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Combining protein and metabolic engineering strategies for high-level production of O-acetylhomoserine in Escherichia coli Liang Wei, Qian Wang, Ning Xu, Jian Cheng, Wei Zhou, Guoqiang Han, Huifeng Jiang, Jun Liu, and Yanhe Ma ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00042 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019
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Combining protein and metabolic engineering strategies for high-level
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production of O-acetylhomoserine in Escherichia coli
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Liang Wei 1, 2, 3, Qian wang 1, 2, 3, Ning Xu 1, 2, Jian Cheng 1, 2, Wei Zhou 1, 2, Guoqiang
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Han 4, Huifeng Jiang 1, 2*, Jun Liu 1, 2*, Yanhe Ma 1, 2
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1. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,
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Tianjin 300308, China.
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2. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of
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Sciences, Tianjin 300308, China.
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3. University of Chinese Academy of Sciences, Beijing 100049, China.
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4. School of Life Science and Biotechnology, Yangtze Normal University, Chongqing
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408100, China.
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*To whom correspondence should be addressed:
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Jun Liu; E-mail:
[email protected].
18
Huifeng Jiang; E-mail:
[email protected].
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Abstract
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O-acetylhomoserine (OAH) is a promising platform chemical for the production
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of L-methionine and other valuable compounds. However, the relative low titer and
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yield of OAH greatly limit its industrial production and cost-effective application. In
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this study, we successfully constructed an efficient OAH-producing strain with high
6
titer and yield by combining protein and metabolic engineering strategies in E. coli.
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Initially, an OAH-producing strain was created by reconstruction of biosynthetic
8
pathway and deletion of degradation and competitive pathways, which accumulated
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1.68 g/L of OAH. Subsequently, several metabolic engineering strategies were
10
implemented to improve the production of OAH. The pathway flux of OAH was
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enhanced by eliminating byproduct accumulation, increasing oxaloacetate supply and
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promoting the biosynthesis of precursor homoserine, resulting in a 1.79-fold increase
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in OAH production. Moreover, protein engineering was applied to improve the
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properties of the rate-limiting enzyme homoserine acetyltransferase (MetXlm) based
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on evolutionary conservation analysis and structure-guided engineering. The resulting
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triple F147L-M182I-M240A mutant of MetXlm exhibited a 12.15-fold increase in
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specific activity, and the optimized expression of the MetXlm mutant led to a 57.14%
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improvement in OAH production. Furthermore, the precursor acetyl-CoA supply and
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NADPH generation were also enhanced to facilitate the biosynthesis of OAH by
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promoting CoA biosynthesis, overexpressing heterogeneous acetyl-CoA synthetase
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(ACS) and introducing NADP-dependent pyruvate dehydrogenase (PDH). Finally, the
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engineered strain OAH-7 produced 62.7 g/L of OAH with yield and productivity of
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0.45 g/g glucose and 1.08 g/L/h, respectively, in a 7.5 L fed-batch fermenter, which
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was the highest OAH production ever reported.
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Keywords: O-acetylhomoserine; metabolic engineering; protein engineering;
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homoserine acetyltransferase; homoserine; acetyl-CoA.
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In recent years, bio-manufacturing for sustainable production of valuable
2
chemicals from renewable resources has been attracting more and more attention due
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to serious environmental problems caused by the petrochemical industry.1,
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O-acetylhomoserine (OAH) is an important platform chemical for the production of
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L-methionine, L-homoserine, γ-butyrolactone and other valuable compounds.3-6 In
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biological systems, OAH does not directly participate in protein synthesis, but is the
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crucial precursor for the biosynthesis of the important sulfur-containing compounds
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for cell metabolism, such as L-methionine and S-adenosylmethionine (SAM).7
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Moreover, OAH can also be applied as an important feedstock to directly combine
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with methanethiol through a high-yield enzymatic conversion for the production of
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L-methionine in industry.6 L-methionine is an essential amino acid which has been
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widely used in food, pharmaceutical, cosmetics and feed industries, and the global
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L-methionine market is over 1.6 million tons per year.8 Through the route from OAH,
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a L-methionine industrial production line with capacity of 80,000 tons per year has
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been constructed in Malaysia, and increases gradually.4 Thus, OAH, as a promising
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platform chemical, has drawn considerable interest due to its increasing market
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demand and application.
2
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Escherichia coli, as an important industrial microorganism, has been used for the
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production of L-aspartate family amino acids, such as L-threonine and L-isoleucine, 9,
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10
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of OAH, L-aspartate is first phosphorylated by aspartokinase (AK) and then
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deoxidized
and thus showed great potential for OAH production. In the biosynthetic pathway
to
generate
L-aspartyl
semialdhyde
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aspartyl
semialdehyde
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dehydrogenase (ASD). Following, L-aspartyl semialdhyde is catalyzed by homoserine
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dehydrogenase (HD) to form homoserine.11 In the final step, homoserine
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acetyltransferase (MetX) catalyzes homoserine and acetyl-CoA to synthetize OAH
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(Fig. 1). E. coli does not contain MetX, but possesses homoserine succinyltransferase
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(MetA)
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O-succinylhomoserine (OSH).3 OSH shows similar structure with OAH, and can also
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be applied for the bioconversion of L-methionine.6 But the route for L-methionine
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production from OSH is less productive due to the low yield (0.68 gMet/gOSH)
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compared with that from OAH (0.93 gMet/gOAH).4 Recently, several bacterial MetXs
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have been identified to catalyze homoserine acetylation, such as MetXs from
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Corynebacterium glutamicum, Haemophilus influenza, Leptospira interrogans and
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Leptospira meyeri.4 However, as the key enzyme for OAH and L-methionine
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biosynthesis in microorganism, MetX was strictly controlled by multi-level regulation
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mechanisms.12,
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rigorously down-regulated by the transcriptional regulator McbR, and the activity of
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MetX was inhibited by SAM and cysteine.13,
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MetX was subjected to great feedback inhibition by the end-product OAH,4 which
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vastly limited the biosynthesis of OAH. Therefore, seeking an efficient MetX is
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extremely important for the production of OAH in E. coli.
which
13
catalyzes
the
similar
acylation
reaction
to
generate
For example, the expression of MetX in C. glutamicum was
14
More importantly, the activity of
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Given the important industrial application of OAH, many efforts have been
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devoted to engineering of microorganism for efficient biosynthesis of OAH.4, 6 Kase
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and Nakayama isolated a L-methionine analog-resistant mutant of Corynebacterium
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glutamicum, which produced 10.5 g/L of OAH.15 Kim et al. engineered E. coli
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through deletion of degradation pathways and overexpression of homoserine
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acetyltransferase (MetX) to constructed an OAH-producing strain with titer of 1.8
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g/L.6 Moreover, an L-threonine-producing strain, E. coli CJM002, was also
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engineered for OAH production, and the resulting strain accumulated 6.5 g/L of OAH
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with a yield of 0.16 g/g glucose in shake flask culture and 55 g/L of OAH in fed-batch
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fermentation.6 Nevertheless, the relative low titer and yield of OAH greatly limits the
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large-scale industrial production of OAH and cost-effective application of OAH for
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the biotransformation of L-methionine. Therefore, it is of great significance to
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engineer an efficient microbial cell factory for OAH production.
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Protein engineering has become a powerful tool for the production of useful
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enzymes with desirable properties in synthetic biology and metabolic engineering.5
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As one of the approaches for protein engineering, semi-rational design has recently
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drawn increasing interest owing to the combined advantages of directed evolution and
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rational design. This approach has the potential to construct smaller and smarter
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libraries based on sequence and structure information of enzymes, and creates
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enzymes possessing desirable properties, such as improved thermostability, increased
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activity and modified selectivity.16-18 As the final step in the OAH biosynthesis
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pathway, the poor characteristics of MetX severely restricts the production of OAH.4
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However, few studies have reported the engineering of MetX to increase activity
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or/and reduce feedback inhibition to date although several crystal structures of MetXs
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have been analyzed. Therefore, modification of MetX to improve its properties by
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protein engineering is of immediate necessity for OAH production.
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In this study, a high-level OAH-producing strain was constructed by combining
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protein and metabolic engineering strategies in E. coli (Fig. 1). Firstly, the initial
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OAH-producing strain was created by deletion of competitive and degradation
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pathways and overexpression of the preferred metXlm and feedback-insensitive thrA
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encoding aspartokinase-homoserine dehydrogenase. Subsequently, the pathway flux
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of OAH was enhanced by eliminating the byproduct L-lysine accumulation,
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modulating expression of phosphoenolpyruvate carboxylase (PPC) to increase
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oxaloacetate supply and promoting the biosynthesis of precursor homoserine.
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Moreover, protein engineering of the rate-limiting enzyme MetXlm was performed
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to improve activity and reduce feedback inhibition for OAH production based on
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evolutionary conservation analysis of sequence and structure-guided engineering.
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Additionally, the intracellular acetyl-CoA level and NADPH generation were
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improved
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acetyl-CoA synthetase (ACS) from Salmonella enterica and introducing
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NADP-dependent
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OAH-producing strain with high titer and yield was achieved, which showed great
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potential for the large scale industrial production.
by
promoting
CoA
pyruvate
biosynthesis,
dehydrogenase
overexpressing
(PDH).
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heterogeneous
an
efficient
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Fig. 1. Metabolic engineering strategies for the construction of an OAH-producing strain. Bold red arrows represent that the genes are overexpressed, and the red “X” indicate that the genes are knocked out. Blue dashed arrow represents the feedback inhibition of enzyme, and triangle symbol means the attenuation of feedback inhibition. Abbreviations: G6P, Glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6P, Fructose-1,6-diphosphate; GA3P, glyceraldehyde 3-phosphate; Ru5P, ribulose 5-phosphate; X5P, xylulose 5-phosphate; S7P, sedoheptulose 7-phosphate; AcCoA, acetyl-CoA; Pan, pantothenate; P-Pan, phosphopantothenate; P-PanSH, 4-phosphopantetheine; CoA, coenzyme A; PEP, phosphoenolpyruvate; PYR, pyruvate; CIT, citrate; ICL, isocitrate; 2-KG, α-ketoglutarate; SUCC, succinyl-CoA; SUC, succinate; OAA, oxaloacetate; FUM, fumarate; MAL, L-malate; ASP, aspartate; LYS, L-lysine; HSE, L-homoserine; THR, L-threonine; MET, L-methionine; ACE, acetate; OSH, O-succinylhomoserine; OAH,
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O-acetylhomoserine.
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Results and Discussion
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Construction of an initial OAH-producing strain
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As the terminal enzyme in the biosynthetic pathway, MetX plays a vital role in
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OAH production.4 Thus, in order to determine a better candidate for OAH production,
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two heterogeneous MetXs from C. glutamicum (MetXcg) and L. meyeri (MetXlm)
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were purified and characterized, respectively (Fig. S1). As a result, the specific
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activity of MetX from L. meyeri was 2.32-fold higher than that from C. glutamicum
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(1.16 ± 0.06 and 0.35 ± 0.02 U/mg protein, respectively) (Fig. S2). In addition,
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enzyme activities of the two MetXs were further measured in the presence of potential
11
inhibitors at different concentrations. The result indicated that the activities of two
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MetXs were little affected by L-methionine, but both of them were subjected to
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significant feedback inhibition by the end-product OAH. In the presence of 10 mM
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OAH, MetXlm retained more residual activity (25%) than that of MetXcg (22.86%).
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These results suggested that MetXlm might be a more promising candidate for OAH
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biosynthesis.
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It is well known that disruption of degradation pathways, elimination of
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competing pathways and enhancement of metabolic fluxes are efficient methods for
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the production of desired amino acids.2 For construction of the OAH-producing strain,
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degradation and competitive reactions-related genes (metB encoding cystathionine
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gamma-synthase, thrB encoding homoserine kinase and metA encoding homoserine
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o-succinyltransferase) were successively knocked out, and the metabolic pathway was
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reconstructed by overexpression of superior metXlm and feedback-insensitive thrA.
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The fermentation performance of the initial OAH-producing strain OAH-1 was
2
analyzed using the wild type (WT) harboring the empty vector as the control (Fig. 2a,
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2c and 2e). As a result, OAH-1 exhibited slightly lower growth and glucose
4
consumption than those of WT. Meanwhile, OAH-1 produced 1.68 g/L OAH with
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specific production of 0.47 g/g CDW in the fermentation medium after 48 h, while the
6
control strain did not accumulate OAH. Moreover, the amino acids homoserine and
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L-lysine, as well as acetate, were observed as the main byproducts, and their titers
8
reached 0.87 g/L, 0.95 g/L and 2.31 g/L, respectively (Fig. 2c). Although the OAH
9
synthesis in E. coli was achieved, the titer was still very low. Hence, more metabolic
10
engineering strategies should be further implemented to enhance the biosynthesis of
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OAH.
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Redistribution of metabolic flux to promote the biosynthesis of OAH
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As one of the main byproducts, the accumulation of L-lysine could diminish the
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carbon metabolic flux for OAH biosynthesis. As noted in the previous study,
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knockout of diaminopimelate decarboxylase encoded by lysA is an effective strategy
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to eliminate the formation of L-lysine.19 With the similar strategy, the lysA gene was
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deleted to construct OAH-2. The fermentation result indicated that OAH-2
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accumulated 2.25 g/L of OAH, a 33.93% increase relative to that of OAH-1 (Fig. 2c
19
and 2e). Accordingly, an obvious increase in yield and specific production of OAH
20
was achieved. These results suggested that preventing the generation of L-lysine
21
could channel increased carbon flux into the OAH biosynthesis pathway.
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Fig. 2. Comparison of the fermentation performances of the engineered OAH-producing strains. Biomass (OD600) (a, b), titers of OAH and byproducts (c, d), glucose consumption, specific production and yield of OAH (e, f) are shown. All data shown are means from three independent experiments and error bars indicate the standard deviation from the means.
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It has been demonstrated that the supply of oxaloacetate is one of the limiting
9
factors for the production of L-aspartate family amino acids.1 Thus, in order to
10
optimize the oxaloacetate supply and improve OAH production, the expression of ppc
11
(encoding phosphoenolpyruvate carboxylase) was modulated using artificial promoter
12
regulatory components. Four promoter regulatory components (PM1-12, PM1-46, PM1-37
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and PM1-93) selected from the promoter element library20 were used to replace the
2
native promoter of ppc in OAH-2 by the Cas9-mediated genomic editing system,
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generating the strains of OAH-3-1, OAH-3-2, OAH-3-3 and OAH-3-4, respectively.
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Of particular interest, OAH-3-2 harboring medium intensity promoter PM1-46 showed
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highest titer of OAH (3.29 g/L), a 46.22% increase compared to that of OAH-2 (Table
6
S1), which suggested that moderate expression of ppc was favorable for OAH
7
biosynthesis, but excessive PPC activity has negative effect, which was consistent
8
with the report described previously.10 Besides, the biomass (OD600) and glucose
9
consumption of OAH-3-2 were slightly increased in comparison to those of OAH-2,
10
while the concentration of acetate was decreased by 43.2% to 1.63 g/L (Table S1).
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Improving supply of precursors is regarded as an important approach for the
12
biosynthesis of target products.2, 9 The increased biosynthesis of precursor homoserine
13
might be beneficial for OAH biosynthesis. It has been proven that aspartokinase is the
14
key enzyme to direct the carbon flux into the aspartate branch.11 Therefore, the two
15
feedback-insensitive aspartokinase lysCec from E. coli and lysCcg from C. glutamicum
16
were overexpressed in OAH-3-2 strain to create OAH-4-1 and OAH-4-2, respectively.
17
The specific activities of aspartokinase in OAH-4-1 and OAH-4-2 were greatly
18
improved by 66.67% and 103.47%, respectively, compared to that of OAH-3-2 (Fig.
19
S3). Accordingly, the titers of OAH in OAH-4-1 and OAH-4-2 significantly increased
20
to 4.23 g/L and 4.61 g/L (Fig. 2d), respectively. In addition, the concentrations of
21
homoserine in OAH-4-1 and OAH-4-2 were also improved to 2.31 g/L and 2.44 g/L,
22
respectively. These results suggested that the aspartokinase from C. glutamicum
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performed better than that from E. coli. In order to further enhance the flux of
2
homoserine, the native asd gene and feedback-resistant hom mutant encoding
3
homoserine dehydrogenase from C. glutamicum were successively overexpressed in
4
OAH-4-2 to create OAH-4-3 and OAH-4-4, respectively. As a result, the resulting
5
strain OAH-4-3 showed a marginal increase in the titer of OAH to 4.69 g/L, but the
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titer of OAH and biomass (OD600) in OAH-4-4 were significantly decreased to 2.57
7
g/L and 7.2, respectively (Fig. 2b). Interestingly, the concentration of homoserine was
8
dramatically increased by 69.48% to 4.22 g/L (Fig. 2d). The considerable homoserine
9
accumulation indicated that the downstream homoserine acetylation catalyzed by
10
MetXlm was insufficient, and the high concentration of homoserine was further
11
detrimental for OAH production. Li et al. demonstrated that the high concentration of
12
homoserine greatly inhibited the cell growth and enzyme activity of hybrid ThrA in E.
13
coli.21 Therefore, the low activity of MetXlm was the main bottleneck for the
14
biosynthesis of OAH.
15
Protein engineering of MetXlm to improve its properties
16
The low activity and feedback inhibition of the key enzymes in biosynthesis
17
pathways are regarded as basic regulation mechanisms to avoid the excessive
18
accumulation of amino acids.11, 22 As the key enzyme for OAH biosynthesis, MetXlm
19
was subjected to great feedback inhibition by the end-product OAH (Fig. S2b), which
20
caused the accumulation of undesirable toxic intermediate metabolites and restricted
21
the biosynthesis of OAH. Therefore, improving enzyme activity and removing
22
feedback inhibition of MetXlm are of vital significance for the production of OAH.
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Evolutionary conservation analysis is an effective approach to improve catalytic
2
properties with low risk of losing catalytic activity though mutations of highly
3
variable positions.23 Hence, in order to improve the properties of MetXlm, the
4
evolutionary conservation analysis was performed to measure the positional
5
conservation of individual residues (Fig. S4). The amino acid residues with less
6
conservation were recognized and the corresponding residues with highest frequency
7
were recommended (Table S2). According to the ranking list of the frequencies, the
8
top 24 least conserved residues were selected to mutate as the suggestion,
9
constructing a small and smart mutant library. Then, the catalytic properties of the 24
10
mutant were respectively measured. Among of them, five mutants including I98V,
11
G118P, F147L, M182I, and Y315F displayed clearly higher activities than others
12
(Fig. 3a). Specifically, the M182I mutant exhibited the highest specific activity of 6.1
13
± 0.31 U/mg protein, 6.09-fold higher than that of MetXlm (0.86 ± 0.05 U/mg
14
protein). Nevertheless, the five obtained mutants were still greatly inhibited by OAH.
15
The M182I mutant retained 25.57% residual enzyme activity (1.56 ± 0.11 U/mg
16
protein) with 10 mM OAH addition (Fig. 3b). In particular, the Y315F mutant only
17
maintained 6.19% residual activity in the presence of 10 mM OAH, and was excluded
18
from further analysis. Interestingly, the four potential residues were all distributed on
19
the surface of the MetXlm protein (Fig. 4a). It was speculated that these beneficial
20
mutants located on the surface of protein might change the thermostability of the
21
enzymes.24 In order to verify this hypothesis, the thermal stabilities of four mutants
22
were analyzed using differential scanning calorimetry.25 The transition midpoint (Tm)
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values of the four mutants I98V, G118P, F147L and M182I were 48.74 °C, 49.37 °C,
2
49.18 °C and 52.71 °C, respectively, all of which were higher than that of MetXlm
3
(47.76 °C) (Fig. 4b). The Tm value is an indicator of thermostability, and the higher Tm
4
value of protein, the less vulnerable the protein is to unfolding and denaturation.25 In
5
order to further improve the catalytic activity of MetXlm, the four favorable
6
mutations of I98V, G118P, F147L, and M182I were systematically combined (Fig.
7
3c). As a result, the combinational mutations F147L-M182I, I98V-F147L-M182I, and
8
G118P-F147L-M182I showed better performance, and the highest specific activity
9
was reached with the I98V-F147L-M182I mutant at 9.55 ± 0.41 U/mg protein, a
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10.1-fold increase compared with MetXlm, which indicated that protein engineering
11
based on the evolutionary conservation analysis was a promising approach to improve
12
activity of enzymes.
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Fig. 3. Determination of the specific activity of various mutated MetXlm. (a, b) Enzyme specific activities in the crude extracts of native MetXlm and 24 mutants are assayed in the absence or presence of OAH. (c) Enzyme specific activities in the crude extracts of mutational combinations of four favorable point mutations are measured with or without addition of OAH. (d) Enzyme specific activities in the crude extracts of M240A mutant and relevant mutational combinations are assayed in the absence or presence of OAH. All data shown are means from three independent experiments and error bars indicate the standard deviation from the means.
10 11
Although the enzyme activities of the MetXlm mutants were significantly
12
improved, they still experienced serious feedback inhibition by OAH (Fig. 3c). The
13
catalysis mode of MetXlm had been demonstrated as a ping-pong kinetic mechanism
14
in which the acetyl group of acetyl-CoA was first transferred to the active-site amino
15
acid residue, and then transfered to the γ-hydroxyl group of homoserine 26. We thusly
16
speculated that the product OAH might be easily stuck in the catalytic pocket to block
17
substrate access tunnels, which would lead to serious feedback inhibition. Based on
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this speculation, the structure-guided engineering of MetXlm was performed to
2
relieve the feedback inhibition. The homology structure of MetXlm was developed
3
using RosettaCM,27 and CoA was docked into the active center of the predicted
4
structure. The 15 residues (Y235, K244, Y264, L265, Q268, G269, D352 L236,
5
M240, Y267, E270, P348, A349, L356, and P357) located within 3Å distance in
6
substrate access tunnels were detected (Fig. S5). According to evolutionary
7
conservation analysis, seven highly conserved residues were excluded and eight
8
variable position residues were chosen as mutational candidates (L236, M240, Y267,
9
E270, P348, A349, L356 and P357). Saturation mutations of the eight positions were
10
respectively performed by high throughput screening method as previously reported.28
11
Interestingly, only the M240A mutant displayed better performance than that of the
12
control, and all other saturation mutations showed lower or no activity. These results
13
might be due to the fact that the active site of MetXlm was relatively conserved.26, 29 It
14
was noteworthy that the M240A mutant exhibited a 2.09-fold increase in specific
15
activity, and retained 49.8% residual activity in the presence of 10 mM OAH,
16
suggesting that the M240A mutant suffered from less feedback inhibition (Fig. 3d).
17
The structure analysis showed that M240A mutation could enlarge the substrate
18
tunnel and reduce the steric hindrance, which might accelerate the release of the
19
product OAH from the catalytic pocket and reduce the feedback inhibition (Fig. 4c
20
and 4d). Consequently, the affinities of M240A mutant for homoserine and
21
acetyl-CoA were also significantly improved compared to those of MetXlm, as
22
indicated by the decreased Km values (Table 2).
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1 2 3 4 5 6 7
Fig. 4. Effects of the beneficial mutations on MetXlm. (a) The structural distribution of 5 beneficial mutations in MetXlm. The native and mutants residues are colored by cyan and purple, respectively. (b) Thermograms of MetXlm (WT) and 4 mutants are determined using differential scanning microcalorimetry. (c, d) The changes of substrate access tunnels in the MetXlm (M240) and mutant (A240) are analyzed by surface representation.
8 9
Subsequently, the M240A mutation was combined with the favorable
10
combinational mutants F147L-M182I, I98V-F147L-M182I and G118P-F147L-M182I
11
obtained above to further improve the properties of enzyme (Fig. 3d). As a result, the
12
triple F147L-M182I-M240A mutant displayed the highest specific activity of 11.31 ±
13
0.82 U/mg protein, a 12.15-fold increase compared to that of MetXlm, and this
14
mutant retained 42.7% residual activity with the addition of 10 mM OAH.
15
Furthermore, binding affinities of the F147L-M182I-M240A mutant for the substrates
16
homoserine and acetyl-CoA were much higher than those of MetXlm as suggested
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from the Km values (Table 2). The catalytic efficiency (kcat/Km) of the
2
F147L-M182I-M240A mutant with homoserine and acetyl-CoA were about 9.81-fold
3
and 7.73-fold higher than those of MetXlm, respectively (Table 2). These results
4
revealed that the triple F147L-M182I-M240A mutant of MetXlm showed significant
5
improvement in enzyme activity and feedback resistance by combining the
6
evolutionary conservation analysis and structure-guided engineering, which also
7
provided an effective strategy for the evolution of enzymes to improve activity and
8
relieve inhibition.
9
Effects of optimized expression of favorable MetXlm mutant on OAH production
10
In order to investigate the effect of triple F147L-M182I-M240A mutant of
11
MetXlm on OAH production, the mutant was overexpressed in OAH-4-3 to create
12
OAH-5. The specific activity of MetXlm mutant in OAH-5 reached 4.14 ± 0.27 U/mg
13
protein, 10.2-fold higher than that of MetXlm in OAH-4-3 (0.36 ± 0.07 U/mg protein)
14
(Fig. 5a). Moreover, the titer of OAH in OAH-5 showed a 44.6% increase to 6.78 g/L
15
with the yield and specific production of 0.21 g/g glucose and 2.08 g/g CDW,
16
respectively (Fig. 5b), and the concentration of byproduct homoserine was decreased
17
by 41.4% to 1.46 g/L compared with OAH-4-3, indicating that the MetXlm mutant
18
had great advantage for transform the precursor homoserine into OAH.
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1 2 3 4 5 6 7
Fig. 5. Effects of optimized expression of MetXlm mutant on OAH production. (a) Enzyme specific activities of MetXlm mutant in the engineered strains are regulated by ribosome bind site (RBS) engineering. (b, c) Titers of OAH and byproducts, biomass, glucose consumption, specific production and yield of OAH in the engineered strains are shown. All data shown are means from three independent experiments and error bars indicate the standard deviation from the means.
8 9
In order to further enhance the positive effect of MetXlm mutant on OAH
10
production, the expression of MetXlm mutant was optimized using ribosome bind site
11
(RBS) engineering. Five RBS artificial elements including RBS6, RBS12, RBS18,
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RBS24 and RBS29 were selected from the reported RBS library,30 and their strengths
2
were about 1.4, 3.2, 4.1, 5.2 and 8.6 folds relative to the induced native E. coli lacZ
3
promoter, respectively. Subsequently, the five RBS artificial elements were applied to
4
modulate the expression of MetXlm mutant in OAH-5,30 generating the strains of
5
OAH-5-1, OAH-5-2, OAH-5-3, OAH-5-4, and OAH-5-5, respectively. As a result,
6
the specific activities of MetXlm mutant in OAH-5-1, OAH-5-2, OAH-5-3, OAH-5-4
7
and OAH-5-5 were 4.32 ± 0.34, 4.91 ± 0.46, 5.63 ± 0.35, 5.92 ± 0.41 and 6.51 ± 0.39
8
U/mg protein, which were obviously higher than that of OAH-5, suggesting that the
9
expression regulation of MetXlm mutant by RBS engineering was effective (Fig. 5a).
10
Moreover, the titers of OAH in OAH-5-1, OAH-5-2, OAH-5-3, OAH-5-4 and
11
OAH-5-5 reached 6.75, 6.89, 7.37, 7.21 and 6.91 g/L, respectively (Fig. 5b).
12
Specifically, the specific production and yield of OAH in OAH-5-3 were increased by
13
66.9% and 54.7% (2.32 g/g CDW and 0.22 g/g glucose), respectively, compared to
14
those of OAH-4-3 (Fig. 5c), which suggested that the fine-tuning expression of
15
MetXlm was conducive to enhanced OAH production. These results highlighted the
16
importance of protein engineering as a key strategy to improve the biosynthesis of
17
desirable product in metabolic engineering.
18
Increasing intracellular acetyl-CoA level to enhance the production of OAH
19
Acetyl-CoA, as the acetyl group donor, is another key precursor for OAH
20
biosynthesis.4, 31 Thus, the acetyl-CoA might be another important limiting factor for
21
OAH production. Under aerobic condition, the major routes for acetyl-CoA
22
biosynthesis have been shown to be the decarboxylation of pyruvate by PDH complex
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1
and the activation of acetate via reversible phosphotransacetylase and acetate kinase
2
(PTA-ACK) or irreversible ACS pathways in E. coli.31 In addition, it has been proven
3
that the increased supply of CoA is helpful for the biosynthesis of acetyl-CoA.31
4
Therefore, in order to increase the intracellular acetyl-CoA level to enhance OAH
5
production, promoting CoA biosynthesis, enhancing PDH flux and overexpressing the
6
ACS were successively performed.
7
In CoA biosynthesis pathway, coaA encoding pantothenate kinase and coaD
8
encoding P-PanSH adenylyltransferase were two rate-limiting enzymes. Therefore,
9
the genes of coaA (R106A, feedback-insensitive mutant) and coaD were
10
combinational expressed in OAH-5-3 to promote the CoA biosynthesis, generating
11
OAH-6-1. The intracellular concentration of acetyl-CoA in OAH-6-1 was noticeably
12
improved to 0.15 nmol/mg CDW, a 2.75-fold increase over that of OAH-5-3 (0.04
13
nmol/mg CDW) (Fig. 6a). Moreover, the fermentation result indicated that OAH-6-1
14
accumulated 8.31 g/L of OAH with specific production and yield of 2.73 g/g CDW
15
and 0.24 g/g glucose, respectively, which was a 12.7% improvement compared to that
16
of OAH-5-3. As well, the concentration of byproduct homoserine was further
17
decreased to 0.75 g/L (Fig. 6b), indicating that the increasing intracellular acetyl-CoA
18
level could drive the conversion of homoserine into OAH.
19
In order to further improve the level of acetyl-CoA, the PDH complex containing
20
pyruvate dehydrogenase (AceE), dihydrolipoamide acetyltransferase (AceF) and
21
dihydrolipoamide dehydrogenase (Lpd) (A358V, feedback-insensitive mutant), were
22
combinational expressed in OAH-6-1 to generate OAH-6-2. The specific activity of
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PDH in OAH-6-2 was increased by 2.26-fold to 0.49 ± 0.05 U/mg protein compared
2
to that of OAH-6-1 (0.15 ± 0.02 U/mg protein) (Fig. S6). Unexpectedly, the
3
concentration of acetyl-CoA in OAH-6-2 showed a marginal increase (Fig. 6a). The
4
titer of OAH in OAH-6-2 was reduced to 6.72 g/L, and the biomass was also slightly
5
decreased (Fig. 6b and 6c). On the other hand, the concentrations of acetate and
6
homoserine were considerably improved to 3.51 and 1.62 g/L, respectively. These
7
results might be due to the complex regulation of acetyl-CoA metabolism. The
8
increasing carbon flux at the acetyl-CoA node by overexpression of the PDH complex
9
could be involved in the formation of acetate via the reversible PTA-ACK pathway.32
10
The high concentration of acetate had detrimental effects on the cell growth and
11
enzyme activities of ThrA and MetXlm,10,
12
production. But deletion of pta would lead to growth suppression and pyruvate
13
excretion, which was not a considered strategy to reduce the formation of acetate.10
14
11, 21, 25
and led to the decline of OAH
Due to the higher affinity for acetate than PTA, overexpression of ACS could
15
effectively reduce the accumulation of acetate.10,
31
16
introducing the gene of ACS (L641P, feedback-insensitive mutant) from S. enterica
17
was selected to decrease the amount of acetate and further promoted the formation of
18
acetyl-CoA. As a result, the titer of acetyl-CoA in the resulting OAH-6-3 strain was
19
significantly increased to 0.67 nmol/mg CDW, a 3.46-fold increase compared with
20
OAH-6-2 (Fig. 6a). Importantly, the OAH titer of OAH-6-3 was increased by 38.9%
21
to 10.94 g/L in comparison to that of OAH-6-2, while the titers of acetate and
22
homoserine were reduced to 1.15 g/L and 0.64 g/L, respectively (Fig. 6b). The
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Therefore, the strategy of
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1
biomass (OD600) in OAH-6-3 was also restored to a comparable level with OAH-6-1.
2
Moreover, in order to explore whether there is synergistic effects between PDH and
3
ACS, the heterogeneous ACS was also overexpressed in OAH-6-1 to construct
4
OAH-6-4. As a result, the intracellular concentration of acetyl-CoA and titer of OAH
5
in OAH-6-4 reached 0.43 nmol/mg CDW and 9.86 g/L, respectively, which were
6
obviously higher than those of OAH-6-1 and OAH-6-2 but lower than those of
7
OAH-6-3 (Fig. 6a, 6b and 6c). These results suggested that PDH and ACS played a
8
synergistic role in the biosynthesis of acetyl-CoA and OAH production. The similar
9
conclusion was also observed in the previous reports.33 The increased activity of PDH
10
could channel more carbon flux to acetyl-CoA node, and overexpression of ACS
11
could compensate for the loss of acetyl-CoA caused by PTA-ACK.32 Consequently,
12
the increased acetyl-CoA as an important precursor further drove the biosynthesis of
13
OAH.
14
To sum up, three strategies were successively performed to regulate the
15
acetyl-CoA level, including promoting CoA biosynthesis, enhancing PDH flux and
16
overexpressing the heterogeneous ACS. As a result, the intracellular concentration of
17
acetyl-CoA was significantly increased by 3.47 folds, which led to higher OAH
18
production along with lower accumulation of acetate. These results also provided
19
promising methods to engineer the acetyl-CoA supply for the production of other
20
valuable chemicals in E. coli.
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Fig. 6. Increasing intracellular concentrations of acetyl-CoA and NADPH to promote OAH production. (a) The intracellular concentrations of acetyl-CoA in the engineered strains are assayed. (b, c) Titers of OAH and byproducts, biomass, glucose consumption, specific production and yield of OAH in the engineered strains are shown. (d) The intracellular NADPH levels and NADPH/NADP ratios of the engineered strains are determined. All data shown are means from three independent experiments and error bars indicate the standard deviation from the means.
9 10
Engineering of NADPH regeneration to improve the biosynthesis of OAH
11
In the OAH biosynthetic pathway, 2 mol of NADPH were required for the
12
formation of 1 mol OAH. Therefore, efficient regeneration of NADPH might be one
13
of the limiting factors for the biosynthesis of OAH. Most of metabolic engineering
14
strategies for enhancement of NADPH supply mainly focused on strengthening
15
pentose phosphate pathway (PPP) and transhydrogenases system, the two major
16
routes for NADPH generation in E. coli.34 However, the increased fluxes of PPP and
17
transhydrogenases system resulted in the inevitable loss of carbon and energy
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1
consumption.35 In PDH pathway, 1 mol NADH would be generated at this enzymatic
2
reaction with decarboxylation of 1 mol pyruvate. Thus, engineering of PDH to switch
3
the coenzyme specificity from NAD+ to NADP+ might be an efficient strategy to
4
increase NADPH availability in E. coli.
5
For engineering of NADPH generation, the NADP-dependent Lpd mutant
6
(Lpdm) was introduced in OAH-6-3 to generate OAH-7.36 The specific activities of
7
PDH in OAH-7 using NAD+ or NADP+ as cofactor were 0.18 ± 0.01 or 0.33 ± 0.02
8
U/mg protein, respectively (Fig. S6). Specially, compared with OAH-6-3, the
9
intracellular NADPH level of OAH-7 was improved from 0.49 μmol/g CDW to 0.71
10
μmol/g CDW and the NADPH/NADP+ ratio was increased from 0.72 to 1.28 (Fig.
11
6d), but the acetyl-CoA level has no visible difference, which suggested the
12
expression of NADP-dependent PDH complex could significantly promote the
13
regeneration of NADPH. More important, OAH-7 could accumulate 12.1 g/L OAH
14
with specific production and yield of 4.15 g/g CDW and 0.37 g/g glucose,
15
respectively, which was 10.6% higher than that of OAH-6-3 (Fig. 6b and 6c).
16
Meanwhile, the titers of byproducts homoserine and acetate reached 1.19 g/L and 0.89
17
g/L, respectively. These results indicated that the increased NADPH level was
18
beneficial to improve the titer and yield of OAH.
19
Fed-batch fermentation in a 7.5 L fermenter
20
A comprehensive evaluation of the characteristic of the OAH-7 strain was
21
implemented using fed-batch fermentation in a 7.5 L fermenter with 4 L working
22
volume. The initial OAH-1 strain was used as the control. When the initial glucose
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was consumed, 80 mL of feeding solution was fed into the fermenter. As shown in
2
figure 7a and 7b, the cell growth steadily increased, and the maximum OD600 of
3
OAH-7 reached 82.8, slightly lower than that of OAH-1 (90.6). The accumulation of
4
OAH in OAH-7 was initially detected at 12 h, and greatly increased to 62.7 g/L at 58
5
h, which was 3.15-fold higher than that of OAH-1 (15.1 g/L). Moreover, the yield and
6
productivity of OAH in OAH-7 were 0.45 g/g glucose and 1.08 g/L/h, respectively,
7
significantly higher than those of OAH-1 (0.11 g/g glucose and 0.24 g/L/h,
8
respectively). However, a number of homoserine was also accumulated during the
9
anaphase of the fermentation although its titer was decreased from 8.63 to 3.42 g/L.
10
The phenomenon may be due to the feedback inhibition of MetXlm by end-product
11
OAH. Despite the improved feedback resistance to OAH, the inhibitory action of the
12
MetXlm mutant still existed (Fig. 3d). Therefore, more works should be further
13
performed to improve the property of MetXlm. Furthermore, the titers of acetate in
14
OAH-7 were considerably decreased from 7.52 to 2.45 g/L in comparison with
15
OAH-1. Additionally, lactate and aspartate were also detected as the byproducts with
16
1.32 g/L and 0.57 g/L, respectively.
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1 2 3 4 5
Fig. 7. Fed-batch fermentation profiles of OAH-1 (a) and OAH-7 (b) in a 7.5 L fermenter. Biomass, glucose consumption, OAH production, HSE and ACE titers are shown. All data shown are means from three independent experiments and error bars indicate the standard deviation from the means.
6 7
Conclusion
8
O-acetylhomoserine (OAH) is a promising platform chemical for the production
9
of L-methionine and other valuable compounds. In this study, we successfully
10
developed an efficient OAH-producing strain with high titer and yield by combining
11
protein and metabolic engineering in E. coli. An initial OAH-producing strain was
12
constructed by reconstruction of biosynthetic pathway and deletion of degradation and
13
competitive pathways to achieve the accumulation of OAH. Subsequently, several
14
metabolic engineering strategies were performed to improve the biosynthesis of OAH.
15
The pathway flux of OAH was enhanced by eliminating the accumulation of L-lysine,
16
modulating the expression of ppc to increase the oxaloacetate supply and promoting
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the biosynthesis of precursor homoserine, resulting in a 1.79-fold increase in OAH
2
production. But the considerable accumulation of intermediate metabolite homoserine
3
indicated that MetX was an important rate-limiting step for the biosynthesis of OAH.
4
Therefore, protein engineering of the key enzyme MetXlm was further performed to
5
improve the enzyme activity and relieve feedback inhibition based on evolutionary
6
conservation analysis and structure-guided engineering. The resulting triple
7
F147L-M182I-M240A mutant of MetXlm exhibited a 12.15-fold increase in specific
8
activity and retained more residual activity in the presence of 10 mM OAH.
9
Moreover, the optimized expression of the triple mutant led to a 57.14% increase in
10
OAH production. As one of the precursors, the acetyl-CoA supply was increased by
11
promoting CoA biosynthesis, enhancing PDH flux and overexpressing the
12
heterogeneous ACS to promote OAH biosynthesis. Furthermore, the NADPH
13
generation was engineered by introducing NADP-dependent PDH complex, which
14
resulted in 10.6% improvement in OAH production. Finally, the engineered strain
15
OAH-7 produced 62.7 g/L of OAH with yield of 0.45 g/g glucose and productivity of
16
1.08 g/L/h in a 7.5 L fed-batch fermenter, which was the highest OAH production
17
reported to date.
18
Materials and methods
19
Strains, plasmids and culture conditions
20
The strains and plasmids used in this study were listed in Table 1. E. coli DH5α
21
and BL21 were used as the host for plasmid construction and protein expression,
22
respectively. E. coli W3110 was used as the parent strain for OAH production. The
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1
plasmids of pTrc99a and pACYC184 were employed for gene expression, and the
2
pET21b vector was used for protein expression. CRISPR/Cas9 based genome editing
3
was applied for deletion of genes by plasmid pRedCas9.37 Luria-Bertani (LB) medium
4
was used for strain cultivation and protein expression. When needed, ampicillin,
5
kanamycin and chloramphenicol were supplemented at final concentrations of 100, 50
6
and 34 μg/mL, respectively.
7
Plasmids construction
8
The plasmids constructed in this study were listed in Table 1. The primers used in
9
this study were listed in Table S3. Golden gate assembly method was used to
10
construct the recombinant plasmid according to the standard protocol.38 For
11
expression of thrA (C1034T, feedback-insensitive mutant), the two partial fragments
12
of thrA were respectively amplified from the genomic DNA of E. coli W3110 using
13
the primers thrA1_F/1_R, 2_F/2_R, and fused by overlap PCR using the primers
14
thrA1_F/2_R to introduce the mutation C1034T. Then, the DNA fragments including
15
metXlm, mutational thrA (C1034T) and plasmid pTrc99a backbone were achieved by
16
PCR amplification using the primers metXlm EF/ER, thrA1_F/2_R and pTrc99a
17
F1/R1, respectively, and assembled by Golden gate cloning reaction to construct the
18
plasmid pTrc-MT.
19
For expression of lysC (C1055T, feedback-insensitive mutant) from E. coli, two
20
partial fragments of lysCec were respectively amplified from the genomic DNA of E.
21
coli W3110 using the primers LysCE1_F/1_R, 2_F/2_R, and fused by overlap PCR
22
using the primers LysCE1_F/2_R. Then the resultant lysCec fragment was assembled
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with the pTrc-MT backbone fragment obtained by the primers pTrc99a-F2/thrA2_R
2
to yield pTrc-MTLec. Meanwhile, the lysCcg (C932T, feedback-insensitive mutant)
3
from C. glutamicum, native asd and hom (G1133A, feedback-insensitive mutant)
4
from C. glutamicum were overexpressed using the same strategy to construct the
5
plasmid pTrc-MTLcg, pTrc-MTLA and pTrc-MTLAH, respectively. In order to
6
optimize the expression of metXlm, five ribosome bind site (RBS) artificial elements
7
with different intensities were selected from the reported library,30 and embedded into
8
the forward primer metXlm EF. Then, the metXlm fragments containing different
9
RBS were achieved by the fused primers, and assembled into the pTrc-MTLA to
10
generate
pTrc-M6TLA,
pTrc-M12TLA,
11
pTrc-M29TLA, respectively.
pTrc-M18TLA,
pTrc-M24TLA
and
12
The low copy plasmid pACYC184 was revised to introduce the Ptrc promoter
13
using golden gate assembly, generating the plasmid pACYC. For expression of coaA
14
(R106A, feedback-insensitive mutant), two partial fragments of coaA were
15
respectively amplified using the primers coaA1_F/1_R, 2_F/2_R, and fused by the
16
primers coaA1_F/2_R. Then, the fragments of coaA and coaD were cloned into the
17
plasmid pACYC using the golden gate assembly to produce pACYC-AD. In order to
18
express PDH complex, three DNA fragments of aceE-aceF, lpd (A358V,
19
feedback-insensitive mutant) and the Ptrc promoter fragment were obtained by the
20
primers aceEF_F/aceEF_R, lpd_F/R and lacI-Ptrc_F2/Ptrc99a-R1, respectively, and
21
then assembled with the plasmid pACYC-AD backbone amplified by the primers
22
pACYC_F2/CoAD_R to generate pACYC-ADEFL. For expression of acs (L641P)
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from S. enterica, the gene was codon optimized and synthesized by Genewiz Biotech
2
Co., Ltd. (Suzhou, China) (Table S4). The acs gene fragment was ligated into the
3
pACYC-ADEFL to yield pACYC-ADAEFL. In order to investigate the interaction of
4
PDH and ACS, the acs gene fragment was cloned into the pACYC-AD to construct
5
pACYC-ADA. In order to switch the coenzyme specificity of PDH, the lpd mutant
6
(lpdm,
7
introduced.39 Three fragments of lpd containing different mutation sites were obtained
8
using the primers lpd_F/1_R, 2_F/2_R and 3_F/_R, respectively, and assembled with
9
the pACYC-ADAEFL backbone generated by the primers pACYC_F2/aceEF_R to
10
G185A/G189A/E203/M204R/F205K/
D206H/P210R/A358V)
was
create the plasmid pACYC-ADAEFLm.
11
For construction of protein expression plasmid, the genes of metX from C.
12
glutamicum and L. meyeri were codon optimized and chemically synthesized by
13
Genewiz Biotech Co., Ltd. (Suzhou, China), respectively (Table S4). Then, the both
14
DNA fragments were amplified using the primers metXcg-PET-F/R and
15
metXlm-PET-F/R, and ligated into pET21b using the restriction enzyme NdeI and
16
XhoI to yield the plasmids pET-metXcg and pET-metXlm, respectively.
17
Genome Manipulation
18
The
gene
deletion
and
promoter
replacement
were
performed
by
19
CRISPR/Cas9-mediated one-plasmid genome editing system using the method
20
described previously.37 For deletion of gene metB, two about 300 bp homologous
21
fragments were respectively amplified from the genome of E. coli W3110 using the
22
primers metB1_F/1_R and metB2_F/2_R, and two functional modules fragments
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1
containing Cas9 fragment and Red recombinase fragment fused with PAM sequence
2
of
3
MetBN20_CASB_F/CASB_R, respectively. Then, the four DNA fragments were
4
assembled in a single golden gate cloning reaction to construct the deletion plasmid
5
pCas-metB. Subsequently, the genes thrB, metA and lysA were all deleted using the
6
same strategies above. For replacement of ppc promoter, four artificial promoter
7
elements, PM1-12, PM1-46, PM1-37 and PM1-93, were embedded into the primers, and the
8
homologous fragments containing different promoters were achieved using these
9
fused primers, and assembled with the two functional modules (Cas9 fragment and
10
Red recombinase fragment fused with PAM sequence of ppc) to yield the plasmid
11
pCas-ppc12, pCas-ppc46, pCas-ppc37 and pCas-ppc93, respectively.
12
Expression and purification of MetX and enzyme activity assays
metB
were
also
obtained
using
the
primers
CASA_F/R,
13
The protein expression and purification were carried out according to the method
14
described previously.40 The purified enzyme were determined by SDS-PAGE, and
15
used to assay the kinetic parameters of enzymes. More detailed descriptions of the
16
experiments were given in the supplementary materials and methods.
17
For measurements of the enzyme activities in the engineered strains, the strains
18
were cultivated in the fermentation medium at 30 °C for 12 h. The cells were
19
harvested by centrifugation, and washed twice with 20 mM Na2HPO4 buffer (pH 7.5).
20
The collected cells were sonicated for 15 min on ice, and centrifuged at 15000 × g for
21
20 min. The supernatants were used for enzyme activities assays. The enzymatic
22
activity of MetX was measured by monitoring the formation of CoA according to the
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1
method described previously.41 The reaction mixture without L-homoserine was used
2
as the blank control. The protein concentration was determined using bicinchoninic
3
acid (BCA) protein assay kit (Pierce, USA) according to the manufacturer’s protocol.
4
The enzymatic activities of aspartokinase (AK), PPC and PDH were assayed using the
5
method described previously.42-44 The specific activity units of enzymes were defined
6
as the amounts of enzymes for the generation of 1 μmol product per min.
7
Evolutionary conservation analysis
8
The homologous sequences of MetXlm were collected through blast in local
9
UniRef90 database for sequence conservation analysis.17 A sequence motif was
10
constructed by HMMER software version 3.2,45 and further used to search in
11
UniRef90 database. The program Cd-hit was applied to filter the high similar proteins
12
with a cutoff value 98%, and the software Muscle was employed for multiple
13
sequence alignment of the remaining homologous sequences.46,
14
quality of sequence analysis, the gaps of sequence were removed using the trimAl
15
program based on the target sequence.48 The final alignment consisted of 974
16
sequences and 378 amino acid positions. The intensity of conservation for each
17
residue and the frequency of amino acids in each position were calculated,
18
respectively.
19
Homology modeling and molecular docking
47
To improve the
20
The three-dimensional model of MetXlm was built using RosettaCM software.27
21
The crystal structures of homoserine acetyltransferase from L. Interrogans (PDB ID:
22
2PL5) and H. influenzae (PDB ID: 2B61) were chosen as templates for homologous
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ACS Synthetic Biology
1
modeling. The model with lowest overall energy was selected for further analysis.
2
The RosettaLigand application was used to dock CoA into the constructed model.49
3
The CoA structure was built based on the geometric orientation observed in the
4
crystal structure of deacetylcephalosporin C acetyltransferase (PDB ID: 2VAT). The
5
docking model with the lowest binding energy and overall energy was selected as
6
ligand-binding model for further analysis. More detailed descriptions about homology
7
modeling and molecular docking were provided in the supplementary materials and
8
methods.
9
Site directed mutagenesis and saturation mutagenesis of MetX
10
Site directed mutagenesis and saturation mutagenesis of MetX were performed
11
according to the method reported previously.28 For saturation mutagenesis, the
12
random primers containing an NNK degenerate codon at desired sites (L236, M240,
13
Y267, E270, P348, A349, L356 and P357) were used to introduce the mutations by
14
golden gate assembly.38 To ensure the 95% coverage of each library, about 95
15
colonies were randomly selected from each single-site mutant library. The cells were
16
cultivated in 96 deep-well plates containing 800 µL of LB medium for 12 h at 30 ºC
17
and 800 rpm. 0.4 mM IPTG was added for inducing protein expression. The cells
18
were harvested by centrifugation, and resuspended in 200 µL of the lysis buffer (20
19
mM Na2HPO4, 200 mM NaCl, pH 7.5) with 2 mg/mL lysozyme and 0.05% Triton
20
X-100 The cell lysate was centrifuged after incubation for 2 h at 37 °C, and the
21
supernatant was usd for enzyme activity assay.
22
Thermal stability measurement
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1
The thermal stability of MetX was determined by differential scanning
2
calorimetry based on the reported method.25 The purified protein was diluted to 0.5
3
mg/mL using 20 mM Na2HPO4 buffer (pH 7.5). A microCal VP-DSC calorimeter
4
(MicroCal Inc.) was employed over a temperature range of 30 to 80 °C at a scan rate
5
of 1°C/min. The phosphate buffer was used as the blank control. For brief comparison
6
of samples, the raw data was processed by subtracting baseline of the buffer.
7
Measurement of intracellular NADPH/NADP+ and acetyl-CoA
8
The E. coli strains were cultivated in the fermentation medium at 30 °C for 12 h,
9
and harvested by centrifugation at 15000 × g for 20 min. The intracellular
10
concentrations of NADPH or NADP+ were determined by the cycling assay method
11
described previously.50 The intracellular acetyl-CoA concentration was measured
12
using Pico-Probe acetyl-CoA assay kit (BioVision, CA, USA) according to the
13
manufacturer’s instructions. The concentrations of NADPH and acetyl-CoA were
14
normalized by the cell dry weight.
15
Batch and fed-batch cultivation
16
For batch fermentation, the single clone of E. coli was cultivated in LB medium
17
at 37 °C for overnight, and then inoculated into 20 mL seed medium with 5% (v/v)
18
inoculum in a 250 mL shake flask at 37 °C for 10 h. Subsequently, the cells were
19
inoculated into a 500 mL shake flask containing 50 mL fermentation medium at the
20
initial optical density (OD) of 0.2, and cultured for 48 h at 30 °C, 200 rpm. IPTG was
21
added to the final concentration of 0.1 mM when OD reached 5. The seed medium
22
contained 10 g/L glucose, 10 g/L yeast extract, 10 g/L (NH4)2SO4, 0.5 g/L citrate, 1.5
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1
g/L KH2PO4, 0.5 g/L MgSO4, 15 mg/L FeSO4, 10 mg/L MnSO4 and 0.8 mg/L VB1,
2
pH 7. The fermentation medium contained 50 g/L glucose, 4 g/L yeast extract, 12 g/L
3
(NH4)2SO4, 1 g/L MgSO4, 2 g/L KH2PO4, 23 mg/L FeSO4, 14 mg/L MnSO4, 0.8
4
mg/L VB1 and 0.1 mg/L VB7, pH 6.5. When necessary, L-methionine, L-threonine,
5
L-lysine and pantothenic acid were supplemented to the final concentrations of 0.1,
6
0.15, 0.1 and 0.05 g/L. 3-morpholinopropanesulfonic acid (MOPS) was used to buffer
7
the pH at a final concentration of 0.2 M.
8
For fed-batch fermentation, the seed and fermentation media in fermenter were
9
the same as those of shake-flask culture. Fed-batch culture was carried out in a 7.5 L
10
fermenter (Shanghai Auzone Bio-Engineering Equipment, Shanghai, China)
11
containing 4 L medium at 30 °C. The pH was kept constantly at 6.5 by automatic
12
feeding of NH4OH (25%, v/v). The dissolved oxygen (DO) was maintained at 20% of
13
air saturation by controlling agitation speed and aeration rate. 0.1 mM IPTG was
14
added to induce expression when OD600 achieved 15. The pH-stat fed-batch
15
fermentation model was performed to control fermentation process.10 When the pH
16
increased higher than 6.8, 80 mL of feeding solution containing 500 g/L glucose was
17
automatically added.
18
Analytical Procedures
19
Cell growth was monitored by the optical density (OD) at 600 nm using an
20
ultraviolet spectrophotometer. Cell dry weight (CDW; in g/L) was determined using
21
the conversion factor described previously, 1 OD600 = 0.33 g/L CDW.51 Glucose
22
concentration was determined using SBA-40D biological sensor (Shandong Academy
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1
Of Sciences, Shandong, China). The concentrations of amino acids were measured
2
using high performance liquid chromatography (HPLC) system (Agilent 1260, USA)
3
with a ZORBAX Eclipse AAA column (4.6 x 150 mm, 5 μm; Agilent, USA)
4
according to the standard method described previously.1 The titers of organic acids,
5
such as acetate and lactate, were analyzed by HPLC with Aminex HPX-87H column
6
(300 x 7.8 mm, Bio-Rad, USA) using the reported standard method.19
7
Author Information
8 9
Corresponding Author
10
Jun Liu; E-mail:
[email protected].
11
Huifeng Jiang; E-mail:
[email protected].
12 13
Author Contribution
14
L.W. and J.L. designed the experiments; Q.W. and H.J. performed the in silico
15
simulation experiments; L.W. performed the experiments; N.X., J.C., W.Z., G.H.,
16
Y.M. provided some help in experiments; L.W. and J.L. drafted the manuscript
17 18
Notes
19
The authors declare no competing financial interest
20 21 22
Acknowledgements We are grateful to Prof. Changhao Bi (Tianjin Institute of Industrial
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ACS Synthetic Biology
1
Biotechnology, Chinese Academy of Sciences, China) for generously providing
2
plasmids. This study was supported by the National Natural Science Foundation of
3
China (No. 31500044, No. 31801526), the Natural Science Foundation of Tianjin
4
(No. 17JCQNJC09600, No. 17JCYBJC24000), the Key Projects in the Tianjin
5
Science and Technology Pillar Program (14ZCZDSY00157), and “Hundred Talents
6
Program” of the Chinese Academy of Sciences.
7 8
Supporting information
9
Table S1. Effects of optimized PPC expression on OAH production;
10
Table S2. The frequency rank lists of residues in MetXlm;
11
Table S3. Primers used in this study;
12
Table S4. The nucleotide sequences of codon-optimized genes used in this study;
13
Fig. S1. SDS-PAGE analysis of MetXs from C. glutamicum and L. meyeri;
14
Fig. S2. Comparison of the specific activities of two MetXs from C. glutamicum and
15
L. meyeri in the presence of inhibitors with different concentrations;
16
Fig. S3. Measurement of the specific activities of aspartokinase in crude enzyme
17
extracts of the engineered strains of OAH-3-2, OAH-4-1 and OAH-4-2;
18
Fig. S4. The evolutionary conservation analysis of MetXlm;
19
Fig. S5. The residues of MetXlm within the 3Å distance in substrate access tunnel;
20
Fig. S6. Determination of the specific activities of PDH with different cofactors
21
(NAD+ or NADP+) in crude enzyme extracts of the engineered strains of OAH-6-1,
22
OAH-6-2, OAH-6-3 and OAH-7.
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1 2 3 4
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Table 1. Strains and plasmids used in this study. Strains or plasmids Characteristics Strains E. coli DH5α Host for plasmid construction E. coli BL21(DE3) Host for protein expression E. coli W3110 Wild type, the starting strain BL21-MetXcg E. coli BL21(DE3) with pET21b-MetXcg BL21-MetXlm E. coli BL21(DE3) with pET21b-MetXlm OAH-1 W3110, ∆metB, ∆thrB, ∆metA; pTrc-MT OAH-2 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; pTrc-MT OAH-3-1 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-12; pTrc-MT OAH-3-2 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-MT OAH-3-3 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-37; pTrc-MT OAH-3-4 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-93; pTrc-MT OAH-4-1 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-MTLec OAH-4-2 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-MTLcg OAH-4-3 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-MTLA OAH-4-4 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-MTLAH OAH-5 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-MmTLA OAH-5-1 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-M6TLA OAH-5-2 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-M12TLA OAH-5-3 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-M18TLA OAH-5-4 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-M24TLA OAH-5-5 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-M29TLA OAH-6-1 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-M18TLA; pACYC-AD OAH-6-2 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-M18TLA; pACYC-ADEFL OAH-6-3 W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; pTrc-M18TLA; pACYC-ADAEFL
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OAH-6-4 OAH-7 Plasmids pTrc99a pACYC184 pRedCas9 pET21b pET21b-MetXcg pET21b-MetXlm pCas-metB pCas-metA pCas-thrB pCas-lysA pCas-ppc12 pCas-ppc46 pCas-ppc37 pCas-ppc93 pTrc-MT pTrc-MTLec pTrc-MTLcg pTrc-MTLA pTrc-MTLAH pTrc-MmTLA pTrc-M6TLA pTrc-M12TLA pTrc-M18TLA pTrc-M24TLA pTrc-M29TLA pACYC pACYC-AD pACYC-ADEFL
W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; This work pTrc-M18TLA; pACYC-ADA W3110, ∆metB, ∆thrB, ∆metA, ∆lysA; Pppc::PM1-46; This work pTrc-M18TLA; pACYC-ADAEFLm AmpR, Ptrc promoter, gene expression vector CmR, gene expression vector KanR, CRISPER/cas9-mediated deletion vector AmpR, expression vector pET21b containing metX from C. glutamicum pET21b containing metX from L. meyeri Vector for deletion of metB Vector for deletion of metA Vector for deletion of thrB Vector for deletion of lysA Vector for replacement of ppc promoter with PM1-12 Vector for replacement of ppc promoter with PM1-46 Vector for replacement of ppc promoter with PM1-37 Vector for replacement of ppc promoter with PM1-93 pTrc99a containing metXlm and mutated thrA (C1034T) pTrc-MT containing mutated lysCec (C1055T) from E. coli pTrc-MT containing mutated lysCcg (C932T) from C. glutamicum pTrc-MTLcg containing native asd pTrc-MTLA containing mutated hom (G1133A) from C. glutamicum pTrc-MTLA containing the triple mutant of MetXlm (F147L-M182I-M240A) pTrc-MTLA containing mutated metXlm under the control of RBS6 pTrc-MTLA containing mutated metXlm under control of RBS12 pTrc-MTLA containing mutated metXlm under control of RBS18 pTrc-MTLA containing mutated metXlm under control of RBS24 pTrc-MTLA containing mutated metXlm under control of RBS29 CmR, pACYC184 containing Ptrc promoter pACYC containing mutated coaA (R106A) and coaD pACYC-AD containing aceE, aceF and mutated
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lpd (A358V) pACYC-ADEFL containing mutated acs (L641P) This study from S. enterica pACYC-ADA pACYC-AD containing mutated acs (L641P) from This study S. enterica pACYC-ADAEFLm pACYC-ADAEFL containing mutated This study NADP-dependent lpd (lpdm) pACYC-ADAEFL
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Table 2. The enzyme kinetics of MetXlm and mutants a Km kcat Enzyme Substrate b (mM) (min-1) MetXlm HSE 0.67 ± 0.02 27.75 ± 1.4 AcCoA 0.36 ± 0.02 27.69 ± 1.7 F147L HSE 0.62 ± 0.05 76.23 ± 3.2 AcCoA 0.38 ± 0.02 76.28 ± 3.5 M182I HSE 0.57 ± 0.04 92.15 ± 3.3 AcCoA 0.31 ± 0.02 92.11 ± 2.9 M240A HSE 0.22 ± 0.02 68.56 ± 2.4 AcCoA 0.14 ± 0.01 68.61 ± 2.5 F147L-M182I-M240A HSE 0.24 ± 0.02 107.48 ± 4.4 AcCoA 0.16 ± 0.01 107.51 ± 4.2 a All data shown are means from three independent experiments enzymes and ± indicate the standard deviation from the means. b HSE, L-homoserine; AcCOA, acetyl-CoA.
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kcat/Km (mM-1 min-1) 41.41 ± 3.5 76.91 ± 8.5 122.95 ± 12.4 200.73 ± 15.8 161.66 ± 14.3 297.13 ± 25.4 311.64 ± 31.1 490.07 ± 41 447.83 ± 40.7 671.94 ± 52.8 using purified
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