concerning the influence of the redox homeostasis and oxidative stress, trans- individual helper targets for cell engi- microgravity effects on microbial cells lation and protein folding-related, and pro- neering. Notably, microgravity effects may receive much attention. Utilizing tein transportation were contributed to can help to obtain more accurate data microgravity effects combined with sys- enhancing production and secretion of the needed for the improvement of certain tems biotechnology based strategy, and recombinant protein from P. pastoris under ground-based processes and products. synthetic biology techniques to find novel SMG condition. It was observed that Huangfu et al. examined the proteomic SMG had a significant effect on P. pastoris profiling of the recombinant P. pastoris target genes will boost metabolic conver- molecular progress (Fig. 2). Bradamante grew under simulated microgravity and sion in bioprocessing technology. et al. demonstrated the cells developed a identified some potential helper genes “multiple ways oxidative stress response” to which could be used for strain improve- Disclosure of Potential Conflicts of Interest prevent oxidative damage created by the ment. Overexpressing the gene encoding microgravity effects. The regulation of the detoxifying enzyme of thiol peroxidase No potential conflicts of interest was pathways appeared to be differentially con- was proved to efficiently counteract oxi- disclosed. trolled during spaceflight and SMG environ- dative stress arising from heterologous Funding ment,comparedtoconventional culture. protein production and improve the The present researches are only at the very productivity of recombinant proteins in This work was financially supported by beginning of clarifying the role of micro- methylotrophic P. pastoris. The utiliza- the National Natural Science Foundation gravity effects at the molecular levels. The tion of deeply “omics” analyzing plat- of China (21425624). important mechanisms have not been forms for engineered microorganism unraveled. under microgravity condition to References The analysis of omic-data can provide improve the metabolic conversion effi- 1. Baumann K, Carnicer M, Dragosits M, Graf AB, Stadl- important platforms to define the molec- cacy of biopharmaceutical products is an mann J, Jouhten P, Maaheimo H, Gasser B, Albiol J, ular mechanisms of response to attractive field (Fig. 3). Mattanovich D, et al. A multi-level study of 254 Bioengineered Volume 6 Issue 4 recombinant Pichia pastoris in different oxygen condi- 13. Demain AL, Fang A. Secondary metabolism in simu- sp. PCC 7120 (Cyanobacterium) under simulated tions. BMC Syst Biol 2010; 22(4):141; lated microgravity. Chem Rec 2001; 1:333-346. microgravity. Acta Astronaut 2004; 55(11):953-7; PMID:20969759; http://dx.doi.org/10.1186/1752- PMID:11893073 PMID:15806733; http://dx.doi.org/10.1016/j. 0509-4-141 14. Fang A, Pierson DL, Mishra SK, Koenig DW, Demain actaastro.2004.04.014 2. Klaus DM. Microgravity and its implication for fer- AL. Secondary metabolism in simulated microgravity: 26. Gaubin Y, Prevost MC, Cariven C, Pianezzi B, Planel mentation biotechnology. Trends Biotechnol 1998; 16 b-lactam production by Streptomyces clavuligerus. J Ind H, Soleilhavoup JP. Enzyme activities and membrane (9):369-73; PMID:9776612 Microbiol Biotechnol 1997; 18(1):22-5; lipids in Artemia cysts after a long duration space. Adv 3. Kim HW, Matin A, Rhee MS. Microgravity alters the PMID:9079284 Space Res 1996; 18(12):221-227; http://dx.doi.org/ physiological characteristics of Escherichia coli O157: 15. Fang A, Pierson DL, Mishra SK, Demain AL. Growth 10.1016/0273-1177(96)00043-9 H7 ATCC 35150, ATCC 43889, and ATCC 43895 of Streptomyces hygroscopicus in rotating-wall bioreactor 27. Qi F, Dai D, Liu Y, Kaleem I, Li C. Effects of low-shear under different nutrient conditions. Appl Environ under simulated microgravity inhibits rapamycin pro- modeled microgravity on the characterization of recom- Microbiol 2014; 80(7):2270-8; PMID:24487539; duction. Appl Microbiol Biotechnol 2000; 54(1):33-6; binant b-D-glucuronidase expressed in Pichia pastoris. http://dx.doi.org/10.1128/AEM.04037-13 PMID:10952002 Appl Biochem Biotechnol 2011; 163(1):162-72; 4. Arunasri K, Adil M, Venu Charan K, Suvro C, Hima- 16. Benoit MR, Li W, Stodieck LS, Lam KS, Winther CL, PMID:20607443; http://dx.doi.org/10.1007/s12010- bindu Reddy S, Shivaji S. Effect of simulated micro- Roane TM, Klaus DM. Microbial antibiotic produc- 010-9025-x gravity on E. coli K12 MG1655 growth and gene tion aboard the International Space Station. Appl 28. Sheehan KB, McInnerney K, Purevdorj-Gage B, Alten- expression. PLoS One 2013; 8(3):e57860; Microbiol Biotechnol 2006; 70(4):403-11; burg SD, Hyman LE. Yeast genomic expression pat- PMID:23472115; http://dx.doi.org/10.1371/journal. PMID:16091928 terns in response to low-shear modeled microgravity. pone.0057860 17. Fang A, Pierson DL, Mishra SK, Demain AL. Relief BMC Genomics 2007; 8:3; PMID:17201921; http:// 5. Raja V, Eric M, Laura L. Changes in gene expression of from glucose interference in microcin B17 biosynthesis dx.doi.org/10.1186/1471-2164-8-3 E. coli under conditions of modeled reduced gravity. by growth in a rotating-wall bioreactor. Lett Appl 29. Crabbe A, Schurr MJ, Monsieurs P, Morici L, Schurr J, Microgravity Sci Technol 2008; 20:41-57; Microbiol 2000; 31(1):39-41; PMID:10886612 Wilson JW, Ott CM, Tsaprailis G, Pierson DL, Stefa- PMID:17343762 18. Fang A, Pierson DL, Koenig DW, Mishra SK, Demain nyshyn-Piper H, et al. Transcriptional and proteomic 6. Johanson K, Allen PL, Lewis F, Cubano LA, Hyman AL. Effect of simulated microgravity and shear stress on responses of Pseudomonas aeruginosa PAO1 to space- LE, Hammond TG. Saccharomyces cerevisiae gene microcin B7 production by Escherichia coli and on its flight conditions involve Hfq regulation and reveal a expression changes during rotating wall vessel suspen- excretion into the medium. Appl Environ Microbiol role for oxygen. Appl Environ Microbiol 2011; 77 sion culture. J Appl Physiol (1985) 2002; 93(6):2171- 1997; 63(10):4090-2; PMID:9327574 (4):1221-30; PMID:21169425; http://dx.doi.org/ 80; PMID:12391061 19. Liu M, Gao H, Shang P, Zhou X, Ashforth E, Zhuo Y, 10.1128/AEM.01582-10 7. Van Mulders SE, Stassen C, Daenen L, Devreese B, Chen D, Ren B, Liu Z, Zhang L. Magnetic field is the 30. Mangala LS, Zhang Y, He Z, Emami K, Ramesh GT, Siewers V, van Eijsden RG, Nielsen J, Delvaux FR, dominant factor to induce the response of Streptomyces Story M, Rohde LH, Wu H. Effects of simulated Willaert R. The influence of microgravity on invasive avermitilis in altered gravity simulated by diamagnetic microgravity on expression profile of microRNA in growth in Saccharomyces cerevisiae. Astrobiology 2011; levitation. PLoS One 2011; 6(10):e24697; human lymphoblastoid cells. J Biol Chem 2011; 286 11(1):45-55; PMID:21345087; http://dx.doi.org/ PMID:22039402; http://dx.doi.org/10.1371/journal. (37):32483-90; PMID:21775437; http://dx.doi.org/ 10.1089/ast.2010.0518 pone 10.1074/jbc.M111.267765 8. Kobayashi Y, Narumi I, Satoh K, Funayama T, Kikuchi 20. Stephen N. Rotary bioreactor for recombinant protein 31. Wilson JW, Ott CM, H€oner zu Bentrup K, Ramamur- M, Kitayama S, Watanabe H. Radiation response production. Cell Technology for Cell Products 2007; thy R, Quick L, Porwollik S, Cheng P, McClelland M, mechanisms of the extremely radioresistant bacterium 3:567-569; http://dx.doi.org/10.1007/978-1-4020- Tsaprailis G, Radabaugh T, et al. Space flight alters Deinococcus radiodurans. Biol Sci Space 2004; 18 5476-1_98 bacterial gene expression and virulence and reveals a (3):134-5; PMID:15858357 21. Xiang L, Qi F, Dai D, Li C, Jiang Y. Simulated micro- role for global regulator Hfq. Proc Nat Acad Sci U S A 9. Rosenzweig JA, Ahmed S, Eunson J Jr, Chopra AK. gravity affects growth of E.coli and recombinant beta- 2007; 104(41):16299-304; PMID:17901201 Low-shear force associated with modeled microgravity D-glucuronidase production. Appl Biochem Biotechnol 32. Qi F, Wang C, Liu Y, Kaleem I, Li Q, Li C. Transcrip- and spaceflight does not similarly impact the virulence 2010; 162(3):654-61; PMID:19921492; http://dx.doi. tional profiling of protein expression related genes of P. of notable bacterial pathogens. Appl Microbiol Biotech- org/10.1007/s12010-009-8836-0 pastoris under simulated microgravity. PLoS One 2011; nol 2014; 98(21):8797-807; PMID:25149449; http:// 22. Qi F, Imdad K, Lv B, Guo X, Li C. Enhancement of 6(11):e26613; PMID:3206813; http://dx.doi.org/ dx.doi.org/10.1007/s00253-014-6025-8 recombinant b-D-glucuronidase production under 10.1371/journal.pone.0026613 10. Brown RB, Klaus D, Todd P. Effects of space flight, cli- low-shear modeled microgravity in P.pastoris. J Chem 33. Bradamante S, Villa A, Versari S, Barenghi L, Orlandi norotation, and centrifugation on the substrate utiliza- Technol Biot 2011; 86(4):505-511; http://dx.doi.org/ I, Vai M. Oxidative stress and alterations in actin cyto- tion efficiency of E. coli. Microgravity Sci Technol 10.1002/jctb.2541 skeleton trigger glutathione efflux in Saccharomyces cere- 2002; 13(4):24-9; PMID:12521048 23. Foster LJ, Catzel D, Atwa S, Zarka M, Mahler SM. visiae. Biochim Biophys Acta 2010; 1803(12):1376-85; 11. Searles SC, Woolley CM, Petersen RA, Hyman LE, Increase in synthesis of human monoclonal antibodies PMID:20708643; http://dx.doi.org/10.1016/j. Nielsen-Preiss SM. Modeled microgravity increases fila- by transfected Sp2/0 myeloma mouse cell line under bbamcr.2010.07.007 mentation, biofilm formation, phenotypic switching, conditions of microgravity. Biotechnol Lett 2003; 25 34. HuangfuJ,QiF,Liu H, ZouH,Ahmed MS,LiC. and antimicrobial resistance in Candida albicans. Astro- (15):1271-4. PMID:14514080 Novel helper factors influencing recombinant pro- biology 2011; 11(8):825-36; PMID:21936634; http:// 24. Boyle D, Montelone B, Cornejo A, Takemoto L. tein production in Pichia pastoris based on proteo- dx.doi.org/10.1089/ast.2011.0664 Effects of Microgravity upon Growth, Morphology, mic analysis under simulated microgravity. Appl 12. Breitling R, Takano E. Synthetic biology advances for and Expression of Recombinant Protein in E. coli. Cos- Microbiol Biotechnol 2015; 99(2):653-65; pharmaceutical production. Curr Opin Biotechnol mic Research 1995; 34:609 PMID:25359479; http://dx.doi.org/10.1007/ 2015; 35C:46-51; PMID:25744872; http://dx.doi.org/ 25. Li GB, Liu YD, Wang GH, Song LR. Reactive oxygen s00253-014-6175-8 10.1016/j.copbio species and antioxidant enzymes activity of Anabaena www.tandfonline.com Bioengineered 255

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BioengineeredTaylor & Francis

Published: Jul 4, 2015

Keywords: bioprocessing technology; cellular process; engineered microorganisms; microgravity effects; metabolic conversion

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