We have two main goals this year. One is to construct an engineered probiotic bacteria
platform; the other is to give the platform several applications. So our design consists of
two dimensions and four parts.
Learn More
Fig. 1 An overview of the project design
Brucella abortus is a hazardous pathogenic bacteria that may lead to severe zoonotic disease, brucellosis, with the help of erythritol. Previous studies have identified the erythritol-utilizing gene clusters in the genome of B. abortus and confirmed that the five enzymes expressed could achieve the utilization from erythritol to D-erythrose-4-P (Barbier T et al., 2014). Therefore, we would like to introduce this pathway into E. coli.
Fig. 2 Metabolic pathway from erythritol to D-erythrose-4-P in Brucella abortus
Considering the known functions of the enzymes in the erythritol utilizing gene clusters, we constructed the pJ23114-eryA-eryB-eryC and pJ23114-eryR-eryH-eryI plasmid, and then got an engineered bacteria of eryABCRHI.
Fig. 3 Plasmid construction method for utilizing erythritol as a carbon source
To test the functions of the erythritol-utilizing cassette, we measured the growth curve under different carbon source conditions. We also did colony competition by both experiments and bioinformatic modulation.Learn More
It is well known that both eukaryotes and prokaryotes have numerous gene regulation systems, and lac operon is one of the most basic and common one. Researchers found a similar structure like the lac operon, regulating gene expression under erythritol control.
Erythritol metabolism pathway is inhibited by EryD protein that is produced from the gene of eryD. Erythritol can bind with EryD, causing its conformation change, which relieves the inhibition of EryD (Fig. 4).
Fig. 4 The structure and visualization of ery operon in Brucella abortus
In this module, we tried to construct an erythritol-EryD-eryO (erythritol operator) induce-response system. Firstly, we tried to construct and optimize the erythritol promoter. We designed different fusion promoters, which is characterized by downstream reporter gene sfGFP. Then, we designed two composite systems to characterize the system.
In the first system (Fig. 5), we use double inducer to test the system, namely, IPTG and erythritol. IPTG is used to triggered the expression of EryD and erythritol is used to triggered the expression of sfGFP. In the second system (Fig. 6), only erythritol is used. We aimed to achieve the erythritol induce-response curves, which is related to the concentration of erythritol.
Fig. 5 The first erythritol induction system
Fig. 6 The second erythritol induction system
To make our projects more relevant to the theme of therapy, we introduced several pathways under the control of erythritol, making it a multi-function platform that benefits human health and cures specific diseases.
L-erythrose is a downstream secondary metabolite of erythritol. It’s noted that L-erythrose can be used to lower blood sugar and blood pressure(Sener, A, et al., 1977), thus has potentially therapeutic effects on some metabolic diseases, like diabetes. Researchers found that erythritol can be transformed into L-erythrulose and L-erythrose under two enzymes, sorbitol dehydrogenase (SDH) and L-ribose isomerase (L-RI) in Gluconobacter oxydans (Zou X et al., 2017). Therefore, we considered the conversion from erythritol to L-erythrose by expressing SDH and L-RI after our engineered probiotics get colonized.
Fig. 7 Conversion from erythritol to L-Erythrulose and L-Erythrose in Gluconobacter oxydans
Considering that pyrroloquinoline quinone (PQQ) is an essential co-enzyme for the conversion process, and, severing as a health care product for benefiting our liver and nerves, we introduced a PQQ synthesis pathway in addition(Zhu W, Klinman JP., 2020).
Fig. 8 Biosynthesis of PQQ in bacteria
To test the genes' functions and validate the products' existence, we used several techniques to qualify and quantify our products. During the course of the assay, we have also been optimising our plasmids and measurement conditions, and the yields have improved to some extent.
Learn MoreIn recent years, cardiovascular disease is becoming the most severe cause of death worldwide. Currently, some therapies indirectly prevent and protect against cardiovascular disease by introducing engineered bacteria that secrete small molecules to lower blood glucose levels. Thus, we got acquainted with HZAU-China through the CCiC and built a solid partnership to give our engineered probiotic platform a specific function.
Learn MoreTrimethylamine (TMA) is the precursor of TMAO (trimethylamine oxide), an important factor that can induce thrombosis. Researchers found that TMA can be demethylated and degraded by TMADH (Roberts AB, et al.,2018). Thus, we introduced this part after characterized successfully, to accomplish a more specific cardiovascular disease prevention effect, broadening functions of our probiotics platform for multi-functional healthcare.
Fig. 9 The relationship between TMADH activity, TMA, and TMAO and thrombosis risk in the host
To test the functions of the enzyme and the existence of products, and to validate the reliability and operations of our ery operon, making coherence with previous parts of our project, we quantify the gene expression under both the constitutive promoter and our ery operon.
Learn More1. Barbier T, Collard F, Zúñiga-Ripa A, et al. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella. Proc Natl Acad Sci U S A. 2014;111(50):17815-17820. doi:10.1073/pnas.1414622111
2. Qiu X, Xu P, Zhao X, Du G, Zhang J, Li J. Combining genetically-encoded biosensors with high throughput strain screening to maximize erythritol production in Yarrowia lipolytica. Metab Eng. 2020;60:66-76. doi:10.1016/j.ymben.2020.03.006
3. Roberts AB, Gu X, Buffa JA, et al. Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential. Nat Med. 2018;24(9):1407-1417. doi:10.1038/s41591-018-0128-1
4. Sener A, Devis G, Somers G, Malaisse WJ. The insulinotropic action of D-erythrose. Diabetologia. 1977 Apr;13(2):125-30. doi: 10.1007/BF00745139.
5. Zhu W, Klinman JP. Biogenesis of the peptide-derived redox cofactor pyrroloquinoline quinone. Curr Opin Chem Biol. 2020;59:93-103. doi:10.1016/j.cbpa.2020.05.001
6. Zou X, Lin J, Mao X, Zhao S, Ren Y. Biosynthesis of L-Erythrose by Assembly of Two Key Enzymes in Gluconobacter oxydans. J Agric Food Chem. 2017;65(35):7721-7725. doi:10.1021/acs.jafc.7b02201