Construction of Halomonas bluephagenesis capable of high cell density growth for efficient PHA production
- 492 Downloads
High-cell-density cultivation is an effective way to improve the productivity of microbial fermentations and in turn reduce the cost of the final products, especially in the case of intracellular products. Halomonas bluephagenesis TD01 is a halophilic platform bacterium for the next generation of industrial biotechnology with a native PHA synthetic pathway, able to grow under non-sterile continuous fermentation conditions. A selection strategy for mutant strains that can grow to a high cell density was developed. Based on an error-prone DNA polymerase III ε subunit, a genome-wide random mutagenesis system was established and used in conjunction with an artificial high cell density culture environment during the selection process. A high-cell-density H. bluephagenesis TDHCD-R3 obtained after 3 rounds of selection showed an obvious enhancement of resistance to toxic metabolites including acetate, formate, lactate and ethanol compared to wild-type. H. bluephagenesis TDHCD-R3-8-3 constructed from H. bluephagenesis TDHCD-R3 by overexpressing an optimized phaCAB operon was able to grow to 15 g/L cell dry weight (CDW) containing 94% PHA in shake flask studies. H. bluephagenesis TDHCD-R3-8-3 was grown to more than 90 g/L CDW containing 79% PHA compared with only 81 g/L with 70% PHA by the wild type when incubated in a 7-L fermentor under the same conditions.
KeywordsHigh-cell-density growth Halomonas bluephagenesis PHB Polyhydroxyalkanoates Next generation industrial biotechnology
SEVA plasmids were kindly donated by Prof. Victor de Lorenzo from the National Centre for Biotechnology in Madrid, Spain.
This study was funded by the Ministry of Sciences and Technology of China (Grant No. 2016YFB0302504), the National Natural Science Foundation of China (Grant No. 31430003 and 31600072) and the Tsinghua President Fund (Grant No. 2015THZ10).
Compliance with ethical standards
Conflict of interest
Yilin Ren declares that he has no conflict of interest.
Chen Ling declares that he has no conflict of interest.
Ivan Hajnal declares that he has no conflict of interest.
Qiong Wu declares that he has no conflict of interest.
Guo-Qiang Chen declares that he has no conflict of interest.
This article does not contain any studies with human participants performed by any of the authors.
- Auriol C, Bestel-Corre G, Claude J-B, Soucaille P, Meynial-Salles I (2011) Stress-induced evolution of Escherichia coli points to original concepts in respiratory cofactor selectivity. Proc Natl Acad Sci U S A 108:1278–1283. https://doi.org/10.1073/pnas.1010431108 CrossRefPubMedPubMedCentralGoogle Scholar
- Chen K, Liu Q, Xie L, Sharp PA, Wang DI (2001) Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production. Biotechnol Bioeng 72:55–61. https://doi.org/10.1002/1097-0290(20010105)72:1%3C55::AID-BIT8%3E3.0.CO;2-4 CrossRefPubMedGoogle Scholar
- Hellmuth K, Korz DJ, Sanders EA, Deckwer WD (1994) Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1. J Biotechnol 32:289–298. https://doi.org/10.1016/0168-1656(94)90215-1 CrossRefPubMedGoogle Scholar
- Herring CD, Raghunathan A, Honisch C, Patel T, Applebee MK, Joyce AR, Albert TJ, Blattner FR, van den Boom D, Cantor CR, Palsson BØ (2006) Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale. Nat Genet 38:1406–1412. https://doi.org/10.1038/ng1906 CrossRefPubMedGoogle Scholar
- Hiroe A, Tsuge K, Nomura CT, Itaya M, Tsuge T (2012) Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly [(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Appl Environ Microbiol 78:3177–3184. https://doi.org/10.1128/AEM.07715-11 CrossRefPubMedPubMedCentralGoogle Scholar
- Horng YT, Chang KC, Chien CC, Wei YH, Sun YM, Soo PC (2010) Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli. Lett Appl Microbiol 50:158–167. https://doi.org/10.1111/j.1472-765X.2009.02772.x CrossRefPubMedGoogle Scholar
- Kato M, Bao H, Kang C-K, Fukui T, Doi Y (1996) Production of a novel copolyester of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids by Pseudomonas sp. 61-3 from sugars. Appl Microbiol Biotechnol 45:363–370. https://doi.org/10.1007/s002530050697
- Knabben I, Regestein L, Marquering F, Steinbusch S, Lara AR, Büchs J (2010) High cell-density processes in batch mode of a genetically engineered Escherichia coli strain with minimized overflow metabolism using a pressurized bioreactor. J Biotechnol 150:73–79. https://doi.org/10.1016/j.jbiotec.2010.07.006 CrossRefPubMedGoogle Scholar
- Kroll J, Steinle A, Reichelt R, Ewering C, Steinbüchel A (2009) Establishment of a novel anabolism-based addiction system with an artificially introduced mevalonate pathway: complete stabilization of plasmids as universal application in white biotechnology. Metab Eng 11:168–177. https://doi.org/10.1016/j.ymben.2009.01.007 CrossRefPubMedGoogle Scholar
- Li T, Ye J, Shen R, Zong Y, Zhao X, Lou C, Chen GQ (2016) Semirational approach for ultrahigh poly (3-hydroxybutyrate) accumulation in Escherichia coli by combining one-step library construction and high-throughput screening. ACS Synth Biol 5:1308–1317. https://doi.org/10.1021/acssynbio.6b00083 CrossRefPubMedGoogle Scholar
- Llamas I, Quesada E, Martínez-Cánovas MJ, Gronquist M, Eberhard A, Gonzalez JE (2005) Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas. Extremophiles 9:333–341. https://doi.org/10.1007/s00792-005-0448-1 CrossRefPubMedGoogle Scholar
- Mairhofer J, Scharl T, Marisch K, Cserjan-Puschmann M, Striedner G (2013) Comparative transcription profiling and in-depth characterization of plasmid-based and plasmid-free Escherichia coli expression systems under production conditions. Appl Environ Microbiol 79:3802–3812. https://doi.org/10.1128/aem.00365-13 CrossRefPubMedPubMedCentralGoogle Scholar
- Ng W-L, Bassler BL (2009) Bacterial quorum-sensing network architectures. Annu Rev Genet 43:197–222. https://doi.org/10.1146/annurev-genet-102108-134304 CrossRefPubMedPubMedCentralGoogle Scholar
- Obruca S, Sedlacek P, Koller M, Kucera D, Pernicova I (2017) Involvement of polyhydroxyalkanoates in stress resistance of microbial cells: biotechnological consequences and applications. Biotechnol Adv. https://doi.org/10.1016/j.biotechadv.2017.12.006
- Ouyang SP, Luo RC, Chen SS, Liu Q, Chung A, Wu Q, Chen G-Q (2007) Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442. Biomacromolecules 8:2504–2511. https://doi.org/10.1021/bm0702307 CrossRefPubMedGoogle Scholar
- Restaino OF, Cimini D, De Rosa M, Catapano A, Schiraldi C (2011) High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production. Microb Cell Factories 10:10. https://doi.org/10.1186/1475-2859-10-10 CrossRefGoogle Scholar
- Roe AJ, McLaggan D, Davidson I, O’Byrne C, Booth IR (1998) Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J Biotechnol 180:767–772Google Scholar
- Silva-Rocha R, Martínez-García E, Calles B, Chavarría M, Arce-Rodríguez A, de las Heras A, Páez-Espino AD, Durante-Rodríguez G, Kim J, Nikel PI (2013) The standard European vector architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res 41:D666–D675. https://doi.org/10.1093/nar/gks1119 CrossRefPubMedGoogle Scholar
- Tapia F, Vázquez-Ramírez D, Genzel Y, Reichl U (2016) Bioreactors for high cell density and continuous multi-stage cultivations: options for process intensification in cell culture-based viral vaccine production. Appl Microbiol Biotechnol 100:2121–2132. https://doi.org/10.1007/s00253-015-7267-9 CrossRefPubMedPubMedCentralGoogle Scholar
- Wang L, Chen X, Wu G, Li S, Zeng X, Ren X, Tang L, Mao Z (2015) Improved ε-poly-l-lysine production of Streptomyces sp. FEEL-1 by atmospheric and room temperature plasma mutagenesis and streptomycin resistance screening. Ann Microbiol 65:2009–2017. https://doi.org/10.1007/s13213-015-1039-8 CrossRefGoogle Scholar