Synthetic biology aims to reprogram the cellular metabolic network to efficiently produce valuable chemicals. L-cysteine, a valuable sulfur-containing amino acid, has been widely used in various fields such as agriculture,...

Aims This study aimed to functionally identify the potential L-homoserine transporters in E. coli, and generate the promising beneficial mutants by targeted directed evolution for improving the robustness and efficiency of microbial cell factories. Methods and results By constructing a series of gene deletion and overexpression strains, L-homoserine tolerance assays revealed that RhtA was an efficient and major L-homoserine exporter in E. coli, whereas RhtB and RhtC exhibited relatively weak transport activities for L-homoserine. Real-time RT-PCR analysis suggested that the expression levels of these three target mRNAs were generally variably enhanced when cells were subjected to L-homoserine stress. Based on in vivo continuous directed evolution and growth-couple selections, three beneficial mutations of RhtA exporter (A22V, P119L, and T235I) with clearly increased tolerance against L-homoserine stress were quickly obtained after two rounds of mutagenesis-selection cycles. L-homoserine export assay revealed that the RhtA mutants exhibited different degrees of improvement in L-homoserine export capacity. Further studies suggested that a combination of these beneficial sites led to synergistic effects on conferring L-homoserine-resistance phenotypes. Moreover, the introducing of RhtA beneficial mutants into the L-homoserine-producing strains could facilitate increased amounts of L-homoserine in the shake-flask fermentation. Conclusions In this study, we provided further evidence that RhtA serves as a major L-homoserine exporter in E. coli, and obtained several RhtA beneficial mutants including A22V, P119L, and T235I that contributed to improving the L-homoserine resistance phenotypes and production efficiency in microbial chassis.

Background Corona Virus Disease 2019 (COVID-19) has adverse effects on patients’ respiratory system. Therefore the pulmonary rehabilitation is particularly necessary for COVID-19 patients. A recent qualitative study indicated that patients perceived the impact of fatigue on their daily lives to be a key factor in decreasing their quality of life. This study aimed to investigate the knowledge and needs of physical fitness and breathing training, and to explore the impact of physical fitness and breathing training on COVID-19 patients. Methods From Feb 16, 2020 to Apr 6, 2020, a self-designed questionnaire was used to investigate the knowledge and needs of physical fitness and breathing training in COVID-19 inpatients. And then the participants received an intervention about physical fitness and breathing training which lasted 2 weeks. The 9-item Functional Assessment of Chronic Illness Therapy-Fatigue scale (FACIT-F) was used to measure COVID-19 related fatigue before and after the intervention. Findings According to a 5-point Likert scale, the 133 COVID-19 patients had only an "not really" and "uncertain" knowledge of physical fitness and breathing training (2.47±1.17). 86.98% of the patients expected to receive guidance of physical fitness and breathing training through video teaching. Differences were observed in fatigue, General fatigue component (15.0(8.0) vs. 19.0(10.0), P<0.01); Functional ability component (4.0 (3.0) vs. 7.0(4.0), P<0.01); Psychological component (6.0(5.0) vs.7.0 (5.0), P<0.01), after the intervention, Moderate degree (36.09% vs. 28.57%) alleviated to mild degree (51.12% vs. 66.91%). SpO2-% 86 ((75, 89) vs. 92(89, 98), P<0.001), and Oxygen flow-L/min (2(0,4) vs. 8(3,9), P<0.001). In our study, 130 healthcare professionals took part in this program. None of the participants reported covid-19 related symptoms. When the participants returned home, they all tested negative for SARSCoV- 2 specific nucleic acids and IgM or IgG antibodies (95% confidence interval 0.0 to 0.7%).Interpretation COVID-19 patients had insufficient knowledge of physical fitness and breathing training for pulmonary rehabilitation. Patients should be guided to receive training, which can be benefit for patients.