Closer look at the matching condition for radiative QCD θ\theta parameter

In this paper, we scrutinize a radiatively generated QCD $\theta$ parameter at the two-loop level based on both full analytical loop functions with the Fock-Schwinger gauge method and the effective field theory approach, using simplified models. We observe that the radiatively generated $\theta$ parameters at the low energy scale precisely match between them. It provides validity to perturbative loop calculations of the QCD $\theta$ parameter with the Fock-Schwinger gauge method. Furthermore, it is also shown that the ordinary Fujikawa method for the radiative $\theta$ parameter by using $\bar\theta = - arg det M_q^{loop}$ does not cover all contributions in the simplified models. But, we also find that when there is a scale hierarchy in $CP$-violating sector, evaluation of the Fujikawa method is numerically sufficient. As an application, we calculate the radiative $\theta$ parameter at the two-loop level in a slightly extended Nelson-Barr model, where the spontaneous $CP$ violation occurs to solve the strong $CP$ problem. It is found a part of the radiative $\theta$ parameters cannot be described by the Fujikawa method.Comment: 24 pages, 4 figures, 1 tabl

A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum

Abstract Background Microbial production of aromatic chemicals is an attractive method for obtaining high-performance materials from biomass resources. A non-proteinogenic amino acid, 4-amino-3-hydroxybenzoic acid (4,3-AHBA), is expected to be a precursor of highly functional polybenzoxazole polymers; however, methods for its microbial production have not been reported. In this study, we attempted to produce 4,3-AHBA from glucose by introducing 3-hydroxylation of 4-aminobenzoic acid (4-ABA) into the metabolic pathway of an industrially relevant bacterium, Corynebacterium glutamicum. Results Six different 4-hydroxybenzoate 3-hydroxylases (PHBHs) were heterologously expressed in C. glutamicum strains, which were then screened for the production of 4,3-AHBA by culturing with glucose as a carbon source. The highest concentration of 4,3-AHBA was detected in the strain expressing PHBH from Caulobacter vibrioides (CvPHBH). A combination of site-directed mutagenesis in the active site and random mutagenesis via laccase-mediated colorimetric assay allowed us to obtain CvPHBH mutants that enhanced 4,3-AHBA productivity under deep-well plate culture conditions. The recombinant C. glutamicum strain expressing CvPHBHM106A/T294S and having an enhanced 4-ABA biosynthetic pathway produced 13.5 g/L (88 mM) 4,3-AHBA and 0.059 g/L (0.43 mM) precursor 4-ABA in fed-batch culture using a nutrient-rich medium. The culture of this strain in the chemically defined CGXII medium yielded 9.8 C-mol% of 4,3-AHBA from glucose, corresponding to 12.8% of the theoretical maximum yield (76.8 C-mol%) calculated using a genome-scale metabolic model of C. glutamicum. Conclusions Identification of PHBH mutants that could efficiently catalyze the 3-hydroxylation of 4-ABA in C. glutamicum allowed us to construct an artificial biosynthetic pathway capable of producing 4,3-AHBA on a gram-scale using glucose as the carbon source. These findings will contribute to a better understanding of enzyme-catalyzed regioselective hydroxylation of aromatic chemicals and to the diversification of biomass-derived precursors for high-performance materials

Specific expression and function of the A-type cytochrome c oxidase under starvation conditions in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has one A-type (caa3) and multiple C-type (cbb3) cytochrome c oxidases as well as two quinol oxidases for aerobic respiration. The caa3 oxidase is highly efficient in creating a proton gradient across the cell membrane, but it is not expressed under normal growth conditions and its physiological role has not been investigated. In the present study, a mutant strain deficient in the coxBA-PA0107-coxC genes encoding caa3 exhibited normal growth under any test conditions, but it had low relative fitness under carbon starvation conditions, indicating that the expression of caa3 is advantageous under starvation conditions. A mutant that lacked four terminal oxidase gene clusters except for the cox genes was unable to grow aerobically because of low expression level of caa3. However, suppressor mutants that grew aerobically using caa3 as the only terminal oxidase emerged after aerobic subculturing. Analyses of the suppressor mutants revealed that a mutation of roxS encoding a sensor kinase of a two-component regulator RoxSR was necessary for the aerobic growth in synthetic medium. Two additional mutations in the 5'-flanking region of coxB were necessary for the aerobic growth in LB medium. Although the expression level of caa3 was higher in the suppressor mutants, their growth rates were lower than when the other terminal oxidases were utilized, suggesting that caa3 was not suited for utilization as the only terminal oxidase. Overexpression of the cox genes also inhibited the aerobic growth of the wild-type strain. These results indicate that caa3 is tightly regulated to be expressed only under starvation conditions at low level and it functions in cooperation with other terminal oxidases to facilitate survival in nutrient starvation conditions

Variants of the industrially relevant protease KP-43 with suppressed activity under alkaline conditions developed using expanded genetic codes

In the present study, we attempted to control the pH profile of the catalytic activity of the industrially relevant alkaline protease KP-43, by incorporating 3-nitro-l-tyrosine and 3-chloro-l-tyrosine at and near the catalytic site. Thirty KP-43 variants containing these non-natural amino acids at the specific positions were synthesized in Escherichia coli host cells with expanded genetic codes. The variant with 3-nitrotyrosine at position 205, near the substrate binding site, retained its catalytic activity at the neutral pH and showed a 60% activity reduction at pH 10.5. This reduction in the alkaline domain is desirable for enhancing the stability of the enzyme in the liquid laundary detergent, whereas the wild-type molecule showed a 20% increase in response to the same pH shift. The engineered pH dependency of the activity of the variant was ascribed partly to a lowered substrate affinity under the alkaline conditions, in which the incorporated 3-nitrotyrosine was probably charged negatively due to the phenolic pKa lower than that of tyrosine. Keywords: Detergent enzyme, Alkaline protease, Nitrotyrosine, Chlorotyrosine, Protein engneering

Effect of the overexpression of the <i>cox</i> genes.

<p>Strains transformed with pMMB67EH or pMMB-cox were cultivated aerobically in 4 ml of LB medium <b>(A)</b> and glutamate medium <b>(B)</b> in test tubes shaken at 200 rpm. The media were supplemented with 200 μg/ml carbenicillin and 0.5 mM IPTG. The results shown are representative based on at least three independent experiments. The maximum specific growth rates (μ_max) of WT (pMMB67EH), WT (pMMB-cox), ROX1 (pMMB67EH), and ROX1 (pMMB-cox) were 0.79 ± 0.055, 0.30 ± 0.041, 0.72 ± 0.067, and 0.34 ± 0.103 h⁻¹, respectively, for LB medium and 0.65 ± 0.034, 0.27 ± 0.014, 0.67 ± 0.042, and 0.50 ± 0.013 h⁻¹, respectively, for glutamate medium. The μ_max value was not calculated for the recombinant strains of PTO5, because they did not show apparent growth.</p

Effects of the suppressor mutations on the transcriptional activity of <i>coxB</i>.

<p>Effects of the suppressor mutations on the transcriptional activity of <i>coxB</i>.</p

Growth profiles of the suppressor mutants.

<p>Strains were cultivated aerobically in 40 ml LB medium or glutamate medium in 100-ml Erlenmeyer flasks with shaking at 300 rpm. The results shown are representative based on three independent experiments. The maximum specific growth rates (μ_max) of WT, QXAaS1, and QXAaS2 were 1.30 ± 0.089, 0.09 ± 0.008, and 0.26 ± 0.041 h⁻¹, respectively, for LB medium and 1.06 ± 0.019, 0.15 ± 0.016, and 0.40 ± 0.034 h⁻¹, respectively, for glutamate medium.</p

Effects of knockout of the <i>cox</i> genes encoding the <i>caa</i>₃ oxidase.

<p><b>(A)</b> Growth profiles of the WT (filled circles) and <i>cox</i> mutant strain SAa (open triangles) under carbon-limited conditions. The strains were grown aerobically in 3 ml of synthetic medium supplemented with 0.6 g/l glucose in test tubes with shaking at 200 rpm. <b>(B)</b> Colony-forming units (CFU) of the strains on LB plates. CFUs were counted immediately after inoculating the cells and after cultivation for 11 h in synthetic medium supplemented with 0.6 g/l glucose. Black and gray bars indicate WT and SAa, respectively. The results represent the means based on three measurements. Error bars indicate standard deviations of the means. Asterisk indicates that the mean values are significantly different by unpaired two-tailed Student’s <i>t</i> test (<i>p</i><0.05). NS indicates that the means of the two samples are not significantly different.</p