Background: The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences. Results: We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Conclusions: Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

Candida albicans, C. glabrata, C. tropicalis, and C. parapsilosis account for approximately 95% of identifiable Candida infections. Other species, including C. krusei, C. lusitaniae, and C. guilliermondii, account for less than 5% of cases of invasive candidiasis. The most common causative agent is still C. albicans, but its incidence is declining and the frequencies of other species are increasing. Recently, Furlaneto et al. 1 noted that non-albicans Candida was the predominant species in different clinical specimens, with the exception of urine samples, in a Brazilian tertiary-care hospital. Invasive candidiasis has a mortality rate that approaches 40% 2,3 . Although most people are colonized by Candida sp., the majority never develop invasive candidiasis. Alterations in host immunity, physiological features, or normal microflora, rather than the acquisition of novel or hypervirulent factors by Candida, are suggested to degenerate the commensal-host interaction and lead to an opportunistic infection 4 .During the course of a systemic infection, Candida cells are engulfed by host phagocytes, where they are exposed to reactive oxygen species (ROS) 5 . ROS contribute to the killing of C. albicans in both cultured cells and entire organisms [6][7][8][9] . Upon incubation with macrophages, C. albicans deoxyribonucleic acid (DNA) repair genes are transcriptionally induced, suggesting that DNA damage indeed occurs in the phagosome and that genotoxic hypersensitivity stress would be disadvantageous to the pathogen 10 . Recently, it was demonstrated that a large proportion of C. albicans cell surface antigens related to acute candidemia are involved in oxidative stress 4 . In C. albicans, hyphal cells ABSTRACT Introduction: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. Methods: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS* + ). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). Results: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. Conclusions: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to resp...

Reproduction is a costly life process, and the reproductive investment by females appears to be greater than males in many species. We have analyzed the effects of reproductive investment during aging with respect to oxidative stress parameters in female Wistar rats. We measured the activity glutathione peroxidase, glutathione S-transferase, superoxide dismutase, consumption of hydrogen peroxide, protein carbonylation, lipid peroxidation, nitrite and nitrate levels, and Vitamin C (Vit. C) and E levels. We traced oxidative profiles at ages 3, 6, 12, and 24 months. Animals were grouped according to reproductive experience: experienced or naive with respect to reproductive activity. We measured aconitase activity and sex hormone levels. The naive animals exhibited an increase with respect to experienced in most parameters studied at 6 and 24 months, whereas experienced animals exhibited a similar increase at 3 and 12 months. At 6 months of age, during the period that would represent peak reproductive activity, naive animals showed higher levels of MDA, Vit. C, consumption of hydrogen peroxide and GPx, aconitase, and SOD activities. In naive elderly rats, we observed an increase in oxidative damage markers and an increase in enzymatic and non-enzymatic antioxidants, with the exception of consumption of hydrogen peroxide and Vit. C. In the long term, the reproductive investment was not sufficient to interfere with antioxidant capacity, and did not contribute to oxidative damage in kidneys of female Wistar rats.

The results suggest that the antioxidant response varies among evaluated supplementations and all supplements were able to alter enzymatic and non-enzymatic antioxidants in the livers of ovariectomized rats. DHA presented the most evident antioxidant effect, decreasing protein and lipid damage.

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster‐growing non‐producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. The stability of E. coli expression systems under long‐term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations are investigated. Iterated fed‐batch‐like microbioreactor cultivations are conducted under production conditions. The easy‐to‐produce green fluorescent protein (GFP) and a challenging antigen‐binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21Q strains as expression hosts. In comparative whole‐genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. A T7 RNA polymerase expression system is only conditionally suitable for long‐term cultivation under production conditions. Mutations leading to non‐producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase‐based BL21Q expression system remains stable in the production of GFP in long‐term cultivations. For the production of Fab, mutations in lacI of the BL21Q derivatives have positive effects on long‐term stability. The results indicate that adaptive evolution carried out with genome‐integrated E. coli expression systems in microtiter cultivations under industrial‐relevant production conditions is an efficient strain development tool for production hosts.