Identification and application of a novel strong constitutive promoter in Corynebacterium glutamicum

Wei, Hongbo; Ma, Yuechao; Chen, Qixin; Cui, Yi; Du, Lihong; Ma, Qian; Li, Yanjun; Xie, Xiulan; Chen, Ning

doi:10.1007/s13213-018-1344-0

Cited by 8 publications

(8 citation statements)

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“…Under aerobic conditions, pyruvate entering the TCA cycle is mainly catalyzed by PDHC and citrate synthase [ 21 ]. The inactivation of PDHC and citrate synthase can cause an increase in l -valine production, however, it also has an irreversible negative impact on cell growth [ 15 , 18 ]. Since most of the inducible promoters are tightly regulated with low basal expression before induction, it could not control the transcription of the gltA or aceE genes at a high level in the exponential phase and a low level in the stationary phase.…”

Section: Resultsmentioning

confidence: 99%

“…The transcriptional strength of the P CP_2454 promoter was about 80% that of the tuf and sod promoters. This promoter could be used to strengthen gene expression and increased the production of l -valine in C. glutamicum [ 18 ]. In this study, statistical analysis of the RNA-seq data of C. glutamicum CP was used to identify genes with significant differences in transcription levels between the exponential and stationary phases.…”

Section: Introductionmentioning

confidence: 99%

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Identification and application of a growth-regulated promoter for improving l-valine production in Corynebacterium glutamicum

Cui

et al. 2018

Microb Cell Fact

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BackgroundPromoters are commonly used to regulate the expression of specific target genes or operons. Although a series of promoters have been developed in Corynebacterium glutamicum, more precise and unique expression patterns are needed that the current selection of promoters cannot produce. RNA-Seq technology is a powerful tool for helping us to screen out promoters with expected transcriptional strengths.ResultsThe promoter PCP_2836 of an aldehyde dehydrogenase coding gene from Corynebacterium glutamicum CP was identified via RNA-seq and RT-PCR as a growth-regulated promoter. Comparing with the strong constitutive promoter Ptuf, the transcriptional strength of PCP_2836 showed a significant decrease that from about 75 to 8% in the stationary phase. By replacing the native promoters of the aceE and gltA genes with PCP_2836 in the C. glutamicum ATCC 13032-derived l-valine-producing strain AN02, the relative transcriptional levels of the aceE and gltA genes decreased from 1.2 and 1.1 to 0.35 and 0.3, and the activity of their translation products decreased to 43% and 35%, respectively. After 28 h flask fermentation, the final cell density of the obtained strains, GRaceE and GRgltA, exhibited a 7–10% decrease. However, l-valine production increased by 23.9% and 27.3%, and the yield of substrate to product increased 43.8% and 62.5%, respectively. In addition, in the stationary phase, the intracellular citrate levels in GRaceE and GRgltA decreased to 27.0% and 33.6% of AN02, and their intracellular oxaloacetate levels increased to 2.7 and 3.0 times that of AN02, respectively.ConclusionsThe PCP_2836 promoter displayed a significant difference on its transcriptional strength in different cell growth phases. With using PCP_2836 to replace the native promoters of aceE and gltA genes, both the transcriptional levels of the aceE and gltA genes and the activity of their translation products demonstrated a significant decrease in the stationary phase. Thus, the availability of pyruvate was significantly increased for the synthesis of l-valine without any apparent irreversible negative impacts on cell growth. Use of this promoter can enhance the selectivity and control of gene expression and could serve as a useful research tool for metabolic engineering. Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1031-7) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification and application of a growth-regulated promoter for improving l-valine production in Corynebacterium glutamicum

Cui

et al. 2018

Microb Cell Fact

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“…At present, overexpression of the ilvBNCDE genes, in various combinations, is performed using constructs with strong constitutive promoters. These include, for example, promoters of superoxide dismutase (Psod ) and elongation factor Tu (Ptuf ) genes and a synthetic construct based on trp and lac operon promoters (Ptac) (Tarutina et al, 2016;Wei et al, 2018;Li et al, 2020b;Wang et al, 2020;Zhang et al, 2021). Other efficient promoters have also been described (Tarutina et al, 2016;Wei et al, 2018;Li et al, 2020b).…”

Section: Enhancement Of Valine Biosynthesis Reactionsmentioning

confidence: 99%

“…These include, for example, promoters of superoxide dismutase (Psod ) and elongation factor Tu (Ptuf ) genes and a synthetic construct based on trp and lac operon promoters (Ptac) (Tarutina et al, 2016;Wei et al, 2018;Li et al, 2020b;Wang et al, 2020;Zhang et al, 2021). Other efficient promoters have also been described (Tarutina et al, 2016;Wei et al, 2018;Li et al, 2020b). Modifications of this type lead to an increase in valine production by about 60 % (Wei et al, 2018).…”

Section: Enhancement Of Valine Biosynthesis Reactionsmentioning

confidence: 99%

Rational metabolic engineering of <i>Corynebacterium glutamicum</i> to create a producer of L-valine

et al. 2023

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L-Valine is one of the nine amino acids that cannot be synthesized de novo by higher organisms and must come from food. This amino acid not only serves as a building block for proteins, but also regulates protein and energy metabolism and participates in neurotransmission. L-Valine is used in the food and pharmaceutical industries, medicine and cosmetics, but primarily as an animal feed additive. Adding L-valine to feed, alone or mixed with other essential amino acids, allows for feeds with lower crude protein content, increases the quality and quantity of pig meat and broiler chicken meat, as well as improves reproductive functions of farm animals. Despite the fact that the market for L-valine is constantly growing, this amino acid is not yet produced in our country. In modern conditions, the creation of strains-producers and organization of L-valine production are especially relevant for Russia. One of the basic microorganisms most commonly used for the creation of amino acid producers, along with Escherichia coli, is the soil bacterium Corynebacterium glutamicum. This review is devoted to the analysis of the main strategies for the development of L- valine producers based on C. glutamicum. Various aspects of L-valine biosynthesis in C. glutamicum are reviewed: process biochemistry, stoichiometry and regulation, enzymes and their corresponding genes, export and import systems, and the relationship of L-valine biosynthesis with central cell metabolism. Key genetic elements for the creation of C. glutamicum-based strains-producers are identified. The use of metabolic engineering to enhance L-valine biosynthesis reactions and to reduce the formation of byproducts is described. The prospects for improving strains in terms of their productivity and technological characteristics are shown. The information presented in the review can be used in the production of producers of other amino acids with a branched side chain, namely L-leucine and L-isoleucine, as well as D-pantothenate.

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“…By using different promoters, different transcriptional levels of the target gene can be achieved, and a more suitable transcriptional level for cell growth and metabolism can be obtained. Meanwhile, promoters can be divided into constitutive and inducible types [36,37]. Constitutive promoters continuously promote the transcription of target genes, while inducible promoters switch between 'on' and 'off' states to achieve flexible regulation of gene transcription.…”

Section: Promoter Engineeringmentioning

confidence: 99%

New strategy for removing acetic acid as a by-product during L-tryptophan production

Zhang

et al. 2019

Biotechnology & Biotechnological Equipment

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Acetic acid, an important by-product of L-tryptophan production by Escherichia coli, is detrimental to cell growth and tryptophan accumulation. To reduce acetic acid accumulation and further improve the production of L-tryptophan, an acetyl-coenzyme A (acetyl-CoA) synthase (ACS) with high synthetic activity is needed. In this study, E. coli ACS was found to have a superior catalytic capability compared to that of other bacterial strains. To reduce the accumulation of acetic acid by metabolic overflow, a higher expression level of ACS was obtained by promoter engineering and copy number optimisation. Furthermore, reconstruction of the tri-carboxylic acid cycle (TCA) not only enhanced the supply of phosphoenolpyruvate (PEP), which is one of the precursors required for tryptophan synthesis, but also improved bacterial growth. The final engineered strain T16 accumulated 54.6 g/L of L-tryptophan, 11.4% more than the original strain T03, with an accumulation of only 1.4 g/L acetic acid after fermentation, which reduced by 74.5% compared to T03.

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Identification and application of a novel strong constitutive promoter in Corynebacterium glutamicum

Cited by 8 publications

References 26 publications

Identification and application of a growth-regulated promoter for improving l-valine production in Corynebacterium glutamicum

Identification and application of a growth-regulated promoter for improving l-valine production in Corynebacterium glutamicum

Rational metabolic engineering of <i>Corynebacterium glutamicum</i> to create a producer of L-valine

New strategy for removing acetic acid as a by-product during L-tryptophan production

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