Cell-growth Phase-dependent Promoter Replacement Approach for Improved Poly(lactate-co-3-hydroxybutyrate) Production in Escherichia Coli

Overview

Journal Microb Cell Fact

Publisher Biomed Central

Specialties Biotechnology
Microbiology

Date 2023 Jul 19

PMID 37468909

Authors

Yuki Nagao

Sangho Koh

Seiichi Taguchi

Tomohiro Shimada

Affiliations

Soon will be listed here.

Abstract

Escherichia coli is a useful platform for producing valuable materials through the implementation of synthetic gene(s) derived from other organisms. The production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] was carried out in E. coli using a set of five other species-derived genes: Pseudomonas sp. 61-3-derived phaC1STQK (for polymerization), Cupriavidus necator-derived phaAB (for 3HB-CoA generation), and Megasphaera elsdenii-derived pct (for LA-CoA generation) cloned into pTV118NpctphaC1p(ST/QK)AB. Here, we aimed to optimize the expression level and timing of these genes to improve the production of P(LA-co-3HB) and to manipulate the LA fraction by replacing the promoters with various promoters in E. coli. Evaluation of the effects of 21 promoter replacement plasmids revealed that the phaC1STQK-AB operon is critical for the stationary phase for P(LA-co-3HB) production. Interestingly, the effects of the promoters depended on the composition of the medium. In glucose-supplemented LB medium, the dps promoter replacement plasmid resulted in the greatest effect, increasing the accumulation to 8.8 g/L and an LA fraction of 14.1 mol% of P(LA-co-3HB), compared to 2.7 g/L and 8.1 mol% with the original plasmid. In xylose-supplemented LB medium, the yliH promoter replacement plasmid resulted in the greatest effect, with production of 5.6 g/L and an LA fraction of 40.2 mol% compared to 3.6 g/L and 22.6 mol% with the original plasmid. These results suggest that the selection of an appropriate promoter for expression of the phaC1STQK-AB operon could improve the production and LA fraction of P(LA-co-3HB). Here, we propose that the selection of cell-growth phase-dependent promoters is a versatile biotechnological strategy for effective intracellular production of polymeric materials such as P(LA-co-3HB), in combination with the selection of sugar-based carbon sources.

Citing Articles

Delaying production with prokaryotic inducible expression systems.

De Baets J, De Paepe B, De Mey M Microb Cell Fact. 2024; 23(1):249.

PMID: 39272067 PMC: 11401332. DOI: 10.1186/s12934-024-02523-w.

Bacterial Population Changes during the Degradation Process of a Lactate (LA)-Enriched Biodegradable Polymer in River Water: LA-Cluster Preferable Bacterial Consortium.

Kadoya R, Soga H, Matsuda M, Sato M, Taguchi S Polymers (Basel). 2023; 15(20).

PMID: 37896354 PMC: 10610160. DOI: 10.3390/polym15204111.

References

Jiang G, Nikolova S, Clark D . Regulation of the ldhA gene, encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology (Reading). 2001; 147(Pt 9):2437-2446. DOI: 10.1099/00221287-147-9-2437. View

Ishihama A, Shimada T . Hierarchy of transcription factor network in Escherichia coli K-12: H-NS-mediated silencing and Anti-silencing by global regulators. FEMS Microbiol Rev. 2021; 45(6). DOI: 10.1093/femsre/fuab032. View

Kadoya R, Matsumoto K, Ooi T, Taguchi S . MtgA Deletion-Triggered Cell Enlargement of Escherichia coli for Enhanced Intracellular Polyester Accumulation. PLoS One. 2015; 10(6):e0125163. PMC: 4454544. DOI: 10.1371/journal.pone.0125163. View

Salamanca-Cardona L, Scheel R, Mizuno K, Bergey N, Stipanovic A, Matsumoto K . Effect of acetate as a co-feedstock on the production of poly(lactate-co-3-hydroxyalkanoate) by pflA-deficient Escherichia coli RSC10. J Biosci Bioeng. 2017; 123(5):547-554. DOI: 10.1016/j.jbiosc.2016.12.019. View

Hori C, Yamazaki T, Ribordy G, Takisawa K, Matsumoto K, Ooi T . High-cell density culture of poly(lactate-co-3-hydroxybutyrate)-producing Escherichia coli by using glucose/xylose-switching fed-batch jar fermentation. J Biosci Bioeng. 2018; 127(6):721-725. DOI: 10.1016/j.jbiosc.2018.11.006. View

Nduko J, Matsumoto K, Ooi T, Taguchi S . Enhanced production of poly(lactate-co-3-hydroxybutyrate) from xylose in engineered Escherichia coli overexpressing a galactitol transporter. Appl Microbiol Biotechnol. 2013; 98(6):2453-60. DOI: 10.1007/s00253-013-5401-0. View

Bunch P, Lee N, Clark D . The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology (Reading). 1997; 143 ( Pt 1):187-195. DOI: 10.1099/00221287-143-1-187. View

Sun J, Matsumoto K, Tabata Y, Kadoya R, Ooi T, Abe H . Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp. C34 and Alcaligenes faecalis T1. Appl Microbiol Biotechnol. 2015; 99(22):9555-63. DOI: 10.1007/s00253-015-6756-1. View

Yamada M, Matsumoto K, Shimizu K, Uramoto S, Nakai T, Shozui F . Adjustable mutations in lactate (LA)-polymerizing enzyme for the microbial production of LA-based polyesters with tailor-made monomer composition. Biomacromolecules. 2010; 11(3):815-9. DOI: 10.1021/bm901437z. View

10.

Kadoya R, Kodama Y, Matsumoto K, Taguchi S . Indirect positive effects of a sigma factor RpoN deletion on the lactate-based polymer production in Escherichia coli. Bioengineered. 2015; 6(5):307-11. PMC: 4825821. DOI: 10.1080/21655979.2015.1069449. View

11.

Taguchi S, Yamada M, Matsumoto K, Tajima K, Satoh Y, Munekata M . A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme. Proc Natl Acad Sci U S A. 2008; 105(45):17323-7. PMC: 2582312. DOI: 10.1073/pnas.0805653105. View

12.

Shozui F, Matsumoto K, Nakai T, Yamada M, Taguchi S . Biosynthesis of novel terpolymers poly(lactate-co-3-hydroxybutyrate-co-3-hydroxyvalerate)s in lactate-overproducing mutant Escherichia coli JW0885 by feeding propionate as a precursor of 3-hydroxyvalerate. Appl Microbiol Biotechnol. 2009; 85(4):949-54. DOI: 10.1007/s00253-009-2100-y. View

13.

Koh S, Sato M, Yamashina K, Usukura Y, Toyofuku M, Nomura N . Controllable secretion of multilayer vesicles driven by microbial polymer accumulation. Sci Rep. 2022; 12(1):3393. PMC: 8888611. DOI: 10.1038/s41598-022-07218-z. View

14.

Steinbuchel A, Hein S . Biochemical and molecular basis of microbial synthesis of polyhydroxyalkanoates in microorganisms. Adv Biochem Eng Biotechnol. 2001; 71:81-123. DOI: 10.1007/3-540-40021-4_3. View

15.

Shimada T, Makinoshima H, Ogawa Y, Miki T, Maeda M, Ishihama A . Classification and strength measurement of stationary-phase promoters by use of a newly developed promoter cloning vector. J Bacteriol. 2004; 186(21):7112-22. PMC: 523215. DOI: 10.1128/JB.186.21.7112-7122.2004. View

16.

Salamanca-Cardona L, Scheel R, Bergey N, Stipanovic A, Matsumoto K, Taguchi S . Consolidated bioprocessing of poly(lactate-co-3-hydroxybutyrate) from xylan as a sole feedstock by genetically-engineered Escherichia coli. J Biosci Bioeng. 2016; 122(4):406-14. DOI: 10.1016/j.jbiosc.2016.03.009. View

17.

Yamada M, Matsumoto K, Uramoto S, Motohashi R, Abe H, Taguchi S . Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli. J Biotechnol. 2011; 154(4):255-60. DOI: 10.1016/j.jbiotec.2011.05.011. View

18.

Shimada T, Ogasawara H, Ishihama A . Genomic SELEX Screening of Regulatory Targets of Escherichia coli Transcription Factors. Methods Mol Biol. 2018; 1837:49-69. DOI: 10.1007/978-1-4939-8675-0_4. View

19.

Spiekermann P, Rehm B, Kalscheuer R, Baumeister D, Steinbuchel A . A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol. 1999; 171(2):73-80. DOI: 10.1007/s002030050681. View

20.

de Almeida A, Catone M, Rhodius V, Gross C, Pettinari M . Unexpected stress-reducing effect of PhaP, a poly(3-hydroxybutyrate) granule-associated protein, in Escherichia coli. Appl Environ Microbiol. 2011; 77(18):6622-9. PMC: 3187130. DOI: 10.1128/AEM.05469-11. View