Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosyntheses of menaquinone and 2,3-dihydroxybenzoate.
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ABSTRACT:
SUBMITTER: Rowland BM
PROVIDER: S-EPMC177735 | biostudies-other | 1996 Feb
REPOSITORIES: biostudies-other
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Project description:To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
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Project description:The formation of biofilm facilitates the synthesis of valuable natural product menaquinone-7 (MK-7) in static culture of Bacillus subtilis, whereas the essential role and mechanism of biofilm in MK-7 synthesis have not been revealed. Herein, comparative transcriptomics show that the formation of biofilm affected MK-7 synthesis by changing the transcription levels of signal receptor (BSU02010), transmembrane transporter (BSU29340, BSU03070), and signal transduction (BSU02630). Moreover, we also found that oxalate decarboxylase OxdC has an important effect on electron generation and MK-7 synthesis, when the transcriptional level of NADH dehydrogenase decreases in static culture. Our results revealed that cell membrane and electron transfer are important factors in promoting MK-7 synthesis.
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Project description:The first committed step in the classical biosynthetic route to menaquinone (vitamin K(2)) is a Stetter-like conjugate addition of alpha-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 A) crystal structure of Bacillus subtilis MenD with cofactor and Mn(2+) has been determined. Based on structure-sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of alpha-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure-reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.
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Project description:Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305-310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis.
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Project description:The presence of duplicated genes in organisms is well documented. There is increasing interest in understanding how these genes subfunctionalize and whether functional overlap can explain the fact that some of these genes are dispensable. Bacillus subtilis possesses four DEAD-box RNA helicases (DBRH) genes, cshA, cshB, deaD/yxiN, and yfmL that make a good case to study to what extent they can complement each other despite their subfunctionalization. They possess the highly conserved N-terminal catalytic domain core common to RNA helicases, but different carboxy-terminal ends. All four genes have been shown to have independent functions although all participate in rRNA assembly. None of the B. subtilis DBRH is essential for growth at 37°C, and all single deletion mutants exhibit defective growth at 18°C except for ?deaD/yxiN. Evaluation of double mutants did not reveal negative epistasis, suggesting that they do not have overlapping functions. The absence of any one gene distorts the expression pattern of the others, but not in a specific pattern suggestive of compensation. Overexpression of these paralogous genes in the different mutant backgrounds did not result in cross-complementation, further confirming their lack of buffering capability. Since no complementation could be observed among full sized proteins, we evaluated to what extent the superfamily 2 (SF2) helicase core of the smallest DBRH, YfmL, could be functional when hooked to each of the C-terminal end of CshA, CshB, and DeaD/YxiN. None of the different chimeras complemented the different mutants, and instead, all chimeras inhibited the growth of the ?yfmL mutant, and other combinations were also deleterious. Our findings suggest that the long time divergence between DEAD-box RNA helicase genes has resulted in specialized activities in RNA metabolism and shows that these duplicated genes cannot buffer one another.
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Project description:BackgroundMenaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation.ResultsWe used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion.ConclusionsIn summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.
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| S-EPMC177381 | biostudies-other
Project description:Menaquinone-7 (MK-7) is an important vitamin K2, synthesized from the menaquinone parent ring and seven isoprene side chains. Presently, the synthesis of MK-7 stimulated by environmental stress primarily focuses on oxygen stress, while the effect of alkali stress is rarely studied. Therefore, this study researched the effects of alkali stress on the fermentation performance and gene expression of Bacillus subtilis natto. The organism's growth characteristics, biomass, sporogenesis, MK-7 biosynthesis, and gene expression were analyzed. After a pH 8.5 stress adaptation treatment for 0.5 h and subsequent fermentation at pH 8.5, which promoted the growth of the strain and inhibited the spore formation rate. In addition, biomass was significantly increased (P < 0.05). The conversion rate of glycerol to MK-7 was 1.68 times higher than that of the control group, and the yield of MK-7 increased to 2.10 times. Transcriptomic analysis showed that the MK-7 high-yielding strain had enhanced carbon source utilization, increased glycerol and pyruvate metabolism, enhanced the Embden-Meyerhof pathway (EMP), tricarboxylic acid (TCA) circulation flux, and terpenoid biosynthesis pathway, and promoted the accumulation of acetyl-CoA, the side-chain precursor of isoprene. At the same time, the up-regulation of transketolase increased the metabolic flux of the pentose phosphate (HMP) pathway, which was conducive to the accumulation of D-erythrose 4-phosphate, the precursor of the menadione parent ring. This study's results contribute to a better understanding of the effects of environmental stress on MK-7 fermentation by Bacillus subtilis natto and the molecular regulatory mechanism of MK-7 biosynthesis.
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