Overexpression Of Pathway Genes Research Articles

Design and Construction of a Synthetic Riboregulator-Based Platform for Metabolic Shunting of Pathways in Lactococcus lactis

Microbial cell factories are subjected to rewiring of basic metabolism to enhance the carbon flux towards the desired product pathway. Conventionally, this metabolic engineering approach often involves over-expression of pathway genes and knocking out genes in the competing pathways. However, these approaches result in severe metabolic burden and eventual poor performance of the cells. Particularly, where biomass formation and product synthesis depend on common precursors, permanent changes like knocking out genes will only result in poor titer. Hence, temporary decoupling of biomass formation and product synthesis is considered to be a potential alternative. In this study, we designed synthetic riboregulators, which are RNA-based genetic switches, to shunt metabolic pathways in Lactococcus lactis bacteria, a GRAS organism employed as a cell factory for many biocompounds. The riboregulators are then subjected to evaluation by tagging the cis-repressive sequence (crRNA) to mCherry, a fluorescent reporter and regulated by the constitutive promoter, [Formula: see text]. The trans-activating RNA (taRNA) that interacts with the crRNA is placed under the control of another inducible promoter, [Formula: see text]. First, we observed that when there is no induction of taRNA, there is negligible fluorescence of mCherry indicating the successful repression of translation by the cis-sequence as expected. This has been further verified by comparing this expression level with the expression level of [Formula: see text]-mCherry without the cis-sequence, using fluorometer. Results from this analysis suggest that there is [Formula: see text]% repression by the designed crRNA sequence. Next, we induced the cells with 2[Formula: see text]ng/mL of nisin in the mid-log phase. Upon induction, there is a maximum of three fold increase in the fluorescence levels when compared to the uninduced cells, suggesting that the trans-activation takes place inside the live cells. However, further studies are necessary to optimize the cis-trans ratio to achieve better dynamic range for expression modulation and time window for operating metabolic shunting of competing pathways successfully in L. lactis.

Application of transport engineering to promote catharanthine production in Catharanthus roseus hairy roots

Low accumulation levels of valuable plant secondary metabolites lead to high costs for these compounds production. In order to promote accumulation levels of these molecules, many efforts have been carried out during the past decades, such as elicitation, precursor feeding, tissue cultures and overexpression of pathway genes. However, these engineering strategies could only slightly increase the amounts of target metabolites, since biosynthesis pathways of these compounds are very complex and involving several different organelles and cell types. In this work, we used Catharanthus roseus hairy roots as research material to investigate the effect of transport engineering on monoterpenoid indole alkaloids (MIAs) production. Results showed that overexpresssion of catharanthine transporter, CrTPT2, in C. roseus hairy roots could dramatically increase the accumulation level of catharanthine to fivefold higher than that in control hairy roots, while other MIAs accumulation levels are not affected. Since the expression of pathway genes are at similar level, timely removal of catharanthine from where it is synthesized could be critical for promoting catharanthine production, which exemplifies the application of transport engineering to effective manipulation of plant secondary metabolites biosynthesis. Overexpression of catharanthine transporter, CrTPT2, in Catharanthus roseus hairy roots specifically promotes catharanthine production, which exemplifies an effective manipulation strategy for plant secondary metabolites biosynthesis.

Production of itaconic acid from acetate by engineering acid-tolerant Escherichia coli W.

Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost-effective microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid from acetate. As acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium, and the W strain was selected as the host. Subsequently, the WC strain was obtained by overexpression of cad (encoding cis-aconitate decarboxylase) using a synthetic promoter and 5' UTR. However, the WC strain produced only 0.13 g/L itaconic acid because of low acetate uptake. To improve the production, the acetate assimilating pathway and glyoxylate shunt pathway were amplified by overexpression of pathway genes as well as its deregulation. The resulting strain, WCIAG4 produced 3.57 g/L itaconic acid (16.1% of theoretical maximum yield) after 88 hr of fermentation with rapid acetate assimilation. These efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli.

Optimizing metabolite production using periodic oscillations.

Methods for improving microbial strains for metabolite production remain the subject of constant research. Traditionally, metabolic tuning has been mostly limited to knockouts or overexpression of pathway genes and regulators. In this paper, we establish a new method to control metabolism by inducing optimally tuned time-oscillations in the levels of selected clusters of enzymes, as an alternative strategy to increase the production of a desired metabolite. Using an established kinetic model of the central carbon metabolism of Escherichia coli, we formulate this concept as a dynamic optimization problem over an extended, but finite time horizon. Total production of a metabolite of interest (in this case, phosphoenolpyruvate, PEP) is established as the objective function and time-varying concentrations of the cellular enzymes are used as decision variables. We observe that by varying, in an optimal fashion, levels of key enzymes in time, PEP production increases significantly compared to the unoptimized system. We demonstrate that oscillations can improve metabolic output in experimentally feasible synthetic circuits.