Difference between revisions of "Team:OUC-China/Description"

WHERE WE STARTED

In synthetic biology, microorganisms with modified metabolic pathways are employed as a reaction vessel to natural or unnatural products. It involves the introduction of several genes encoding the enzymes of a metabolic pathway[1,2]. Indeed, pathway optimization requires to adjust expression of multiple genes at appropriately balanced levels, for example the synthesic of poly(3-hydroxybutyrate)[3] and Mevalonate[4]. Similarly, manipulation of multisubunit proteins (for example F1F0-ATPase) also requires coordinated expression of several genes to produce the subunits at the appropriate stoichiometries[5]. As is done in the prokaryotes, grouping a cluster of genes into a single polycistron is a convenient mean for regulating genes simultaneously. Thus, for the sake of tuning the expressions of genes within polycistron, we aim to develop a tightly regulated, predictable components – stem-loop to realize cistrons concerto.

LOOK CLOSELT TO THE EXISTING STRATEGIES

As a matter of fact, regulation of gene expression includes a wide range of strategies that are used by synthetic scientists, for examples copy number, promoters and ribosome binding sites (RBSs)[6]. Despite their prominent advantages, it is nearly impossible to predict the necessary strengths of the promoters and RBSs to balance and coordinate the expression of multiple genes[7]. What's more, when traditional cloning is utilized, constructing libraries of hundreds of configurations of pathway genes with varying copy number, promoter, and RBS strength is a daunting and time consuming task even for small pathways[8].

Ergo, we are enlightened to try a rational design of one regulatory element to mainly reduce the number of trials and transform it into a user-friendly kit. We mainly focus on the prior design of the DNA sequences to work on the post-transcriptional level which can directly determine the relative levels of gene expression. Thus, stem-loop catches our attention.

---

INSPIRATION FROM NATURE

Nature is uncanny workmanship. Recently, in Clostridium cellulolyticum, within a polycistron encoding cellulosome, Xu reported that stem-loop structures associated with those intergenic post-transcriptional processed sites located at 3' end of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes[9]. Thus we consider the possibility of stem-loops inserted in the intergenic region for tuning expression in E.coli which is more widely utilized as an engineered strain.
Fortunately, Keasling has identified that stem-loops function well in the post-transcriptional process in E.coli[10], which confirmed our thought. Thus, we decided to develop stem-loops with different folding free energy inserted in the intergenic region of two genes to coordinate expressions.

---

FOCUS ON THE DEEP MECHANISM

The operon is transcribed by its sole promoter and the primary transcript is cleaved into several secondary transcripts by RNase E, a single-stranded, nonspecific endonuclease with preference for cleaving A/U-rich sequence . However, the stability of these secondary transcripts against exonuclease degradation from the 3’ end varied due to their distinct terminal structure. When stem-loops inserted in the 3’ end of the upstream gene, it protects its mRNA against the degradation of exonuclease, increasing the ratio of abundance of the first gene product relative to that of the second gene product. Furthermore, the lower free energy of stem-loops are, the more stable the secondary transcripts of the upstream are, tuning the expression of multiple genes.

---

OUR CREATIVE DESIGN

For further developing stem-loops as useful regulatory parts, we racked our brains and divided our project into two parts. First we exploited stem-loops as new parts to tune the expression and transformed it into a user-friendly toolkit. SOLID data are carried out to confirmed its function. And then we explored more of this novel regulation method though modeling and software. More details in Design

---

FURTHER APPLICATION

After visiting some local industrial enterprises, we aimed to devoted our design into a practical industrial production, for improving product titers, yields, and productivity. In the fermentation of beer, the regulation of GSH, a reducing substance that is resistant to beer aging, contributes to increasing the productivity of beer. GSH is composed of two subunits GSHⅠand GSHⅡ. Regulating the ratio of GSHⅠand GSHⅡ at proper stoichiometries results in higher yields . Thus, we are going to insert the stem-loops with required folding free energy between the coding regions of GSHⅠand GSHⅡ to coordinate their relative expression.

REFERENCE

[1]. Khosla, C. & Keasling, J.D. Metabolic engineering for drug discovery and development.Nat. Rev. Drug Discov. 2, 1019–1025 (2003).
[2]. Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D. & Keasling, J.D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802 (2003).
[3]. Li T, Ye J, Shen R, et al. Semi-rational approach for ultra-high poly (3-hydroxybutyrate) accumulation in Escherichia coli by combining one-step library construction and high-throughput screening[J]. ACS synthetic biology, 2016.
[4]. Dudley Q M, Anderson K C, Jewett M C. Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis[J]. ACS Synthetic Biology, 2016.
[5]. White, M.M. Pretty subunits all in a row: using concatenated subunit constructs to force the expression of receptors with defined stoichiometry and spatial arrangement. Mol. Pharmacol. 69, 407–410 (2006).
[6]. Song C W, Lee J, Ko Y S, et al. Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid[J]. Metabolic engineering, 2015, 30: 121-129.
[7]. Pfleger B F, Pitera D J, Smolke C D, et al. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes[J]. Nature biotechnology, 2006, 24(8): 1027-103
[8]. Jones, J.A., O.D. Toparlak, and M.A. Koffas, Metabolic pathway balancing and its role in the production of biofuels and chemicals. Curr Opin Biotechnol, 2015. 33: p. 52-9
[9]. Xu C, Huang R, Teng L, et al. Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization[J]. Nature communications, 2015, 6.
[9]. Smolke C D, Keasling J D. Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon[J]. Biotechnology and bioengineering, 2002, 80(7): 762-776.
[10]. Mackie, George A. "RNase E: at the interface of bacterial RNA processing and decay." Nature Reviews Microbiology 11.1 (2013): 45-57.
[10]. Liao X Y, Shen W, Chen J, et al. Improved glutathione production by gene expression in Escherichia coli[J]. Letters in Applied Microbiology, 2006, 43(2):211-214.