A dynamic metabolite valve for the control of central carbon metabolism

Solomon, Kevin; Sanders, Tarielle M.; Prather, Kristala L. J.

doi:10.1016/j.ymben.2012.08.006

Cited by 96 publications

(77 citation statements)

References 55 publications

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“…Previous approaches to the redirection of endogenous malonyl-CoA into heterologous pathways have relied on conventional gene knockout strategies (3,5,6). However, these strategies typically cause poor biomass accumulation and reduced cofactor regeneration (28). Here, an efficient asRNA-based strategy for cascading regulation of genes at the posttranscriptional level was developed.…”

Section: Discussionmentioning

confidence: 99%

“…However, similar tools for bacteria that are as reliable and efficient as eukaryotic RNAi have been elusive (15). There has been increasing interest in the use of asRNA for various applications in bacteria (15,22,28). Although many studies find that the 5=-UTR, especially the ribosomal binding site, is the most sensitive for as-RNA silencing (13,15,22), it is unclear how the start point, binding location, and RNA structure affect the inhibitory efficiency.…”

Section: Discussionmentioning

confidence: 99%

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Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2 S )-Naringenin Production from l -Tyrosine in Escherichia coli

et al. 2014

Appl Environ Microbiol

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cMalonyl coenzyme A (malonyl-CoA) is an important precursor for the synthesis of natural products, such as polyketides and flavonoids. The majority of this cofactor often is consumed for producing fatty acids and phospholipids, leaving only a small amount of cellular malonyl-CoA available for producing the target compound. The tuning of malonyl-CoA into heterologous pathways yields significant phenotypic effects, such as growth retardation and even cell death. In this study, fine-tuning of the fatty acid pathway in Escherichia coli with antisense RNA (asRNA) to balance the demands on malonyl-CoA for target-product synthesis and cell health was proposed. To establish an efficient asRNA system, the relationship between sequence and function for asRNA was explored. It was demonstrated that the gene-silencing effect of asRNA could be tuned by directing asRNA to different positions in the 5=-UTR (untranslated region) of the target gene. Based on this principle, the activity of asRNA was quantitatively tailored to balance the need for malonyl-CoA in cell growth and the production of the main flavonoid precursor, (2S)-naringenin. Appropriate inhibitory efficiency of the anti-fabB/fabF asRNA improved the production titer by 431% (391 mg/liter). Therefore, the strategy presented in this study provided a useful tool for the fine-tuning of endogenous gene expression in bacteria.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2 S )-Naringenin Production from l -Tyrosine in Escherichia coli

et al. 2014

Appl Environ Microbiol

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“…Table 1 shows a comparison of the itaconate titers reported in the literature. Dynamic control has already been applied in E. coli (40) and S. cerevisiae (6) for the synthesis of products that are toxic to cells. In addition to metabolite concentrations (41, 42), other triggers, e.g., heat shock (43), quorum sensing (44), and stress response to intermediate toxicity (5), have served as tools for dynamic control.…”

Section: Discussionmentioning

confidence: 99%

P gas , a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

Yin

Shin

et al. 2017

Appl Environ Microbiol

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The dynamic control of gene expression is important for adjusting fluxes in order to obtain desired products and achieve appropriate cell growth, particularly when the synthesis of a desired product drains metabolites required for cell growth. For dynamic gene expression, a promoter responsive to a particular environmental stressor is vital. Here, we report a low-pH-inducible promoter, Pgas, which promotes minimal gene expression at pH values above 5.0 but functions efficiently at low pHs, such as pH 2.0. First, we performed a transcriptional analysis of Aspergillus niger, an excellent platform for the production of organic acids, and we found that the promoter Pgas may act efficiently at low pH. Then, a gene for synthetic green fluorescent protein (sGFP) was successfully expressed by Pgas at pH 2.0, verifying the results of the transcriptional analysis. Next, Pgas was used to express the cis-aconitate decarboxylase (cad) gene of Aspergillus terreus in A. niger, allowing the production of itaconic acid at a titer of 4.92 g/liter. Finally, we found that Pgas strength was independent of acid type and acid ion concentration, showing dependence on pH only. IMPORTANCE The promoter Pgas can be used for the dynamic control of gene expression in A. niger for metabolic engineering to produce organic acids. This promoter may also be a candidate tool for genetic engineering.KEYWORDS Aspergillus niger, low-pH-inducible promoter, itaconic acid, dynamic gene expression, metabolic engineering A spergillus niger is an excellent cell factory for the production of organic acids (1). The rational engineering of A. niger has attracted increasing attention, and great achievements have been made in this area (2, 3). Nevertheless, because heterologous pathways are controlled by constitutive promoters, e.g., the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA), most of this research has focused on static metabolic engineering; that is, the gene expression level is set without sensing changes in the pathway output or cellular environment (4). One disadvantage of static control is related to the trade-off between growth and the production of a desired compound; suboptimal productivity is obtained because these pathways can drain metabolites required for biomass synthesis (5). Metabolic engineering approaches are being developed to tackle this problem via a dynamic control system to compensate for changing conditions (6). In this system, the cell modulates its metabolic pathways dynamically to adjust fluxes such that the required metabolic intermediates are delivered at the appropriate levels and times to optimize growth (7).

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“…15 Recently, the antisense RNA (asRNA)based repression techniques were applied in E. coli to directly control glycolytic flux via repression of glk to enhance flavonoid production via repression of fab operon and to increase polyketide 6deoxyerythronolide B production via repression of guaB and zwf. [110][111][112][113] Thus, the RNAbased methods are complementary to gene knockout method and possesses some advantages in controlling multigenes includ ing essential genes. The newly developed CRISPR/dCas9 repression sys tem (CRISPRi) holds more promising applications in metabolic engineering because of its higher repression efficacy than sRNA or asRNA method.…”

Section: Strategies In Metabolic Engineering Of Glycolysismentioning

confidence: 99%

Glycolysis and Its Metabolic Engineering Applications

Wang¹,

Yan²

2017

Engineering Microbial Metabolism for Chemical Synthesis

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IntroductionMicroorganisms play a pivotal role in the degradation of complex carbon sources in nature and could survive in different conditions due to the genetically coined cellular metabolism. Cellular metabolism of microorg anisms is defined as the sum of biochemical processes that involves the conversion of environmental nutrients into simple biosynthetic building blocks and energy in the cells. Within these metabolic processes, catabo lism is responsible for breakdown of complex molecules into simple molecules, producing energy, reducing power, and precursor intermedi ates for other metabolic processes like anabolism. The central catabolic pathways include Embden-Meyerhof-Parnas (EMP) pathway, pentose phosphate (PP) pathway, Entner-Doudoroff (ED) pathway, and the tricar boxylic acid (TCA) cycle. The EMP pathway, also known as glycolysis

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A dynamic metabolite valve for the control of central carbon metabolism

Cited by 96 publications

References 55 publications

Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2 S )-Naringenin Production from l -Tyrosine in Escherichia coli

Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2 S )-Naringenin Production from l -Tyrosine in Escherichia coli

P gas , a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

Glycolysis and Its Metabolic Engineering Applications

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