2014 ; Vol.24-1: 59~69
|Author||Ignacio Poblete-Castro, Andre Luis Rodriguez, Carolyn Ming Chi Lam, Wolfgang Kessler|
|Place of duty||Microbial Drugs Group, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany,Department of Loss Prevention and Environmental Engineering, Universidad Tecnológica Metropolitana, Dieciocho 390, Santiago, Chile|
|Title||Improved Production of Medium-Chain-Length Polyhydroxyalkanoates in Glucose-Based Fed-Batch Cultivations of Metabolically Engineered Pseudomonas putida Strains|
J. Microbiol. Biotechnol.2014 ;
|Abstract||One of the major challenges in metabolic engineering for enhanced synthesis of value-added
chemicals is to design and develop new strains that can be translated into well-controlled
fermentation processes using bioreactors. The aim of this study was to assess the influence of
various fed-batch strategies in the performance of metabolically engineered Pseudomonas
putida strains, Δgcd and Δgcd-pgl, for improving production of medium-chain-length polyhydroxyalkanoates
(mcl-PHAs) using glucose as the only carbon source. First we developed a
fed-batch process that comprised an initial phase of biomass accumulation based on an
exponential feeding carbon-limited strategy. For the mcl-PHA accumulation stage, three
induction techniques were tested under nitrogen limitation. The substrate-pulse feeding was
more efficient than the constant-feeding approach to promote the accumulation of the
desirable product. Nonetheless, the most efficient approach for maximum PHA synthesis was
the application of a dissolved-oxygen-stat feeding strategy (DO-stat), where P. putida Δgcd
mutant strain showed a final PHA content and specific PHA productivity of 67% and
0.83 g·l-1·h-1, respectively. To our knowledge, this mcl-PHA titer is the highest value that has
been ever reported using glucose as the sole carbon and energy source. Our results also
highlighted the effect of different fed-batch strategies upon the extent of realization of the
intended metabolic modification of the mutant strains.|
|Key_word||Dissolved-oxygen-stat, Fed-batch process, Medium-chain-length polyhydroxyalkanoates, Metabolic engineering, Glucose, Pseudomonas putida|
Chen G-Q. 2009. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem. Soc. Rev. 38: 24342446.
del Castillo T, Ramos JL, RodrÃ-guez-Herva JJ, Fuhrer T, Sauer U, Duque E. 2007. Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida:genomic and flux analysis. J. Bacteriol. 189: 5142-5152.
Diniz S, Taciro M, Cabrera Gomez JGr, da Cruz Pradella JG. 2004. High-cell-density cultivation of Pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates. Appl. Biochem. Biotechnol. 119:51-70.
Escapa I, Morales V, Martino V, Pollet E, Averous L, Garcia J, Prieto M. 2011. Disruption of B-oxidation pathway in Pseudomonas putida KT2442 to produce new functionalized PHAs with thioester groups. Appl. Microbiol. Biotechnol. 89:1583-1598.
Follonier S, Panke S, Zinn M. 2011. A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate. Microb. Cell Factor. 10: 25.
Frazzetto G. 2003. White biotechnology. EMBO Rep. 4: 835837.
Fujita Y, Matsuoka H, Hirooka K. 2007. Regulation of fatty acid metabolism in bacteria. Mol. Microbiol. 66: 829-839.
Hartmann R, Hany R, Geiger T, Egli T, Witholt B, Zinn M. 2004. Tailored biosynthesis of olefinic medium-chain-length poly[(r)-3-hydroxyalkanoates] in Pseudomonas putida GPo1 with improved thermal properties. Macromolecules 37: 67806785.
Huijberts GN, Eggink G, de Waard P, Huisman GW, Witholt B. 1992. Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers. Appl. Environ. Microbiol. 58: 536-544.
Kim GJ, Lee IY, Yoon SC, Shin YC, Park YH. 1997. Enhanced yield and a high production of medium-chainlength poly(3-hydroxyalkanoates) in a two-step fed-batch cultivation of Pseudomonas putida by combined use of glucose and octanoate. Enzyme Microb. Technol. 20: 500-505.
Klinke S, Dauner M, Scott G, Kessler B, Witholt B. 2000. Inactivation of isocitrate lyase leads to increased production of medium-chain-length poly(3-hydroxyalkanoates) in Pseudomonas putida. Appl. Environ. Microbiol. 66: 909-913.
Koller M, Atlic A, Dias M, Reiterer A, Braunegg G. 2010. Microbial PHA production from waste raw materials, p. 85-119. Plastics from Bacteria, Springer Berlin Heidelberg.
Lee H-J, Choi MH, Kim T-U, Yoon SC. 2001. Accumulation of polyhydroxyalkanoic acid containing large amounts of unsaturated monomers in Pseudomonas fluorescens BM07 utilizing saccharides and its inhibition by 2-bromooctanoic acid. Appl. Environ. Microbiol. 67: 4963-4974.
Lee SY, Wong HH , Choi J I, L ee SH , Lee SC, H an CS. 2000. Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation. Biotechnol. Bioeng. 68: 466-470.
Liu Q, Luo G, Zhou XR, Chen GQ. 2011. Biosynthesis of poly(3-hydroxydecanoate) and 3-hydroxydodecanoate dominating polyhydroxyalkanoates by β-oxidation pathway inhibited Pseudomonas putida. Metab. Eng. 13: 11-17.
Mozejko J, Ciesielski S. 2013. Saponified waste palm oil as an attractive renewable resource for mcl-polyhydroxyalkanoate synthesis. J. Biosci. Bioeng. 116: 485-492.
Muhr A, Rechberger EM, Salerno A, Reiterer A, Malli K, Strohmeier K, et al. 2013. Novel description of mcl-PHA biosynthesis by Pseudomonas chlororaphis from animalderived waste. J. Biotechnol. 165: 45-51.
Muller C, Petruschka L, Cuypers H, Burchhardt G, and Herrmann H. 1996. Carbon catabolite repression of phenol degradation in Pseudomonas putida is mediated by the inhibition of the activator protein PhlR. J. Bacteriol. 178:2030-2036.
Ouyang SP, Luo RC, Chen SS, Liu Q, Chung A, Wu Q, Chen GQ. 2007. Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442. Biomacromolecules 8: 2504-2511.
Poblete-Castro I, Becker J, Dohnt K, dos Santos V, Wittmann C. 2012. Industrial biotechnology of Pseudomonas putida and related species. Appl. Microbiol. Biotechnol. 93: 2279-2290.
Poblete-Castro I, Binger D, Rodrigues A, Becker J, Martins dos Santos VAP, Wittmann C. 2013. In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates. Metab. Eng. 15: 113-123.
Poblete-Castro I, Escapa I, Jager C, Puchalka J, Lam CMC, Schomburg D, et al. 2012. The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach. Microb. Cell Factor. 11:34.
Puchalka J, Oberhardt MA, Godinho M, Bielecka A, Regenhardt D, Timmis KN, et al. 2008. Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology. PLoS Comput. Biol. 4: e1000210.
Rai R, Keshavarz T, Roether JA, Boccaccini AR, Roy I. 2011. Medium chain length polyhydroxyalkanoates, promising new biomedical materials for the future. Mater. Sci. Eng. R Reports 72: 29-47.
Simon-Colin C, Raguenes G, Crassous P, Moppert X, Guezennec J. 2008. A novel mcl-PHA produced on coprah oil by Pseudomonas guezennei biovar. tikehau, isolated from a kopara mat of French Polynesia. Int. J. Biol. Macromolec. 43:176-181.
Smyth PF, Clarke PH. 1975. Catabolite repression of Pseudomonas aeruginosa amidase: the effect of carbon source on amidase synthesis. J. Gen. Microbiol. 90: 81-90.
Solaiman DY, Ashby R, Hotchkiss Jr A, Foglia T. 2006. Biosynthesis of medium-chain-length poly(hydroxyalkanoates) from soy molasses. Biotechnol. Lett. 28: 157-162.
Sun Z, Ramsay J, Guay M, Ramsay B. 2006. Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas putida KT2440. Appl. Microbiol. Biotechnol. 71:423-431.
Sun Z, Ramsay J, Guay M, Ramsay B. 2009. Enhanced yield of medium-chain-length polyhydroxyalkanoates from nonanoic acid by co-feeding glucose in carbon-limited, fed-batch culture. J. Biotechnol. 143: 262-267.
Sun Z, Ramsay JA, Guay M, Ramsay B. 2007. Increasing the yield of mcl-PHA from nonanoic acid by co-feeding glucose during the PHA accumulation stage in two-stage fed-batch fermentations of Pseudomonas putida KT2440. J. Biotechnol. 132: 280-282.
Sun Z, Ramsay JA, Guay M, Ramsay BA. 2007. Carbonlimited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by Pseudomonas putida KT2440. Appl. Microbiol. Biotechnol. 74: 69-77.
Sun Z, Ramsay JA, Guay M, Ramsay BA. 2007. Fermentation process development for the production of medium-chainlength poly-3-hydroxyalkanoates. Appl. Microbiol. Biotechnol. 75: 475-485.
van Duuren JBJH. 2011. Optimization of Pseudomonas putida KT2440 as Host for the Production of Cis, Cis-muconate from Benzoate. Wageningen University.
Wang B, Sharma-Shivappa R, Olson J, Khan S. 2012. Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies. Bioprocess Biosyst. Eng. 35: 15911602.
Wang Q, Tappel RC, Zhu C, Nomura CT. 2012. Development of a new strategy for production of medium-chain-length polyhydroxyalkanoates by recombinant Escherichia coli via inexpensive non-fatty acid feedstocks. Appl. Environ. Microbiol. 78: 519-527.
Witholt B, Kessler B. 1999. Perspectives of medium chain length poly(hydroxyalkanoates), a versatile set of bacterial bioplastics. Curr. Opin. Biotechnol. 10: 279-285.