Encoded sensors And Metabolite production--论文代写范文精选

丙烯酸酯生物合成途径是在这项研究中开发的,据我们所知,第一个不等的路线从葡萄糖到丙烯酸酯。生物传感器结合各自的生产监控产品的形成。下面的paper代写范文进行详述。

Abstract
The framework for real-time observation of metabolite production consists of two components: the biosensor and the pathway (Fig. 1). The biosensor is a small-molecule inducible system that produces a fluorescent readout proportional to the amount of product inside of the cell. The pathway consists of all the genes necessary to produce the product molecule from a desired starting point, typically a low-cost feedstock such as glucose or biomass. Rates of product formation vary depending on the amount of intermediate supplied, the number of reactions leading from that intermediate to the final product, and how fast those reactions take place. Final titers depend on these factors as well as the amount of starting material that is shunted into side reactions or used for energy by the cell. We selected pathways for the production of 3HP, glucarate, and muconate from the literature. 

The acrylate biosynthesis pathway was developed in this study and represents, to our knowledge, the first heterologous route from glucose to acrylate. Previously characterized muconate and glucarate biosensors were combined with their respective production pathways to monitor product formation. Because there are no existing biosensors for 3HP, novel sensors were developed and evaluated for their ability to sense 3HP production in real time. The first 3HP biosensor was developed from the Escherichia coli 2-methylcitrate responsive transcriptional regulator prpR (17). Because no 3HP-responsive allosteric transcriptional regulator is known, it was necessary to use a transcriptional regulator that binds to a molecule that 3HP can be converted to intracellularly. The principle of relying on a downstream molecule to affect a response from a nonbinding target compound was pioneered in the Keasling laboratory, when prpR and two endogenous enzymes were used to construct a propionate biosensor (18). Here, we use the endogenous enzyme, 2-methylcitrate synthase (prpC) and the heterologous multifunctional enzyme propionyl-CoA synthase (pcs) from the carbon fixation pathway of Chloroflexus aurantiacus (19) to produce 2-methylcitrate from 3HP. Together the system of three genes (pcs, prpC, and prpR) comprise the prpR-based 3HP biosensor.

The second 3HP biosensor was developed from acuR, an acrylate responsive transcriptional regulator found in the aquatic bacterium Rhodobacter sphaeroides (20). A pathway was constructed that converts 3HP to acrylate (Fig. 2A), allowing the acrylate biosensor to report intracellular 3HP concentration. In this case, a truncated version of the multifunctional enzyme pcs is used to convert 3HP into acrylyl-CoA, which is subsequently hydrolyzed to acrylate by the acrylyl-CoA hydrolase (ach) from Acinetobacter baylyi (21). In Chloroflexus aurantiacus, pcs catalyzes three subsequent reactions: 3HP to 3HP-CoA to acrylylCoA to propionyl-CoA (19). We made use of all three reactions in the prpR-based biosensor, but for the acuR-based biosensor, accumulation of acrylyl-CoA rather than propionyl-CoA is necessary. 

Separation of pcs into its functional domains has been shown to increase the rates of the individual reactions (22). Because of this finding, we reasoned that we could remove the domain responsible for conversion of acrylyl-CoA to propionylCoA while preserving the activity of the other two domains. We refer to the truncated enzyme as pcsΔ3 , and its coexpression with ach and acuR constitute the acuR-based 3HP biosensor (Fig. 2A). Increasing concentrations of 3HP in the medium resulted in increasing levels of fluorescence when pcsΔ3 and ach were present, but resulted in no biosensor activation in their absence (Fig. 2B). A 90-fold increase in fluorescence was obtained when the acuR-based biosensor was induced with 10 mM 3HP. This result is a much more dramatic activation than that achieved with the prpR-based biosensor. The induction kinetics of 3HP and the authentic activator acrylate were compared by monitoring biosensor activation in real time. The 3HP-mediated induction only slightly lagged the time course of acrylate induction (Fig. 2C). Fluorescence remained at background levels for >16 h in the absence of pcsΔ3 and ach (Fig. S2).(paper代写)

We coexpressed the 3HP biosensors with the 3HP production pathway to monitor 3HP production in real time (Fig. 3A). The production pathway consists of the endogenous biosynthesis of malonyl-CoA and the bifunctional enzyme malonyl-CoA reductase (mcr) from the carbon fixation pathway of Chloroflexus aurantiacus (19). Mcr shunts malonyl-CoA away from fatty acid biosynthesis by catalyzing the conversion of malonyl-CoA, first into malonate semialdehyde, and then into 3HP, at the expense of two NADPH. 

This route from glucose to 3HP has been published, with the authors achieving titers of 60 mg/L with expression of mcr alone (23). Titers were increased to 180 mg/L with overexpression of the ACC complex and pntAB, increasing availability of malonylCoA and NADPH, respectively. For our study, we chose to increase the amount of malonyl-CoA available for 3HP production by use of the fatty acid inhibitor cerulenin, rather than through genetic manipulations. Fatty acid biosynthesis is the primary sink for malonyl-CoA and operates at a much higher velocity than heterologously expressed mcr (23). Because cerulenin inhibits the activities of fabB and fabF, increasing its concentration results in lower fatty acid biosynthesis rates and a higher concentration of available malonyl-CoA (24). In each of the 3HP implementations, the biosensor helper enzymes, pcs and pcsΔ3 /ach, were constitutively expressed, whereas mcr was expressed conditionally with the addition of IPTG.

Conclusions 
Through this work, we have developed a framework for tracking the formation of metabolic products in real time using fluorescent biosensors. We demonstrated that the fluorescence achieved by a cell is indicative of its productivity—higher fluorescence indicates higher product titers. Fluorescence as a proxy for product titer not only allows for real-time observation of product formation, but also enables cells to report their own individual progress in producing a chemical. Although we have deployed our biosensors for production optimization in 96-well plates, the greatest potential for biosensors is in pathway evaluation via fluorescence-activated cell sorting (FACS). (paper代写)

FACS has been used for the optimization of biosynthetic pathways for more than two decades (31). Historically, the metabolic product must have possessed fluorescent properties or been amenable to fluorescent staining (31–33). In contrast, genetically encoded biosensors have enabled FACS to be used for inconspicuous compounds. These studies have shown that biosensor-based FACS can be successful with maximum dynamic ranges as low as 12-fold, indicating that even poorly operating biosensor systems enable multiplexed phenotype evaluation (34). Implementing FACS with our techniques for real-time monitoring of whole pathway biosynthesis will enable greater versatility for phenotype evaluation in metabolic engineering. Furthermore, we have shown that the breadth of metabolic products sensed is not limited by the set of biosensors on hand—one or more downstream reactions can be used to transform the produced compound into the sensed compound. This strategy is complemented by emerging technologies that enable construction of biosensors for arbitrary compounds (35).(paper代写)

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