scholarly journals In Silico-guided Metabolic Engineering of Bacillus Subtilis for Efficient Biosynthesis of Purine Nucleosides by Blocking the Key Backflow Nodes

2021 ◽  
Author(s):  
Aihua Deng ◽  
Qidi Qiu ◽  
Qinyun Sun ◽  
Zhenxiang Chen ◽  
Junyue Wang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Engineering ◽  
In Silico ◽  
Metabolic Flux ◽  
De Novo ◽  
Feedback Inhibition ◽  
Purine Nucleotides ◽  
Metabolic Switch ◽  
Purine Synthesis ◽  
Wide Range

Abstract Background: Purine nucleosides play essential roles in cellular physiological processes and have a wide range of applications in the fields of antitumor/antiviral drugs and food. However, microbial overproductions of purine nucleosides by de novo metabolic engineering have been a great challenge due to their strict and complex regulatory machinery involved in the biosynthetic pathways. Results: In this study, we designed an in silico-guided strategy for overproducing purine nucleosides based on the genome-scale metabolic network model in Bacillus subtilis. The metabolic flux was analyzed to predict two key backflow nodes Drm (Purine nucleotides toward PPP) and YwjH (PPP-EMP) for resolving the competitive relationship between biomass and purine nucleotides synthesis. In terms of the purine synthesis pathway, the first backflow node Drm was inactivated to block the degradation of purine nucleotides and greatly increased the inosine production to 13.98–14.47 g/L without affecting cell growth. Furthermore, releasing feedback inhibition of purine operon by promoter replacement further enhanced the accumulation of purine nucleotides. In terms of the central carbon metabolic pathways, deleting the second backflow node YwjH and overexpressing Zwf were combined to increase the inosine production to 22.01±1.18 g/L by enhancing the metabolic flow of PPP. Through switching on the flux node of the glucose-6- phosphate to PPP or EMP, the final inosine engineered strain produced up to 25.81±1.23 g/L of inosine by a pgi-based metabolic switch in shake-flask cultivation, suggesting the highest yield in de novo engineered inosine bacteria. Under the guidance of the in silico-designed strategy, a general chassis bacterium was generated for the first time to efficiently synthesize inosine, adenosine, guanosine, IMP, and GMP, providing the sufficient precursor for the synthesis of various purine intermediates. Conclusions: Overall, in silico-guided metabolic engineering successfully optimized the purine synthesis pathway by exploring the efficient targets, representing a superior strategy for efficient biosynthesis of the biotechnological products.

scholarly journals Constraint-based metabolic control analysis for rational strain engineering

2020 ◽  
Author(s):  
Sophia Tsouka ◽  
Meric Ataman ◽  
Tuure Hameri ◽  
Ljubisa Miskovic ◽  
Vassily Hatzimanikatis
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Metabolic Control ◽  
Metabolic Flux ◽  
Strain Engineering ◽  
Metabolic Control Analysis ◽  
Physiological Data ◽  
Response Analysis ◽  
Control Analysis ◽  
Wide Range

AbstractThe advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering interventions on flows in metabolic networks and metabolic concentrations, it fails to account for the physiological limitations of the cellular environment and metabolic engineering design constraints. We report here a constraint-based framework based on MCA, Network Response Analysis (NRA), for the rational genetic strain design that incorporates biologically relevant constraints, as well as genome editing restrictions. The NRA core constraints being similar to the ones of Flux Balance Analysis, allow it to be used for a wide range of optimization criteria and with various physiological constraints. We show how the parametrization and introduction of biological constraints enhance the NRA formulation compared to the classical MCA approach, and we demonstrate its features and its ability to generate multiple alternative optimal strategies given several user-defined boundaries and objectives. In summary, NRA is a sophisticated alternative to classical MCA for rational metabolic engineering that accommodates the incorporation of physiological data at metabolic flux, metabolite concentration, and enzyme expression levels.

Increased de novo purine synthesis by insulin through selective enzyme induction in primary cultured rat hepatocytes

1990 ◽  
Vol 258 (5) ◽  
pp. C841-C848 ◽  
Author(s):  
M. Tsuchiya ◽  
H. Yoshikawa ◽  
M. Itakura ◽  
K. Yamashita
Keyword(s):  
Enzyme Induction ◽  
De Novo ◽  
Feedback Inhibition ◽  
Rat Hepatocytes ◽  
Purine Metabolism ◽  
Hepatocyte Proliferation ◽  
Adenylate Energy Charge ◽  
Purine Synthesis ◽  
Primary Cultured Rat Hepatocytes ◽  
Cultured Rat Hepatocytes

The proliferative effect of insulin on de novo purine synthesis and on the expression of various enzymes of purine metabolism were studied in primary cultured rat hepatocytes. Insulin greater than 1.5 x 10(-8) M increased DNA and de novo purine synthesis to 260-390 and 270-420%, respectively, 24 and 8 h after the administration. Insulin at 1.5 x 10(-7) M increased the specific activity of amidophosphoribosyltransferase (ATase) to 154-180%, hypoxanthine-guanine phosphoribosyltransferase to 129%, and adenine phosphoribosyltransferase (APRT) to 205%, in contrast to unchanged xanthine dehydrogenase at 80%. Enzyme induction was supported by the results of kinetic analysis and the inhibition of the insulin-induced increase in enzyme activities by protein synthesis inhibitors. Insulin increased ATP to 127% and decreased AMP, ADP, 5'-guanylic acid (GMP), and guanosine 5'-diphosphate (GDP), respectively, to 73, 69, 73, and 69%. Insulin increased adenylate energy charge from 0.83 to 0.90 without changing total feedback inhibitory potential on ATase. No obvious increase of 5-phosphoribosyl-1-pyrophosphate supply was suggested, although its apparent availability for purine ribonucleotide synthesis was increased to 208-245%, reflecting mainly induced APRT activity to 205%. It is concluded that hepatocyte proliferation by insulin, as evidenced by purine metabolism, is mediated by the selective gene activation of anabolic enzymes and increased ATP as the basis to activate multiple metabolic pathways without remarkable changes of substrate availability or feedback inhibition.

De novo engineering and metabolic flux analysis of inosine biosynthesis in Bacillus subtilis

2011 ◽  
Vol 33 (8) ◽  
pp. 1575-1580 ◽  
Author(s):  
Haojian Li ◽  
Guoqiang Zhang ◽  
Aihua Deng ◽  
Ning Chen ◽  
Tingyi Wen
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
De Novo ◽  
Flux Analysis

scholarly journals Metabolic Engineering of the Purine Pathway for Riboflavin Production in Ashbya gossypii

2005 ◽  
Vol 71 (10) ◽  
pp. 5743-5751 ◽  
Author(s):  
Alberto Jiménez ◽  
María A. Santos ◽  
Markus Pompejus ◽  
José L. Revuelta
Keyword(s):  
De Novo ◽  
Feedback Inhibition ◽  
Site Directed Mutagenesis ◽  
Transcriptional Level ◽  
Ashbya Gossypii ◽  
Purine Nucleotides ◽  
Phosphoribosyl Pyrophosphate ◽  
Triple Mutant ◽  
Riboflavin Production ◽  
Purine Pathway

ABSTRACT Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid310, lysine333, and alanine417) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter).

scholarly journals Biocomputational genome-wide analysis of micro RNA genetic variability in some vertebrates

Genetika ◽  
2013 ◽  
Vol 45 (3) ◽  
pp. 799-810
Author(s):  
Abdulmojeed Yakubu ◽  
Ibrahim Musa-Azara ◽  
Blessing Yakubu ◽  
Sylvester Daikwo ◽  
Samuel Vincent ◽  
...  
Keyword(s):  
Genetic Variability ◽  
In Silico ◽  
Target Genes ◽  
De Novo ◽  
Experimental Studies ◽  
Micro Rna ◽  
Seed Region ◽  
Livestock Species ◽  
Human Mirnas ◽  
Wide Range

MicroRNAs (miRNAs) are small endogenously expressed single-stranded RNAs that regulate gene expression post transcriptionally and shape diverse cellular pathways. miRNAs regulate a wide range of biological processes through the recognition of complementary sequences between miRNAs and their target genes. The present investigation aimed at determining in-silico the genetic variability of miRNA genes in some livestock and non-livestock species. Effects of single nucleotide polymorphisms (SNPs) in genes? 3'UTR on target gain/loss of human miRNAs were also explored. A total of twenty four mature miRNA sequences and genomic coordinates in three livestock [chicken (5), pig (1) and cattle (9)] and two non-livestock (human (6) and mouse (3)] species were retrieved from the miRBase 15 release. Computational scanning of polymorphisms in the miRNAs revealed 33 and 20 polymorphic sites in livestock and non-livestock species, respectively. Of this, 7 (chicken), 11 (cattle) and 2 (mouse) were located within the seed region. The de novo computational prediction revealed that SNPs rs1042725 (C/U) and rs1044129 (A/G) in genes? 3'UTR of human miRNAs positively influenced the target site thereby resulting in target gain. However, the effects of SNPs rs56109847 (A/G), rs28927680, rs12720208 (G/A) and rs5186 (A/C) were negative. The evolutionary tree showed that the relationship between miRNA consensus sequences of livestock (pig, chicken and cattle) was closer compared to non-livestock species (mouse and human), which could be implicated in morphological complexity among vertebrates. Although the function of miRNA is only beginning to be understood, future in-silico research evaluating the functional effect of miRNA in gene translation and subsequent biological pathways especially in livestock is of paramount importance; and this should be complemented with hypothesis-driven experimental studies to evaluate the phenotypic effect of identified miRNA genetic polymorphisms in animals.

scholarly journals Identification of sugarcane genes involved in the purine synthesis pathway

2001 ◽  
Vol 24 (1-4) ◽  
pp. 251-255 ◽  
Author(s):  
Mario A. Jancso ◽  
Susana A. Sculaccio ◽  
Otavio H. Thiemann
Keyword(s):  
Expressed Sequence Tag ◽  
De Novo ◽  
Statistical Significance ◽  
Local Alignment ◽  
Central Importance ◽  
Purine Nucleotides ◽  
Nucleotide Synthesis ◽  
Purine Synthesis ◽  
Search Tool ◽  
Expressed Sequence

Nucleotide synthesis is of central importance to all cells. In most organisms, the purine nucleotides are synthesized de novo from non-nucleotide precursors such as amino acids, ammonia and carbon dioxide. An understanding of the enzymes involved in sugarcane purine synthesis opens the possibility of using these enzymes as targets for chemicals which may be effective in combating phytopathogen. Such an approach has already been applied to several parasites and types of cancer. The strategy described in this paper was applied to identify sugarcane clusters for each step of the de novo purine synthesis pathway. Representative sequences of this pathway were chosen from the National Center for Biotechnology Information (NCBI) database and used to search the translated sugarcane expressed sequence tag (SUCEST) database using the available basic local alignment search tool (BLAST) facility. Retrieved clusters were further tested for the statistical significance of the alignment by an implementation (PRSS3) of the Monte Carlo shuffling algorithm calibrated using known protein sequences of divergent taxa along the phylogenetic tree. The sequences were compared to each other and to the sugarcane clusters selected using BLAST analysis, with the resulting table of p-values indicating the degree of divergence of each enzyme within different taxa and in relation to the sugarcane clusters. The results obtained by this strategy allowed us to identify the sugarcane proteins participating in the purine synthesis pathway.

scholarly journals Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Arun S. Rajkumar ◽  
John P. Morrissey
Keyword(s):  
Kluyveromyces Marxianus ◽  
Minimal Medium ◽  
Biosynthetic Pathway ◽  
Metabolic Flux ◽  
De Novo ◽  
Feedback Inhibition ◽  
Industrial Biotechnology ◽  
Branch Points ◽  
Specific Product ◽  
Rational Engineering

Abstract Background The yeast Kluyveromyces marxianus offers unique potential for industrial biotechnology because of useful features like rapid growth, thermotolerance and a wide substrate range. As an emerging alternative platform, K. marxianus requires the development and validation of metabolic engineering strategies to best utilise its metabolism as a basis for bio-based production. Results To illustrate the synthetic biology strategies to be followed and showcase its potential, we describe a comprehensive approach to rationally engineer a metabolic pathway in K. marxianus. We use the phenylalanine biosynthetic pathway both as a prototype and because phenylalanine is a precursor for commercially valuable secondary metabolites. First, we modify and overexpress the pathway to be resistant to feedback inhibition so as to overproduce phenylalanine de novo from synthetic minimal medium. Second, we assess native and heterologous means to increase precursor supply to the biosynthetic pathway. Finally, we eliminate branch points and competing reactions in the pathway and rebalance precursors to redirect metabolic flux to a specific product, 2-phenylethanol (2-PE). As a result, we are able to construct robust strains capable of producing over 800 mg L−1 2-PE from minimal medium. Conclusions The strains we constructed are a promising platform for the production of aromatic amino acid-based biochemicals, and our results illustrate challenges with attempting to combine individually beneficial modifications in an integrated platform.

scholarly journals Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid

2020 ◽  
Author(s):  
Pingping Zhou ◽  
Chunlei Yue ◽  
Bin Shen ◽  
Yi Du ◽  
Nannan Xu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Caffeic Acid ◽  
Acid Production ◽  
De Novo ◽  
Feedback Inhibition ◽  
Biological Activities ◽  
Acid Product ◽  
Enhanced Production ◽  
Microbial Biosynthesis

Abstract Background As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently, Saccharomyces cerevisiae has been reported sporadically for biosynthesis of caffeic acid via free plasmid‑mediated pathway assembly. However, the production was far from satisfactory and even relied on the addition of precursor. Results In this study, we first established a controllable caffeic acid pathway by employing a modified GAL regulatory system in S. cerevisiae and realized de novo biosynthesis of 313.8 mg/L caffeic acid from glucose. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback inhibition, deleting genes involved in competing pathways and overexpressing rate-limiting enzymes led to about 2.5-fold improvement in the caffeic acid production, reaching up to 769.3 mg/L in shake-flask cultures. To our knowledge, this is the highest ever reported titer of caffeic acid de novo synthesized by engineered yeast. Conclusions Caffeic acid production in S. cerevisiae strain was successfully improved by adopting a glucose-regulated GAL system and comprehensive metabolic engineering strategies. This work showed the prospect for microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of its derived metabolites.

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