Abstract
Objectives
To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHAMCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways.
Results
Pseudomonas mendocina NK-01 produces PHAMCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHAMCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(∆alg), NK-U-2 (∆alg∆galU), NK-U-3(alg∆galU∆rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHAMCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHAMCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHAMCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHAMCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %.
Conclusion
The PHAMCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.
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References
Borrero-de Acuna JM et al (2014) Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida. Microb Cell Fact 13:88
Byrd MS et al (2009) Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production. Mol Microbiol 73:622–638
de Eugenio LI, Escapa IF, Morales V, Dinjaski N, Galan B, Garcia JL, Prieto MA (2010) The turnover of medium-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance. Environ Microbiol 12:207–221
Escapa IF, Morales V, Martino VP, Pollet E, Averous L, Garcia JL, Prieto MA (2011) Disruption of beta-oxidation pathway in Pseudomonas putida KT2442 to produce new functionalized PHAs with thioester groups. Appl Microbiol Biot 89:1583–1598
Guo W, Song C, Kong M, Geng W, Wang Y, Wang S (2011a) Simultaneous production and characterization of medium-chain-length polyhydroxyalkanoates and alginate oligosaccharides by Pseudomonas mendocina NK-01. Appl Microbiol Biotechnol 92:791–801
Guo W et al (2011b) Complete genome of Pseudomonas mendocina NK-01, which synthesizes medium-chain-length polyhydroxyalkanoates and alginate oligosaccharides. J Bacteriol 193:3413–3414
Guo W, Feng J, Geng W, Song C, Wang Y, Chen N, Wang S (2012) Augmented production of alginate oligosaccharides by the Pseudomonas mendocina NK-01 mutant. Carbohyd Res 352:109–116
Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86
Jendrossek D, Pfeiffer D (2014) New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3-hydroxybutyrate). Environ Microbiol 16:2357–2373
Leong YK, Show PL, Ooi CW, Ling TC, Lan JC (2014) Current trends in polyhydroxyalkanoates (PHAs) biosynthesis: insights from the recombinant Escherichia coli. J Biotechnol 180:52–65
Liu Q, Luo G, Zhou XR, Chen GQ (2011) Biosynthesis of poly(3-hydroxydecanoate) and 3-hydroxydodecanoate dominating polyhydroxyalkanoates by beta-oxidation pathway inhibited Pseudomonas putida. Metab Eng 13:11–17
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 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 Fact 11:34
Poblete-Castro I, Binger D, Rodrigues A, Becker J, Martins Dos Santos VA, Wittmann C (2013) In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates. Metab Eng 15:113–123
Qiu YZ, Han J, Guo JJ, Chen GQ (2005) Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from gluconate and glucose by recombinant Aeromonas hydrophila and Pseudomonas putida. Biotechnol Lett 27:1381–1386
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 Microb 78:519–527
Wang Y et al (2015) An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440. J Microbiol Methods 113:27–33
Acknowledgments
This work was supported by the National Key Basic Research Program of China (“973”-Program) 2012CB725204, the National Key Technology Support Program 2015BAD16B04, the Natural Science Foundation of China Grant Nos. 31470213 and 31170030, and the Project of Tianjin, China (13JCZDJC27800, 13JCYBJC24900, 13TXSYJC40100 and 14ZCZDSF00009).
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Supplementary Table 1—Primers used in this study.
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Wang, Y., Zhao, F., Fan, X. et al. Enhancement of medium-chain-length polyhydroxyalkanoates biosynthesis from glucose by metabolic engineering in Pseudomonas mendocina . Biotechnol Lett 38, 313–320 (2016). https://doi.org/10.1007/s10529-015-1980-4
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DOI: https://doi.org/10.1007/s10529-015-1980-4