Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a <i>Bacillus subtilis</i> strain

Yeh, Chuan-Mei; Wang, Jyh-Perng; Lo, Shih-Ching; Chan, Wen-Chia; Lin, Ming-Yi

doi:10.1002/btpr.417

Cited by 25 publications

(8 citation statements)

References 34 publications

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“…). It is proposed that the copy number obtained by integration is highly dependent on the size and content of the expression cassette (Lee and Silva, ; Yamada et al, ; Yeh et al, ).…”

Section: Discussionmentioning

confidence: 99%

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Shi

Valle-Rodríguez

Siewers

et al. 2014

Biotech & Bioengineering

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In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast.

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“…). It is proposed that the copy number obtained by integration is highly dependent on the size and content of the expression cassette (Lee and Silva, ; Yamada et al, ; Yeh et al, ).…”

Section: Discussionmentioning

confidence: 99%

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Shi

Valle-Rodríguez

Siewers

et al. 2014

Biotech & Bioengineering

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“…Although the constructed recombinant bacteria Bacillus WB600- pgs BCA showed the capacity to synthesize γ -PGA, our results still could not match the highest synthetic yield of γ -PGA (40–50 g/L) that is induced by the fermentation of mutated bacteria [ 25 , 26 ]. Therefore, our next research study will focus on introducing hemoglobin, other exogenous genes, or certain control sequences to efficiently synthesize and express γ -PGA and to increase the bacterial concentration, oxygen uptake, or endogenous synthase expression, thereby ultimately increasing γ -PGA yield [ 27 , 28 ]. Alternatively, we will knock out genes of degrading enzymes in γ -PGA-producing strains to reduce γ -PGA degradation, thereby increasing γ -PGA yield [ 29 ].…”

Section: Discussionmentioning

confidence: 99%

Cloning and Expression of theγ-Polyglutamic Acid Synthetase GenepgsBCA inBacillus subtilisWB600

Lin

Zhang

et al. 2016

BioMed Research International

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To clone and express the -polyglutamic acid ( -PGA) synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize -PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. -PGA samples from the fermentation broth were purified and characterized. Hydrolysates of -PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the -PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500-600 kDa. Expressing the -PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications.

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“…The two most commonly studied natural poly(amino acids) used as biomaterials are poly(γ-glutamic acid) and poly(L-lysine). Poly(γ-glutamic acid) (γPGA) is composed of both enantiomeric D-and L-glutamic acid units produced by several bacteria [345]. The reactive side carboxylate of γPGA allows the covalent attachment of other functional groups or drugs (e.g., chemotherapeutics) [346] and the formation of particle delivery systems for antibiotics, vaccines, DNA, and proteins [347].…”

Section: Proteins and Poly(amino Acids)mentioning

confidence: 99%

Biodegradable polymers for dental tissue engineering and regeneration

Conte¹,

Riccitiello²,

Luise³

et al. 2017

Biodegradable Polymers: Recent Developments and New Perspectives

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Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Cited by 25 publications

References 34 publications

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Cloning and Expression of theγ-Polyglutamic Acid Synthetase GenepgsBCA inBacillus subtilisWB600

Biodegradable polymers for dental tissue engineering and regeneration

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