Team:Virginia/Engineering

Manifold

MANIFOLD

Index:

Engineering

Asking New Questions

Manifold introduces many significant improvements to the efficiency and capability of biosynthesis. Bacterial compartmentalization increases metabolic yield by decreasing the available reaction space and controlling metabolite passage. However, this advancement introduces a difficulty derived from the use of bacterial microcompartments for a role outside of their native application. The compartmentalization capabilities, which serve a crucial role in Manifold’s design, are due to the ability of BMCs to serve as selective barriers for metabolite diffusion. While this selective diffusion is crucial to the functionality of Manifold, it also requires all precursors and coenzymes be able to diffuse through the BMC pore and enter the reaction space. This requirement is an integral consideration in our continued development of Manifold and our proof of concept, resveratrol production system.

Our initial design utilized only the two enzymes responsible for resveratrol production,

4-coumarate-CoA ligase (4CL)4CL converts p-Coumaric acid to CoA-ester as the first step in synthesizing resveratrol.

and

stilbene synthase (STS)STS converts CoA-ester to resveratrol to complete the biosynthesis of resveratrol.

, from p-Coumaric as shown in Figure 1. In order for this reaction to occur, malonyl-CoA must be present in the reaction space

[1]C. Lim, Z. Fowler, T. Hueller, S. Schaffer, and M. Koffas, “High-Yield Resveratrol Production in Engineered Escherichia coli,” Appl. Environ. Microbiol., vol. 77, pp. 3451–60, Mar. 2011, doi: 10.1128/AEM.02186-10.

. Thus, our original design required the diffusion of malonyl-CoA through the BMC pore. An investigation of this metabolite in relation to the pore (for more information see the Modeling page) introduced concerns about the ability of malonyl-CoA to freely diffuse into the BMC. Malonyl-CoA is much larger than metabolites which have previously been shown to move through the pore and has significant steric hindrances stemming from the bulky CoA group

[2]C. Chowdhury, S. Sinha, S. Chun, T. O. Yeates, and T. A. Bobik, “Diverse Bacterial Microcompartment Organelles,” Microbiol. Mol. Biol. Rev. MMBR, vol. 78, no. 3, pp. 438–468, Sep. 2014, doi: 10.1128/MMBR.00009-14.

. Malonyl-CoA is also utilized in the fatty acid biosynthesis pathway; therefore, our system will be competing with native pathways for the limited molecule

[1]C. Lim, Z. Fowler, T. Hueller, S. Schaffer, and M. Koffas, “High-Yield Resveratrol Production in Engineered Escherichia coli,” Appl. Environ. Microbiol., vol. 77, pp. 3451–60, Mar. 2011, doi: 10.1128/AEM.02186-10.

. The Manifold system is unable to create significant increases in yield if the availability of malonyl-CoA serves to limit the reaction rate. Thus, we were forced to take a step back and redesign our system to address this new need.

Fig 1. Resveratrol biosynthesis pathway shown alongside the competing fatty acid biosynthesis pathway

[1]C. Lim, Z. Fowler, T. Hueller, S. Schaffer, and M. Koffas, “High-Yield Resveratrol Production in Engineered Escherichia coli,” Appl. Environ. Microbiol., vol. 77, pp. 3451–60, Mar. 2011, doi: 10.1128/AEM.02186-10.

.

Engineering Solutions

We choose to focus on acetate, a molecule found two steps upstream of malonyl-CoA in the glucose metabolism pathway as shown in Figure 2

[3]W. Zha, S. B. Rubin-Pitel, Z. Shao, and H. Zhao, “Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering,” Metab. Eng., vol. 11, no. 3, pp. 192–198, May 2009, doi: 10.1016/j.ymben.2009.01.005.

. The size of and functional groups present in acetate closely resemble 1,2-propanediol which exhibits free pore diffusion as it is the natural precursor of the reactions which occur in PDU BMCs. This led us to conclude that acetate will be able to diffuse through the pore more readily than malonyl-CoA.

Fig 2. Malonyl-CoA biosynthesis pathway shown

[3]W. Zha, S. B. Rubin-Pitel, Z. Shao, and H. Zhao, “Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering,” Metab. Eng., vol. 11, no. 3, pp. 192–198, May 2009, doi: 10.1016/j.ymben.2009.01.005.

.

Based on our understanding of pore structure we felt confident that acetate introduced into the culture media would diffuse into the BMC, but once there it still needs to be converted to malonyl-CoA. This requires the recruitment of the additional enzymes acetyl-CoA carboxylase (ACC) and acetyl-CoA synthetase (ACS) to the lumen of the BMC

[3]W. Zha, S. B. Rubin-Pitel, Z. Shao, and H. Zhao, “Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering,” Metab. Eng., vol. 11, no. 3, pp. 192–198, May 2009, doi: 10.1016/j.ymben.2009.01.005.

. Thus, we introduced a second, unique scaffold to our design with different zinc-finger motifs when compared to the original resveratrol scaffold. By introducing ACC and ACS zinc-finger fusion enzymes, in addition to our 4CL and STS zinc-finger fusion enzymes, we were able to design a way to target ACC and ACS to the interior of the BMC at controlled concentrations. The introduction of ACC and ACS to the interior of the BMC allows malonyl-CoA to be recycled within the BMC ensuring that it will always be present in sufficient quantities for the synthesis of resveratrol from p-Coumaric acid.

Testing Solutions

Due to COVID-19 restrictions we were unable to test our designs in between the redesign steps; however, we did include a plan to compare our original and redesigned system in our wet lab planning (for more information see the Experiments page). We will assemble and test the resveratrol production in capabilities of multiple versions of the system, including two with both the resveratrol and the CoA scaffolds in either a 1:1 ratio or a 1:3 ratio and one with just the resveratrol scaffold. By analyzing the resveratrol production capabilities of both our original system and our redesigned system we will be able to determine the most efficient system and determine what harmful effects may arise from including additional enzymes. Thus, we have implemented a somewhat adapted version of the Engineering Design Cycle to fit our current situation.

Extending Applications

The need to address molecular diffusion will undoubtedly be an issue in other pathways to which the Manifold system is applied. As such an engineering process similar to the one discussed above will have to be performed for each pathway to ensure that all required precursors and coenzymes are present in the reaction space. For many pathways this may be addressed by recruitment of additional enzymes to the BMC as described here. For others a solution may require the use of other BMCs such as EUT BMCs or carboxysomes, and still others will likely require even more inventive solutions. As research into the field of BMCs continues to advance, the understanding of and ability to control selective diffusion across BMCs will extend the set of pathways to which Manifold can be applied.

References

[1] C. Lim, Z. Fowler, T. Hueller, S. Schaffer, and M. Koffas, “High-Yield Resveratrol Production in Engineered Escherichia coli,” Appl. Environ. Microbiol., vol. 77, pp. 3451–60, Mar. 2011, doi: 10.1128/AEM.02186-10.
[2] C. Chowdhury, S. Sinha, S. Chun, T. O. Yeates, and T. A. Bobik, “Diverse Bacterial Microcompartment Organelles,” Microbiol. Mol. Biol. Rev. MMBR, vol. 78, no. 3, pp. 438–468, Sep. 2014, doi: 10.1128/MMBR.00009-14.
[3] W. Zha, S. B. Rubin-Pitel, Z. Shao, and H. Zhao, “Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering,” Metab. Eng., vol. 11, no. 3, pp. 192–198, May 2009, doi: 10.1016/j.ymben.2009.01.005.