Whole-cell biosensors (WCBs) have been designed to detect As(III), but most suffer from poor sensitivity and specificity. In this paper, we developed an arsenic WCB with a positive feedback amplifier in Escherichia coli DH5α. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier. The sensitivity of the WCB with positive feedback is about 1 order of magnitude higher than that without positive feedback when evaluated using a half-saturation As(III) concentration. The minimum detection limit for As(III) was reduced by 1 order of magnitude to 0.1 µM, lower than the World Health Organization standard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 µM. Due to the amplification of the output signal, the WCB was able to give detectable signals within a shorter period, and a fast response is essential for in situ operations. Moreover, the WCB with the positive feedback amplifier showed exceptionally high specificity toward As(III) when compared with other metal ions. Collectively, the designed positive feedback amplifier WCB meets the requirements for As(III) detection with high sensitivity and specificity. This work also demonstrates the importance of genetic circuit engineering in designing WCBs, and the use of genetic positive feedback amplifiers is a good strategy to improve the performance of WCBs. IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simple methods for the sensitive and specific monitoring of arsenic concentration in drinking water are needed. In this study, we designed an arsenic WCB with a positive feedback amplifier. It is highly sensitive and able to detect arsenic below the WHO limit level. In addition, it also significantly improves the specificity of the biosensor toward arsenic, giving a signal that is about 10 to 20 times stronger in response to As(III) than to other metals. This work not only provides simple but effective arsenic biosensors but also demonstrates the importance of genetic engineering, particularly the use of positive feedback amplifiers, in designing WCBs.

Pseudomonas putida was metabolically engineered to produce medium chain length polyhydroxyalkanoate (mcl-PHA) from acetate, a promising carbon source to achieve cost-effective microbial processes. As acetate is known to be harmful to cell growth, P. putida KT2440 was screened from three Pseudomonas strains (P. putida KT2440, P. putida NBRC14164, and P. aeruginosa PH1) as the host with the highest tolerance to 10 g/L of acetate in the medium. Subsequently, P. putida KT2440 was engineered by amplifying the acetate assimilation pathway, including overexpression of the acs (encoding acetyl-CoA synthetase) route and construction of the ackA-pta (encoding acetate kinase-phosphotransacetylase) pathway. The acs overexpressing P. putida KT2440 showed a remarkable increase of mcl-PHA titer (+ 92%), mcl-PHA yield (+ 50%), and cellular mcl-PHA content (+ 43%) compared with the wild-type P. putida KT2440, which indicated that acetate could be a potential substrate for biochemical production of mcl-PHA by engineered P. putida.

Background: FK506 is an important immunosuppressant, which can be produced by Streptomyces tsukubaensis. However, the production capacity of the strain is very low. Hereby, a computational guided engineering approach was proposed in order to improve the intracellular precursor and cofactor availability of FK506 in S. tsukubaensis. Results: First, a genome-scale metabolic model of S. tsukubaensis was constructed based on its annotated genome and biochemical information. Subsequently, several potential genetic targets (knockout or overexpression) that guaranteed an improved yield of FK506 were identified by the recently developed methodology. To validate the model predictions, each target gene was manipulated in the parent strain D852, respectively. All the engineered strains showed a higher FK506 production, compared with D852. Furthermore, the combined effect of the genetic modifications was evaluated. Results showed that the strain HT-ΔGDH-DAZ with gdhA-deletion and dahp-, accA2-, zwf2-overexpression enhanced FK506 concentration up to 398.9 mg/L, compared with 143.5 mg/L of the parent strain D852. Finally, fed-batch fermentations of HT-ΔGDH-DAZ were carried out, which led to the FK506 production of 435.9 mg/L, 1.47-fold higher than the parent strain D852 (158.7 mg/L). Conclusions: Results confirmed that the promising targets led to an increase in FK506 titer. The present work is the first attempt to engineer the primary precursor pathways to improve FK506 production in S. tsukubaensis with genome-scale metabolic network guided metabolic engineering. The relationship between model prediction and experimental results demonstrates the rationality and validity of this approach for target identification. This strategy can also be applied to the improvement of other important secondary metabolites.

Background: Isobutanol is considered as a leading candidate for the replacement of current fossil fuels, and expected to be produced biotechnologically. Owing to the valuable features, Bacillus subtilis has been engineered as an isobutanol producer, whereas it needs to be further optimized for more efficient production. Since elementary mode analysis (EMA) is a powerful tool for systematical analysis of metabolic network structures and cell metabolism, it might be of great importance in the rational strain improvement. Results: Metabolic network of the isobutanol-producing B. subtilis BSUL03 was first constructed for EMA. Considering the actual cellular physiological state, 239 elementary modes (EMs) were screened from total 11,342 EMs for potential target prediction. On this basis, lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHC) were predicted as the most promising inactivation candidates according to flux flexibility analysis and intracellular flux distribution simulation. Then, the in silico designed mutants were experimentally constructed. The maximal isobutanol yield of the LDH- and PDHC-deficient strain BSUL05 reached 61% of the theoretical value to 0.36 ± 0.02 C-mol isobutanol/C-mol glucose, which was 2.3-fold of BSUL03. Moreover, this mutant produced approximately 70 % more isobutanol to the maximal titer of 5.5 ± 0.3 g/L in fed-batch fermentations. Conclusions: EMA was employed as a guiding tool to direct rational improvement of the engineered isobutanol-producing B. subtilis. The consistency between model prediction and experimental results demonstrates the rationality and accuracy of this EMA-based approach for target identification. This network-based rational strain improvement strategy could serve as a promising concept to engineer efficient B. subtilis hosts for isobutanol, as well as other valuable products.

Mineralization catalyzed by carbonic anhydrase (CA) is one of the most promising technologies for capturing CO₂. In this work, Escherichia coli BL21(DE3) was used as the host, and the N-terminus of ice nucleation protein (INPN) was used as the carrier protein. Different fusion patterns and vectors were used to construct CA surface display systems for α-carbonic anhydrase (HPCA) from Helicobacter pylori 26695 and α-carbonic anhydrase (SazCA) from Sulfurihydrogenibium azorense. The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity, while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system. The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA. Cell fractionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell's outer membrane as a fusion protein. The enzyme activity of the surface display strain E-22b-I_RLS (11.43 U·mL⁻¹OD₆₀₀⁻¹) was significantly higher than that of the intracellular expression strain E-22b-S (8.355 U·mL⁻¹OD₆₀₀⁻¹) under optimized induction conditions. Compared with free SazCA, E-22b-I_RLS had higher thermal and pH stability. The long-term stability of SazCA was also significantly improved by surface display. When the engineered strain and free enzyme were used for CO₂ mineralization, the amount of CaCO₃ deposition catalyzed by the strain E-22b-I_RLS on the surface (241 mg) was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S (173 mg). These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO₂ capture and mineralization.