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