Team:NAU-CHINA/Project/Design
DESIGN
Flavoenzyme: ssuE
SefA Protein
Genetic circuit
Our high-efficiency selenium recovery bioreactor program used Escherichia coli BL21 as the chassis bacteria and utilized synthetic biology methods to efficiently recover selenium-containing waste in industry. Next we introduced the key parts, including a flavoenzyme called SsuE which improves the efficiency of E. coli's reduction of selenite, the SefA protein that guarantees the ability to encapsulate elemental selenium, and promoter families of different intensities into SeNPs.
Flavoenzyme: ssuE
In fact, many microorganisms are capable to reduce high-valence selenium to elemental selenium, but their efficiency is quite different. Our chassis bacteria Escherichia coli BL21 is a promising but not perfect one. So we engineered it to fit our needs better. Primarily, we transferred a flavoenzyme into Escherichia coli BL21 to improve the reduction ability to selenite.
Figure 1. Ping-pong bisubstrate bioproduct reaction mechanism
Flavoenzyme is a type of enzyme that contains a heterocyclic isoalloxazine chromophore with FMN or FAD as a cofactor. These enzymes are colourful oxidoreductases that catalyze a large variety of different types of reactions. Flavoenzymes have been shown to be responsible for the reduction of some inorganic salts such as sulfate, nitrite, chromate, arsenate and ferric iron. Some NAD(P)H dependent flavin oxidoreductases were reported to reduce selenite in bacteria under aerobic conditions. A NAD(P)H dependent flavin oxidoreductase CsrF of Alishewanella sp. WH16-1 was identified as a novel aerobic selenite reductase. The enzyme ssuE is a NAD(P)H dependent flavin oxidoreductase of the Escherichia coli sulfur starvation utilization(ssu) operon. According to a ping-pong bisubstrate biproduct reaction mechanism common to other NAD(P)H dependent FMN reductases, it can reduce FMN to FMNH2 and provide FMNH2 for other oxidoreductases of the flavodoxin-like superfamily. In conclusion, we speculate that SsuE could help bacteria reduce selenite by providing reductive power.
SefA Protein
When selenite is reduced to zero-valent selenium, a problem occurs. The zero-valent selenium atom is bare and unstable, and is easy to accumulate into other forms of elemental selenium which is harmful to chasis cell. In order to avoid the accumulation of zero-valent selenium and bacterial necrosis, cells must have a mechanism to stabilize these unstable zero-valent selenium. SefA is such a protein, which is relevant to the assembly and secretion of selenium spheres.
Its functions mainly include:
①Combining and stabilizing zero-valent selenium. SefA can wrap the reduced selenium atoms to form SeNPs;
②Enhancing the selenite resistance of Escherichia coli. In addition, the literature shows that 1 unit of SefA protein can combine
with 320 units of Se0 to produce SeNPs.
Genetic circuit
On this basis, we optimized the codons of SefA protein and SsuE to make them suitable for expression in our chassis organism Escherichia coli BL21. We are hoping that under the joint action of these parts, our chassis Escherichia coli BL21 can efficiently reduce selenite and wrap the selenium atoms produced by the reduction to form stable SeNPs.
By changing the combination of the promoter and RBS, we tried the expression of SefA and SsuE at low, medium, and high, 9 different intensities in total to figure out a combination of high yield, high tolerance, and low bacterial load, so that the engineered bacteria can achieve the best SeNPs production efficiency. The left figure shows the 9 sets of gene pathways we designed in the experiment.
Xia X, Wu S, Li N, et al. Novel bacterial selenite reductase CsrF responsible for Se (IV) and Cr (VI) reduction that produces nanoparticles in Alishewanella sp. WH16-1[J]. Journal of hazardous materials, 2018, 342: 499-509.
Driggers C M, Dayal P V, Ellis H R, et al. Crystal structure of Escherichia coli SsuE: defining a general catalytic cycle for FMN reductases of the flavodoxin-like superfamily[J]. Biochemistry, 2014, 53(21): 3509-3519.
Debieux C M, Dridge E J, Mueller C M, et al. A bacterial process for selenium nanosphere assembly[J]. Proceedings of the National Academy of Sciences, 2011, 108(33): 13480-13485.