POLA1

Para-hydroxybenzoic acid (PHBA) is extensively used as an additive in the food and cosmetics industries, significantly enhancing product shelf life and stability. While microbial fermentation offers an environment-friendly and sustainable method for producing PHBA, the titer and productivity are limited due to product toxicity and complex metabolic flux distributions. Here, we initially redesigned a L-phenylalanine-producing Escherichia coli by employing rational metabolic engineering strategies, resulting in the production of PHBA reached the highest reported level of 14.17 g/L. Subsequently, a novel accelerated evolution system was devised comprising deaminase, the alpha subunit of RNA polymerase, an uracil-DNA glycosylase inhibitor, and the PHBA-responsive promoter P_yhcN. This system enabled us to obtain a mutant strain exhibiting a 47% increase in the half-inhibitory concentration (IC₅₀) for PHBA within 15 days. Finally, the evolved strain achieved a production of 21.35 g/L PHBA in a 5-L fermenter, with a yield of 0.19 g/g glucose and a productivity rate of 0.44 g/L/h. This engineered strain emerges as a promising candidate for industrial production of PHBA through an eco-friendly approach.

The ATR-CHK1 pathway plays a fundamental role in the DNA damage response and is therefore an attractive target in cancer therapy. The antitumorous effect of ATR inhibitors is at least partly caused by synthetic lethality between ATR and various DNA repair genes. In previous studies, we have identified members of the B-family DNA polymerases as potential lethal partner for ATR, i.e. POLD1 and PRIM1. In this study, we validated and characterized the synthetic lethality between ATR and POLA1. First, we applied a model of ATR-deficient DLD-1 human colorectal cancer cells to confirm synthetic lethality by using chemical POLA1 inhibition. Analyzing cell cycle and apoptotic markers via FACS and Western blotting, we were able to show that apoptosis and S phase arrest contributed to the increased sensitivity of ATR-deficient cancer cells towards POLA1 inhibitors. Importantly, siRNA-mediated POLA1 depletion in ATR-deficient cells caused similar effects in regard to impaired cell viability and cumulation of apoptotic markers, thus excluding toxic effects of chemical POLA1 inhibition. Conversely, we demonstrated that siRNA-mediated POLA1 depletion sensitized several cancer cell lines towards chemical inhibition of ATR and its main effector kinase CHK1. In conclusion, the synthetic lethality between ATR/CHK1 and POLA1 might represent a novel and promising approach for individualized cancer therapy: First, alterations of POLA1 could serve as a screening parameter for increased sensitivity towards ATR and CHK1 inhibitors. Second, alterations in the ATR-CHK1 pathway might predict in increased sensitivity towards POLA1 inhibitors.

Chloro-haloacetonitrile (Cl-HAN), belongs to a group of nitrogenous disinfection by-products (N-DBPs) found in surface water, and are known to pose a major risk to the safety of human drinking water. However, the exact biological toxicity mechanism and the extent of the stress response caused by Cl-HAN remain unclear, resulting in a lack of effective measures to control its presence. Thus, the quantitative toxicological genomics and bioinformatics methods were applied to explore the effects of three chloro-haloacetonitriles (Cl-HANs) on the transcription of fusion genes under varying concentrations of stress in E. coli over 2-hour period. The initial stress response and their toxic mechanism were analyzed. The study also identified the molecular toxicity endpoint, and the core genes that are responsible for the specific toxicity of different Cl-HANs. Cl-HANs exhibited concentration-dependent characteristics of toxic effects, and caused changes in gene expression related oxidative and membrane stress. The stress response results showed that dichloroacetonitrile (dCAN) still caused significant DNA damage under the lowest concentration stress. Chloroacetonitrile (CAN) and trichloroacetonitrile (tCAN) exhibited lower genetic toxicity levels at 513 μg/L and 10.7 μg/L, respectively. The toxic effects of tCAN were widespread. And there was a good correlation between the molecular endpoint (EC-TELI_1.5) and the phenotypic endpoint (LD50) with r_p=-0.8634 (P=0.0593). In all concentrations of stress in CAN, dCAN, and tCAN, the number of overexpressed genes shared was 15, 2, and 14, respectively. Furthermore, bioinformatics analysis demonstrated that Cl-HANs affected genes associated with general stress pathways, such as cell biochemistry and physical homeostasis, resulting in changes in biological processes. And for CAN-induced DNA damage, polA played a dominant role, while katG, oxyR, and ahpC were the core genes involved in oxidative stress induced by dCAN and tCAN, respectively. These findings provide valuable data for the toxic effect of Cl-HANs.