Using the inner membrane of Escherichia coli as a scaffold to anchor enzymes for metabolic flux enhancement
You Wang1, 2, Yushu Wang1, 2, Yuqi Wu2, Yang Suo2, Huaqing Guo2, Yineng Yu2, Ruonan Yin2, Rui Xi2, Jiajie Wu2, Nan Hua2, Yuehan Zhang2, Shaobo Zhang2, Zhenming Jin2, Lin He2, and Gang Ma1, 2*
1 Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, P.R. China;
2Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, P.R. China
*Corresponding author: Dr. Ma Gang, magang@sjtu.edu.cn, Fax: 86-21-34207232.
#You Wang and Yushu Wang contributed equally to this work and considered as the first author.
ABSTRACT:Clustering enzymes in the same metabolic pathway is a natural strategy to enhance productivity. Synthetic protein, RNA and DNA scaffolds have been designed to artificially cluster multiple enzymes in the cell, which require complex construction processes and possess limited slots for target enzymes. We utilized the Escherichia coli inner cell membrane as a native scaffold to cluster four fatty acid synthases and achieved to improve the efficiency of fatty acid synthesis in vivo. The construction strategy is as simple as fusing target enzymes to the N-terminus or C-terminus of the membrane anchor protein (Lgt), and the number of anchored enzymes is not restricted. This novel device not only presents a similar efficiency in clustering multiple enzymes to that of other artificial scaffolds but also promotes the product secretion, driving the entire metabolic flux forward and further increasing the gross yield compared with that in a cytoplasmic scaffold system.
KEYWORDS: cell membrane, scaffold, metabolic flux, fatty acid synthesis

Introduction

In cells, many enzymes undergo energy uptake and produce materials essential for daily life processes. A number of enzymes in a metabolic flux naturally cluster as multi-enzyme “sequential” or “cascade” reactions, such as glycolysis and Krebs cycle [1, 2]. The existence of these natural “flow lines” indicates that clustering relevant enzymes can improve the efficiency of metabolic flux and thus save biological energy.
To mimic a natural multi-enzyme complex and to organize functional-related enzymes, researchers developed several approaches, such as designing an artificial protein scaffold for the generation of the desired metabolic flux [3]. Using well-characterized and widespread protein–protein interaction domains from metazoan signaling proteins (SH3-, PDZ-, and GBD-binding domains), the author constructed a modular genetically encoded scaffold system, where enzyme localization was predefined and programmable. With this system, the amount of the target product increased by 77-fold, demonstrating the advantages of artificial scaffolds[3, 4].
Nevertheless, protein and other scaffold systems with predefined artificial scaffolds are generally limited by the length of scaffolds or the number of artificially clustered modules [5, 6]. To simplify the clustering system and expand the number of enzymes that can be exerted to the system, we proposed that the inner cell membrane could be a good candidate because of its several properties. First, unlike previously synthesized scaffolds, the cell membrane is an innate organelle that has no limitation on the number of scaffolds. Second, the membrane has a much more compact space than the cytoplasm, suggesting the presence of unlimited slots for scaffolding proteins. Third, the membrane structure restricts the reaction space to a 2D plane compared with discrete scaffolds, thereby facilitating the interaction among the anchored proteins. Moreover, the enzymes can be organized in a 2D pattern on the membrane to further enhance the metabolic flux. The proposed membrane scaffold could be used to effectively increase the concentration of the final synthesized products near the membrane, thereby facilitating the transmembrane transportation of products and further simplifying the post-processing procedure. Thus, we decided to develop the potential of the cell membrane as a native scaffold for the clustering of enzyme systems.
To verify this concept, we selected key enzymes of the fatty acid metabolic pathway in Escherichia coli and anchored them onto the inner cell membrane. E. coli has nine fatty acid synthases (FAS), namely, FabA, FabB, FabD, FabF, FabG, FabH, FabI, FabZ, and ACP (Fig. 1). Besides, TesA, a periplasmic thioesterase, can release free fatty acids from acyl-ACP species [7]. Previous studies suggested that FabG, FabI, FabZ, and TesA control the rate-limiting steps in fatty acid biosynthesis in E. coli[7-9]. By conducting a systematic kinetic analysis on the fully reconstituted E. coli (FAS), X. Yu suggested that different combinations of the molar ratios of the four rate-limiting enzymes of FAS, namely, FabZ, FabG, FabI, and TesAʹ, remarkably influenced the overproduction of fatty acids. Therefore, we determined to fuse FabG, FabI, FabZ, and TesAʹ, which is a TesA mutant without a signal sequence peptide that redirects it to become localized in the cytoplasm and thus increase the accessibility of substrates to the active site, with phosphatidylglycerol::prolipoprotein diacylglyceryl transferase, which is a well-studied E. coli inner transmembrane protein [10], to confirm the viability of the membrane scaffold. By anchoring these enzymes, we observe increased final products yield and dramatically enhanced products exportation. Collectively, our results provide novel insight into the potential application of cell membrane as a scaffold for important metabolism pathway to produce valuable bioproducts.

Experimental section