Enhanced extracellular gamma glutamyl transpeptidase production by overexpressing of PrsA lipoproteins and improving its mRNA stability in Bacillus subtilis and application in biosynthesis of L-theanine

Yang, Taowei; Komera, Irene; Liu, Huiling; Liu, Shuanying; Zhang, Xian; Rao, Zhiming

doi:10.1016/j.jbiotec.2019.06.302

Cited by 20 publications

(15 citation statements)

References 34 publications

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“…In eukaryotes, poly A tails are stretch adenine bases in the 3' end of RNA, and it was found that when the poly(A) tails become short enough, the mRNA could be degraded. It was also reported that the different of poly(A) tails had a great effect on the stability of mRNA of the enzyme, which plays a key role in its overproduction process [25][26][27].…”

Section: Resultsmentioning

confidence: 98%

Metabolic Engineering of Bacillus subtilis for 2.3-BDO production by introducing an exogenous NADPH/NADP+ regeneration system

Liu¹,

Liu²,

Xie³

et al. 2019

Preprint

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Background: Generally, glucose is transformed into pyruvate from glycolysis before the target products acetoin and 2,3-butanediol (2,3-BDO) are formed. Pentose Phosphate Pathway (PPP) is an inefficient synthetic pathway for pyruvate production from glucose in Bacillus subtilis. Previously, it was found that engineered PPP in B. subtilis unbalanced NADH and NADPH regeneration systems and affected acetoin and 2,3 -BDO production.Results: In this study, metabolic engineering strategies were proposed to redistribute carbon flux to 2,3-BDO via reconstructing intracellular cofactors regeneration systems. Firstly, extra copies of glucose dehydrogenase (GDH)and an exogenous NADPH-dependent 2,3-BDO dehydrogenase (TDH) were introduced into the GRAS strain B. subtilis 168 to introduce an exogenous NADPH/NADP + regeneration system and broaden 2,3-BDO production pathway. It was found that overexpressing the NADPH/NADP + regeneration system effectively improved 2,3-BDO production and inhibited NADH-dependent by-products accumulation. Subsequently, the disruption of lactate dehydrogenase (encoded by ldh ) by insertion of the transcriptional regulator ALsR, essential for the expression of alsSD (encoding two key enzymes for the conversion of pyruvate to acetoin) in B. subtilis, resulted in the recombinant strain in which alsSD was overexpressed and the pathway to lactate was blocked simultaneously. On fermentation by the result engineered strain, the highest 2,3-BDO concentration increased by18.43%, while the titers of main byproducts acetoin and lactate decreased by 22.03% and 64%, respectively.Conclusion: In this study, it shows that engineering PPP and reconstructing intracellular cofactors regeneration system could be an alternative strategy in the metabolic engineering of 2,3-BDO production in B. subtilis .

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Section: Resultsmentioning

confidence: 98%

Metabolic Engineering of Bacillus subtilis for 2.3-BDO production by introducing an exogenous NADPH/NADP+ regeneration system

Liu¹,

Liu²,

Xie³

et al. 2019

Preprint

Self Cite

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show abstract

“…RT-qPCR analysis was performed using iCycler iQ Real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, USA) with a 20 μL reaction volume and using 2 × ChamQ™ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China). Primers were designed using Primer Premier 5 software [19]. Gene expression was calculated by 2 -ΔΔCt method as follows: 2 -ΔΔCt = 2 -[(CtA-CtB) treated-(CtA-CtB) control] , where A denotes target gene and B denotes control gene [22].…”

Section: Real-time Quantitative Pcr (Rt-qpcr)mentioning

confidence: 99%

“…Furthermore, it is generally noted that Bacillus is a large genus of the gram-positive, heterotrophic, endospore-forming bacteria and belongs to Class: Bacilli, Phylum: Firmicutes [19]. Members of the Bacillus genus provide a model system for the study of metal ions and exhibit broad resistance to heavy metals [20].…”

Section: Introductionmentioning

confidence: 99%

Complete genome sequencing and comparative genomic analyses of Bacillus sp. S3, a novel hyper Sb(III)-oxidizing bacterium

et al. 2020

BMC Microbiol

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Background: Antimonite [Sb(III)]-oxidizing bacterium has great potential in the environmental bioremediation of Sb-polluted sites. Bacillus sp. S3 that was previously isolated from antimony-contaminated soil displayed high Sb(III) resistance and Sb(III) oxidation efficiency. However, the genomic information and evolutionary feature of Bacillus sp. S3 are very scarce. Results: Here, we identified a 5,436,472 bp chromosome with 40.30% GC content and a 241,339 bp plasmid with 36.74% GC content in the complete genome of Bacillus sp. S3. Genomic annotation showed that Bacillus sp. S3 contained a key aioB gene potentially encoding As (III)/Sb(III) oxidase, which was not shared with other Bacillus strains. Furthermore, a wide variety of genes associated with Sb(III) and other heavy metal (loid) s were also ascertained in Bacillus sp. S3, reflecting its adaptive advantage for growth in the harsh eco-environment. Based on the analysis of phylogenetic relationship and the average nucleotide identities (ANI), Bacillus sp. S3 was proved to a novel species within the Bacillus genus. The majority of mobile genetic elements (MGEs) mainly distributed on chromosomes within the Bacillus genus. Pan-genome analysis showed that the 45 genomes contained 554 core genes and many unique genes were dissected in analyzed genomes. Whole genomic alignment showed that Bacillus genus underwent frequently large-scale evolutionary events. In addition, the origin and evolution analysis of Sb(III)-resistance genes revealed the evolutionary relationships and horizontal gene transfer (HGT) events among the Bacillus genus. The assessment of functionality of heavy metal (loid) s resistance genes emphasized its indispensable role in the harsh eco-environment of Bacillus genus. Real-time quantitative PCR (RT-qPCR) analysis indicated that Sb(III)-related genes were all induced under the Sb(III) stress, while arsC gene was down-regulated. Conclusions: The results in this study shed light on the molecular mechanisms of Bacillus sp. S3 coping with Sb(III), extended our understanding on the evolutionary relationships between Bacillus sp. S3 and other closely related species, and further enriched the Sb(III) resistance genetic data sources.

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“…Quantitative real time polymerase chain reaction (qRT-PCR) analyses was performed using iCycler iQ Real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, USA) with a 20mL reaction volume and using 2×ChamQ TM Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, China). Primers were designed by using Primer Premier 5 software (Table S9) [19]. Gene expression was calculated by -∆∆Ct method as follows: 2 -∆∆Ct = 2 -[(CtA-CtB) treated-(CtA-CtB) control] , where A denotes target gene and B denotes control gene [22].…”

Section: Quantitative Real-time Pcr (Qrt-pcr)mentioning

confidence: 99%

“…Furthermore, it is generally noted that Bacillus is a large genus of the gram-positive, heterotrophic, endospore-forming bacteria and belongs to class bacilli, phylum firmicutes [19]. Members of genus Bacillus provide a model system for the study of metal ions and exhibit broad resistance to heavy metals [20].…”

mentioning

confidence: 99%

Complete genome sequencing and comparative genomic analyses of Bacillus sp. S3, a novel hyper Sb(III)-oxidizing bacterium

Zeng

et al. 2020

Preprint

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Background: Antimonite [Sb(III)]-oxidizing bacterium has great potential in the environmental bioremediation of Sb-polluted sites. Bacillus sp. S3 that was previously isolated from antimony-contaminated soil displayed high Sb(III) resistance and Sb(III) oxidation efficiency. However, the genomic information and evolutionary feature of Bacillus sp. S3 are very scarce. Results: Here, we identified a 5,579,638 bp chromosome with 40.30% GC content and a 241,339 bp plasmid with 36.74% GC content in the complete genome of Bacillus sp. S3. Genomic annotation showed that Bacillus sp. S3 contained a key aioB gene potentially encoding As(III)/Sb(III) oxidase, which was not shared with other Bacillus strains. Further, a series of genes associated with Sb(III) and other heavy metal(loid)s were also ascertained in Bacillus sp. S3, reflecting its adaptive advantage for growth in the harsh eco-environment. Based on the analysis of phylogenetic relationship and the average nucleotide identities (ANI), we found that Bacillus sp. S3 was a novel species within the Bacillus genus. The majority of mobile genetic elements (MGEs) mainly distributed on chromosomes within the Bacillus genus. Pan-genome analysis showed that the 45 genomes contained 554 core genes and many unique genes were dissected in analyzed genomes. Whole genomic alignment showed that Bacillus genus underwent frequently large-scale evolutionary events. In addition, the origin and evolution analysis of Sb(III)-resistance genes revealed that evolutionary relationships and horizontal gene transfer (HGT) events among the Bacillus genus. The assessment of functionality of heavy metal(loid)s resistance genes emphasized its indispensable roles in the harsh eco-environment of Bacillus genus. The real-time Quantitative PCR (RT-qPCR) results of Sb(III)-related genes indicated that the Sb(III) resistance was constantly increased under the Sb(III) stress. Conclusions: The results in this study shed light on the molecular mechanisms of Bacillus sp. S3 coping with Sb(III), extended our understanding on the evolutionary relationship between Bacillus sp. S3 and other closely related species, and further enriched the Sb(III) resistance genetic data sources.

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Enhanced extracellular gamma glutamyl transpeptidase production by overexpressing of PrsA lipoproteins and improving its mRNA stability in Bacillus subtilis and application in biosynthesis of L-theanine

Cited by 20 publications

References 34 publications

Metabolic Engineering of Bacillus subtilis for 2.3-BDO production by introducing an exogenous NADPH/NADP+ regeneration system

Metabolic Engineering of Bacillus subtilis for 2.3-BDO production by introducing an exogenous NADPH/NADP+ regeneration system

Complete genome sequencing and comparative genomic analyses of Bacillus sp. S3, a novel hyper Sb(III)-oxidizing bacterium

Complete genome sequencing and comparative genomic analyses of Bacillus sp. S3, a novel hyper Sb(III)-oxidizing bacterium

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