Increased Isoprene Production by the Recombinant Pantoea ananatis Strain due to the Balanced Amplification of Mevalonate Pathway Genes

Katashkina, J. I.; Kazieva, Ekaterina; Tajima, Yoshinori; Mashko, Sergey V.

doi:10.1134/s0003683819090023

Cited by 4 publications

(7 citation statements)

References 40 publications

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“…As a result of test-tube cultivation under P i -starved conditions using a biphasic fermentation system, SWITCH-PphoC Δ gcd /pAaLINS-ispA* produced 3.4 ± 0.2 g/L of ( S )-linalool from 60 ± 0.0 g/L of glucose in 48 h (Table 1 ), which was greater than the reported linalool titers from yeasts [ 12 , 13 , 29 , 30 ] and bacteria [ 15 , 31 ]. As our previous study showed that additional integration of an mvaES operon expression-cassette led to higher productivity in an isoprene-producing P. ananatis strain [ 32 ], the strain IP03 was constructed by integrating one mvaES operon expression-cassette into the genome of SWITCH-PphoC Δ gcd . The strain IP04 was also constructed from IP03 by integrating one mvk expression-cassette to increase carbon flux to IPP/DMAPP.…”

Section: Resultsmentioning

confidence: 99%

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

et al. 2021

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Background Linalool, an acyclic monoterpene alcohol, is extensively used in the flavor and fragrance industries and exists as two enantiomers, (S)- and (R)-linalool, which have different odors and biological properties. Linalool extraction from natural plant tissues suffers from low product yield. Although linalool can also be chemically synthesized, its enantioselective production is difficult. Microbial production of terpenes has recently emerged as a novel, environmental-friendly alternative. Stereoselective production can also be achieved using this approach via enzymatic reactions. We previously succeeded in producing enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis, a member of the Enterobacteriaceae family of bacteria, via the heterologous mevalonate pathway with the highest linalool titer ever reported from engineered microbes. Results Here, we genetically modified a previously developed P. ananatis strain expressing the (S)-linalool synthase (AaLINS) from Actinidia arguta to further improve (S)-linalool production. AaLINS was mostly expressed as an insoluble form in P. ananatis; its soluble expression level was increased by N-terminal fusion of a halophilic β-lactamase from Chromohalobacter sp. 560 with hexahistidine. Furthermore, in combination with elevation of the precursor supply via the mevalonate pathway, the (S)-linalool titer was increased approximately 1.4-fold (4.7 ± 0.3 g/L) in comparison with the original strain (3.4 ± 0.2 g/L) in test-tube cultivation with an aqueous-organic biphasic fermentation system using isopropyl myristate as the organic solvent for in situ extraction of cytotoxic and semi-volatile (S)-linalool. The most productive strain, IP04S/pBLAAaLINS-ispA*, produced 10.9 g/L of (S)-linalool in “dual-phase” fed-batch fermentation, which was divided into a growth-phase and a subsequent production-phase. Thus far, this is the highest reported titer in the production of not only linalool but also all monoterpenes using microbes. Conclusions This study demonstrates the potential of our metabolically engineered P. ananatis strain as a platform for economically feasible (S)-linalool production and provides insights into the stereoselective production of terpenes with high efficiency. This system is an environmentally friendly and economically valuable (S)-linalool production alternative. Mass production of enantiopure (S)-linalool can also lead to accurate assessment of its biological properties by providing an enantiopure substrate for study.

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Section: Resultsmentioning

confidence: 99%

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

et al. 2021

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“…It allows for the inclusion of large artificial inserts that are difficult to introduce using commonly used PCR-based recombination procedures. Katashkina et al [42] further improved the dual-in/out system to a method that simultaneously incorporates a few DNA fragments into specific loci on the genome. They divided the mevalonic acid pathway into acetyl-CoA to mevalonic acid (MVA), MVA to phosphomevalonate, and the remaining pathway (see Isoprenoid production).…”

Section: Isolation Of P Ananatis Aj 13355 and Genome-editing Toolsmentioning

confidence: 99%

“…Through the improved dual-in/out system and electroporation efficiency in P. ananatis, they were able to handle plasmids for all three parts simultaneously. They showed that their experimental design was sufficient to remove all selection markers [42]. Consequently, it is now possible to evaluate the production of genetically engineered strains that retain the desired multiple genetic traits, resulting in an increase in breeding speed.…”

Section: Isolation Of P Ananatis Aj 13355 and Genome-editing Toolsmentioning

confidence: 99%

“…This result suggests that the promoters of phoC and pstS can be used to produce substances that respond to phosphate concentration. Katashkina et al [42] studied isoprene production via the MVA pathway in P. ananatis. The mvaE and mvaS genes from E. faecalis were introduced under the control of a phosphate responsive promoter.…”

Section: Isoprenoid Productionmentioning

confidence: 99%

“…The expression of the gene encoding mevalonate kinase from M. paludicola was implemented by introducing an expression unit using the tac promoter into a locus separate from the artificial operon. As a result, all genes of the MVA pathway were integrated into the genome of P. ananatis, and all enzymes were functional during isoprene production in P. ananatis [42].…”

Section: Isoprenoid Productionmentioning

confidence: 99%

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Microbial Production Potential of Pantoea ananatis: From Amino Acids to Secondary Metabolites

et al. 2022

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Pantoea ananatis, a gram-negative bacterium belonging to the Erwiniaceae family, is a well-known phytopathogen isolated from many ecological niches and plant hosts. However, this bacterium also provides us with various beneficial characteristics, such as the growth promotion of their host plants and increased crop yield. Some isolated non-pathogenic strains are promising for the microbial production of useful substances. P. ananatis AJ13355 was isolated as an acidophilic bacterium and was used as an excellent host to produce L-glutamic acid under acidic conditions. The genome sequence of P. ananatis AJ13355 was determined, and specific genome-engineering technologies were developed. As a result, P. ananatis was successfully used to construct a bacterial strain that produces cysteine, a sulfur-containing amino acid that has been difficult to produce through fermentation because of complex regulation. Furthermore, by heterologous expression including plant-derived genes, construction of a strain that produces isoprenoids such as isoprene and linalool as secondary metabolites was achieved. P. ananatis is shown to be a useful host for the production of secondary metabolites, as well as amino acids, and is expected to be used as a platform for microbial production of bioactive substances, aromatic substances, and other high-value-added substances of plant origin in the future.

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One-step cloning and targeted duplication of Pantoea ananatis chromosomal fragments

Igonina,

Samsonov,

Stoynova

2024

Journal of Microbiological Methods

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Increased Isoprene Production by the Recombinant Pantoea ananatis Strain due to the Balanced Amplification of Mevalonate Pathway Genes

Cited by 4 publications

References 40 publications

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

Microbial Production Potential of Pantoea ananatis: From Amino Acids to Secondary Metabolites

One-step cloning and targeted duplication of Pantoea ananatis chromosomal fragments

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