Type IIs restriction based combinatory modulation technique for metabolic pathway optimization
© The Author(s) 2017
Received: 6 December 2016
Accepted: 8 March 2017
Published: 16 March 2017
One of the most important research subjects of metabolic engineering is pursuing a balanced metabolic pathway, which is the basis of an efficient cell factory. In this work, we dedicated to develop a simple and efficient technique to modulate expression of multiple genes simultaneously, and select for the optimal regulation pattern.
A Type IIs restriction based combinatory modulation (TRCM) technique was designed and established in the research. With this technique, a plasmid library containing variably regulated mvaE, mvaS, mvaK 1 , mvaD and mvaK 2 of the mevalonate (MVA) pathway were obtained and transformed into E. coli DXS37-IDI46 to obtain a β-carotene producer library. The ratio of successfully assembled plasmids was determined to be 35%, which was increased to 100% when color based pre-screening was applied. Representative strains were sequenced to contain diverse RBSs as designed to regulate expression of MVA pathway genes. A relatively balanced MVA pathway was achieved in E. coli cell factory to increase the β-carotene yield by two fold. Furthermore, the approximate regulation pattern of this optimal MVA pathway was illustrated.
A TRCM technique for metabolic pathway optimization was designed and established in this research, which can be applied to various applications in terms of metabolic pathway regulation and optimization.
KeywordsMetabolic pathway optimization Type IIs restriction β-carotene MVA pathway Terpene
As the development of Synthetic Biology and Metabolic Engineering, various microbial cell factories have been developed for producing value-added chemical compounds. However, engineering of cell metabolism often disturbs the metabolic network,triggers energetic and objective inefficiency inside the cell, and impedes cell metabolism . Hence, one of the most important research subjects of metabolic engineering is pursuing the balanced metabolic network and pathways. Techniques have been developed to analyze metabolic pathways, including genome-scale models and C13-metabolic flux analysis. And there have been strategies developed to relieve the metabolic burden, such as enhancing respiration, co-utilizing nutrient resources, decoupling cell growth with production phases, and dynamic regulatory systems . As for a specific metabolic pathway, gene expression level is the key effector of the pathway efficiency . Lower expression of genes decreases metabolic pathway flux, while overexpressed genes may over-consume building blocks and cause cells metabolic burden . Furthermore, imbalanced pathway may cause accumulation of pathway intermediates, some of which may even be toxic and jeopardize cell growth .
Due to the complexity of metabolic network in organisms and difficulty to precisely control expression of certain gene, it is almost impossible to rationally design and construct an optimized metabolic pathway. In most metabolic engineering projects, one common way was to modulate gene expression one by one . With this strategy, the possibility to achieve an optimized regulation pattern is very low. A better solution was to analyze all possible expression levels of pathway genes in a combinatorial fashion. With this strategy, Yin et al. constructed a plasmid library containing the possible combinations of gene regulation patterns . A similar strategy was employed by Xu et al. to optimize fatty acid pathway. Plasmids of various copy numbers were used to carry expression genes for the first round of optimization, which was followed by fine tuning expression with four RBS elements . However, in their work, regulatory parts were limited and the plasmids were exhaustively constructed one by one, which limited the experiment outcome and made the process laborious. The same group also established a BioBrick based method with specially designed restriction adapters. Genes with regulatory elements could be iteratively integrated into the ePathBrick vectors to create a diversified expression library . Similarly, Zelcbuch et al. created a plasmid library construction method to “span high-dimensional expression space” . In their methods, the libraries were constructed with multiple rounds of plasmid construction, which made the practice very time consuming. In the work of Lee et al., a combinatorial library was established by Gibson assembly based method in one reaction [10, 11]. However, only five regulatory parts were employed, which decreased the diversity of the combinatorial library. Based on the extensive researches and great progress achieved by fellow researchers in this subject, we were able to develop a convenient method for constructing complex combinatorial expression library, which was aimed for optimizing a metabolic pathway with maximal outcome and minimal lab hours.
Strains, medium and growth conditions
Strains and plasmids in this study
E. coli DH5α
F− endA1thi-1 recA1 relA1 gyrA96deoRΦ80dlacΔ(lacZ) M15 Δ (lacZYA-argF) U169hsdR17 (r K − , m K + ) λ− supE44 phoA
Enterococcus faecalis wild-type
Streptococcus Pneumoniae wild-type
ATCC 8739, ldhA::M1-12::crtEXYIB::ldhA, M1-37::dxs, M1-46::idi
E. coli expression vector derived from pACYC184, promoter of gadA, RFP, cat
plasmid library of combinatorically regulated MVA pathway, derived from pACYC184-PgadA-RFP
Genes, vector and primers
MVA pathway genes mvaE, mvaS, mvaK 1 , mvaD, and mvaK 2 were amplified from genomic DNA of Enterococcus faecalis CGMCC No.1.2135 using primer set Ga2-R1-EfmvaE-F/Ga2-R1-EfmvaE-R, Ga3-R1-EfmvaS-F/Ga3-R1-EfmvaS-R, and from genomic DNA of Streptococcus pneumoniae CGMCC No.1.8722 using primer set Ga46-R1-SpmvaK1-F/Ga46-R1-SpmvaK1-R, Ga7-R1-SpmvaD-F/Ga7-R1-SpmvaD-R, and Ga8-R1-SpmvaK2-F/Ga8-R1-SpmvaK2-R respectively (Additional file 1: Table S1). The DNA fragments used for assembly were gel purified and designated as Ga2-mvaE, Ga3-mvaS, Ga46-mvaK1, Ga7-mvaD and Ga8-mvaK2 (Fig. 1b). Vector Fragment Ga91-184A was amplified from pACYC184-PgadA-RFP, and subjected to DpnI digestion (10 U, 16 h, 37 °C) and gel purification. PCR was performed with PrimeSTAR® HS DNA Polymerase (Takara) with primers purchased from GENEWIZ (Suzhou, China). All assembly primers were designed with optimized linkers for Type IIs restriction enzyme based assembly, in which forward primers for amplification of genes were embedded with an RBS library at 5′ ends. Primers used in this study are summarized in Additional file 1: Table S1.
Construction of mva operon variants using TRCM
DNA fragments were assembled by Golden Gate DNA assembly method [22, 23]. 100 nanogram vector fragment Ga91-184A and equimolar amount of PCR amplified genes Ga2-mvaE, Ga3-mvaS, Ga46-mvaK1, Ga7-mvaD and Ga8-mvaK2 were mixed in 20 μL Golden Gate reaction solution with 1 μL BsaI-HF, 1 μL T4 ligase (New England Biolabs, Ipswich, MA) and 1× T4 ligase buffer. The reaction was carried out in a thermocycler using the following program: 37 °C for 5 min, 37 °C for 5 min (step 2), 16 °C for 10 min (step 3), step 2 and 3 for 20 cycles, 16 °C for 20 min, 37 °C for 30 min, 75 °C for 6 min, and 4 °C for hold. After PCR, 0.5 μL plasmid-safe nuclease (Epicenter), and 1 μL of 25 mM ATP was added to the reaction, which was incubated at 37 °C for 15 min. 1.5 μL of the resultant reaction solution were transformed into 80 μL competent DXS37-IDI46 cells to obtain the library .
In order to determine whether the MVA pathway genes were successfully incorporated into vector backbone, recombinant clones were subjected to colony PCR analysis. PCR primers were designed to amplify the region from mvaE (the first gene on plasmid) to mvaK 2 (the last gene on plasmid) by primers fE-JF and pK2-JR (Additional file 1: Table S1), which had a product size of 3.8 Kbp. A master mix with 1× Es Taq MasterMix (CWBio, Peking, China) and 0.4 μM forward and reverse primer (Additional file 1: Table S1) was prepared, and 20 μL master mix was dispensed into each PCR tubes. Colonies were directly transferred from LB agar plates into the PCR tubes with sterile toothpicks. The PCR cycling was started with an initial denaturation temperature at 94 °C for 10 min, followed by 30 cycles (94 °C, 30 s; 61 °C, 30 s; and 72 °C, 2 min) and one fill-up cycle (72 °C, 2 min). The PCR products were analyzed on agarose–TAE gels.
Measurement of β-carotene titer
Production of β-carotene was quantified by measuring absorption of acetone-extracted β-carotene at 453 nm as previously reported . A standard curve was obtained by measuring OD453 of β-carotene standard samples (Cat. No. C4582, Sigma, USA) with varied concentrations using a Shimadzu UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). The results represented the mean ± standard deviation (S.D.) of three independent experiments. Dry cell weight (DCW) was calculated based on optical density at 600 nm (1 OD600 = 323 mg DCW).
Calculation of MVA pathway genes RBS strength of strains from TRCM libraries
RBS sequences of mvaE, mvaS, mvaK 1 , mvaD, and mvaK 2 in representative strains were obtained by PCR and DNA sequencing. Their theoretical RBS strength characterized by the value of translation initiation rate was calculated with the RBS Calculator [25, 26]. The RBS sequence diversity of the combinatory library was analyzed with the Weblogo software .
Results and discussions
Design of a Type IIs restriction based combinatory modulation technique (TRCM) for metabolic pathway optimization
With the purpose of developing a simple technique to modulate and optimize expression of multiple genes simultaneously, we designed a Type IIs restriction based combinatory modulation technique (TRCM) for metabolic pathway optimization as illustrated in Fig. 2. Variably regulated genes were obtained by PCR amplification with extended primers, in which degenerate RBS nucleotides were embedded at the 5′ ends. Specifically designed linkers for Type IIs restriction enzymes were also imbedded in the primers to ensure the assembly pattern and efficiency.
Type IIs restriction based Golden Gate  was employed as DNA assembly method in this work, which has several advantages. First, there is no PCR reaction involved in the assembling process, which reduces the possibility of mutation compared with other PCR based assembly methods such as Gibson and CPEC ; second, the ligase facilitated irreversible ligation greatly improves assembly efficiency compared with homologous arm based method . With this method, gene parts of a pathway were assembled with a vector part to form an expression plasmid. Since each gene part was constructed to carry a collection of regulatory parts, a combinatory plasmid library with variably regulated pathway genes was created, which was subsequently transformed into dedicated host to be screened and selected for strains carrying optimized pathways.
This technique was designed with the modularized strategy to be as simple as possible. The vector part was ready-made for all reactions, providing a stable plasmid backbone. By incorporation of fixed linkers and regulatory elements in primers for amplification of genes, the only variable parts of this method were the actual PCR primer sequences of pathway genes (Fig. 2a).
Development and application of TRCM for MVA pathway optimization
Our lab has constructed a few E. coli β-carotene producers, such as DXS37-IDI46 and CAR001, by modulating several key genes of the MEP pathway module, the pentose phosphate pathway (PPP) module, the ATP module and the tricarboxylic acid cycle (TCA) module . In this work, a heterologous MVA pathway optimized with TRCM technique was introduced into DXS37-IDI46 for further improving its β-carotene production (Fig. 2).
Since MVA pathway upstream genes mvaS, mvaE from Enterococcus faecalis and downstream genes mvaK 1 , mvaK 2 , mvaD from Streptococcus pneumoniae were reported to be successfully expressed in E. coli, they were selected to be used in this research . As designed with the TRCM technique, primers for PCR amplification of vector and MVA genes were embedded with BsaI recognition sites GGTCTC and specific four bp linkers, in order to be assembled in sequence. These linkers were rationally designed and experimentally tested to enable efficient assembly of DNA parts regardless of condition change (Fig. 2a).
To create a library of differently regulated genes, the RBS sequences of each gene were degenerated. For this purpose, forward primers of MVA genes Ga2-R1-EfmvaE-F, Ga3-R1-EfmvaS-F, Ga46-R1-SpmvaK1-F, Ga7-R1-SpmvaD-F and Ga8-R1-SpmvaK2-F were embedded with the random RBS sequence AGGAGRNNNNNN behind the 4 bp linkers. The starting code ATG of each gene was located behind the six Ns, which was also the starting point of actual PCR primers (Fig. 1a).
Gene parts, which carried front and back linkers for assembly in sequence, were obtained with PCR amplification. In Golden Gate assembly reaction, PCR amplified mvaS, mvaE, mvaK 1 , mvaK 2 and mvaD parts were mixed with the ready-made vector part Ga91–184A (Fig. 1b). After reaction, a plasmid library was created, which had differentially regulated MVA genes in various combinations. Theoretically, all patterns of differently expressed MVA pathway could be obtained in such a library (Fig. 1c). With this simple method, we achieved the goal of spanning high-dimensional expression space .
β-carotene production was improved with TRCM optimized MVA pathway
A combinatorial expression library with five genes regulated by diverse RBSs was obtained with TRCM technique
MVA gene RBS sequences with their calculated strength of representative strains from the combinatory expression library DXS37-IDI46 (pACYC184-AL-mva)
The genes RBS of MVA pathway
Relative yield (%)b
An approximate expression pattern of optimal MVA pathway was illustrated
Analysis of the representative strains indicated that an efficient MVA pathway contained genes expressed at a medium level, among which mvaE coordinately expresses with mvaS to avoid HMG-CoA accumulation, and a higher expression level of mvaK 1 is beneficial.
A TRCM technique was designed and established in the research, which could be easily applied to various applications in terms of metabolic pathway regulation and optimization. An optimized MVA pathway was constructed with TRCM to increase β-carotene yield of E. coli cell factory by twofold, and the optimal regulation pattern of MVA pathway was analyzed and illustrated.
dry cell weight
YL and HP planned and performed experiments, analyzed and interpreted the data. LQ, BC and ZX supervised the study, designed experiments and interpreted the results. YL wrote the manuscript. All authors read and approved the final manuscript.
We would like to thank the two great reviewers this manuscript is lucky to have, who took a lot of time and made tremendous efforts to give very precise criticism and very constructive suggestions to help make this manuscript better.
The authors declare that they have no competing interests.
Availability of data and materials
All supporting data is present in the article and the supplemental material documents. Specifically, plasmid maps and DNA sequence data are repent in Additional file 2.
Consent for publication
I hereby give the Journal of Microbial Cell Factories the right and permission to publish this article.
This research was supported by grants from National High Technology Research and Development Program of China (2015AA020202), Tianjin Key Technology RD program of Tianjin Municipal Science and Technology Commission (Y5M2121111), National Natural Science Foundation of China (31522002), Natural Science Foundation of Tianjin (15JCYBJC49400), and Chinese Academy of Sciences (NN-CAS) Research Fund (NNCAS-2015-2).
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