Engineering the pathway in Escherichia coli for the synthesis of medium-chain-length polyhydroxyalkanoates consisting of both even- and odd-chain monomers
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Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) containing various chain length monomers from C6 to C14 have more applications besides sustainable and environmental-friendly biomaterials owing to their superior physical and mechanical properties. We engineered a reversed fatty acid β-oxidation pathway in Escherichia coli that can synthesize mcl-PHA directly from glucose and achieved high yield. However, there were only even-chain monomers in the biosynthetic polymers. The need for mcl-PHA harboring both even- and odd-chain monomers with better and wider utility impels us to develop the biosynthetic routes for the production of the novel and unnatural mcl-PHA through rewiring the basic metabolism.
In the present study, a propionate assimilation and metabolic route was integrated into the reversed fatty acid β-oxidation in order to produce mcl-PHA consisting of both even- and odd-numbered monomers. The content of odd-numbered monomers in mcl-PHA was improved with the increased propionate addition. After further deletion of pyruvate oxidase (PoxB) and pyruvate formate-lyase (PflB), the metabolically engineered chassis E. coli LZ08 harboring pQQ05 and pZQ06 (overexpression of prpP and prpE genes from Ralstonia eutropha H16) innovatively accumulated 6.23 wt% mcl-PHA containing odd-chain monomers ranging from 7 to 13 carbon atoms about 20.03 mol%.
This is the first successful report on production of mcl-PHA harboring both even- and odd-chain monomers (C6–C14) synthesized from glucose and propionate in recombinant E. coli. This present study achieved the highest yield of de novo production of mcl-PHA containing odd-numbered monomers in E. coli at shake-flask fermentation level. Continued engineering of host strains and pathway enzymes will ultimately lead to more economical production of odd-chain monomers based on market demand. The synthetic pathway can provide a promising platform for production of other value-added chemicals and biomaterials that use acetyl-CoA and propionyl-CoA as versatile precursors and can be extended to other microorganisms as intelligent cell factories.
KeywordsEscherichia coli Polyhydroxyalkanoates Odd-chain monomers Reversed fatty acid β-oxidation cycle Metabolic engineering Synthetic biology
cell dry weight
The continuous consumption of resources such as petroleum and fossil fuels along with the increasing environmental pollution caused by petrochemical plastics have generated significant interests in developing and synthesizing bio-based materials. Polyhydroxyalkanoates (PHAs), as a class of environmental-friendly biomaterials, are accumulated by a variety of microbes from renewable carbon resources such as sugars [1, 2]. They have garnered great attention because of their unparalleled properties similar to elastomers and thermoplastics as potential alternatives for petroleum-based polymers [3, 4].
According to the different chain length monomer composition, PHAs can be divided into three main types: short-chain-length PHAs (scl-PHAs) which contain 3–5 carbon atoms, medium-chain-length PHAs (mcl-PHAs) which contain 6–14 carbon atoms, and scl-mcl PHAs which contain 3–14 carbons in length . The composition of copolymers determines the physical and mechanical material properties of the bioplastics. Generally, mcl-PHAs are synthesized via fatty acid de novo biosynthesis pathway or β-oxidation pathway from Pseudomonads in nature . They are semicrystalline and thermoplastic elastomers which are suitable for the materials in biomedical application . The traditional mcl-PHAs with only even-chain monomers have shown to own a desirable set of physical properties, and incorporating the fractions of odd-numbered monomers may lend the plastics more strength and flexibility so as to endow the polyesters novel and favorable properties and utilities. The Pseudomonas putida KT2442 mutant, KTOY06, accumulated a homopolymer of poly-3-hydroxyheptanoate (P3HHp) up to 71 wt% of its cell dry weight (CDW) when heptanoate was added as a single carbon source . In another case, 3-hydroxynonanoate (3HN) monomer (30–80 mol%) was the major constituent of polyhydroxyalkanoates accumulated from odd-numbered fatty acids by microorganisms . Lately, feeding of odd carboxylic acids ranging from valeric acid to pentadecanoic acid resulted in the odd carbon number monomer fractions such as 3HHp, 3HN and 3-hydroxyundecanoate (3HUD) and a small amount (10 mol% or less) of even carbon number monomer fractions was also detected in P. putida Bet001 . Researchers also reported that in N-limited shake flasks using nonanoic acid, P. citronellolis DSM 50332 produced 32% of its dry biomass as mcl-PHA containing 78% 3HN with 22% 3HHp . Therefore, propionate or odd-chain fatty-acid-rich feedstocks have been exogenously supplemented in the culture medium for their direct conversion to propionyl-CoA as the aforementioned studies. However, the high costs and toxicity to microbial cells associated with these fatty acids will limit their practical applications. Besides, the monomer types of mcl-PHA synthesized in the above research were not diversified. In view of this, it is a pressing demand to exploit an efficient metabolic pathway that leads to the formation of corresponding odd-chain (R)-3-hydroxyacyl-CoA as precursors for the acyl-chain elongation to biosynthesize mcl-PHA containing various odd-numbered monomers via adding the inexpensive carbon source-glucose.
For the past few years, rational strategies for metabolic pathway engineering and synthetic biology were exploited to balance the enzyme expression, eliminate the pathway regulatory bottleneck, and facilitate the production of targeted metabolites [12, 13, 14, 15, 16], such as PHA production [17, 18, 19, 20]. The engineered reversal of the fatty acid β-oxidation cycle provides a promising platform that can support the generation of various advanced products at high yields from renewable feedstocks recently with the development of systems metabolic engineering and synthetic biology [21, 22, 23]. Furthermore, there has been no report on the accumulation of odd-chain acyl-CoA for mcl-PHA production using glucose and propionate in E. coli cell factory by far. For this reason, the functional fatty acid β-oxidation reversal was mediated through supplying two-carbon extending acyl-CoA molecules from unrelated and cheap carbon source as biogenic precursors to synthesize different odd-numbered (R)-3-hydroxyacyl-CoA instead of adding only related carbon sources-fatty acids. To synthesize mcl-PHA that contained odd-chain monomers from the reversed fatty acid β-oxidation cycle, the starting precursor propionyl-CoA must be provided. In the previous study, after overexpressing the prpP gene in E. coli, the increasing pool of intracellular propionate facilitated the content of propionyl-CoA and increased the cell biomass . For the production of PHBV, Yang et al. employed the prpE gene from Ralstonia eutropha H16 to synthesize the propionyl-CoA and elevated the 3HV monomer fraction . Regarding PctRe, it can catalyze the transfer of CoA from acetyl-CoA to propionate . At the same time, it is worth mentioning that acetate overflow is the major drawback for production of acetyl-CoA-derived chemicals. Approaches for overcoming acetate overflow may be beneficial for biomass accumulation and the production of acetyl-CoA-derived products; for instance, PHA . This research aimed to construct the metabolic pathway for PHA production by integrating two parallel modules leading to the production of the even-chain monomers and the odd-chain monomers. The results demonstrated that the amount of odd-numbered monomers accumulated in the recombinant E. coli depended on the combination of propionate supplementation and propionyl-CoA supply. This is the first case revealing that engineered E. coli can produce novel and unnatural mcl-PHA consisting of the highest amount of odd-chain ranging from C7 to C13 motieties from glucose with addition of propionate.
Results and discussion
Construction and integration of individual module to enable direct microbial synthesis of even- and odd-chain mcl-PHA
Improvement of odd-chain monomer biosynthesis by simultaneous overexpression of double genes in the metabolic pathway
Effect of propionate concentration on odd-chain monomer production
Improvement of mcl-PHA accumulation by reinforcing acetyl-CoA supply
It has been a challenging task to synthesize mcl-PHA copolymers for a long time, especially for synthesizing even- and odd-chain mcl-PHA monomers equal to or longer than C8. There was no research reported that could make PHA copolyesters consisting of C6–C14 even- and odd-chain monomers. However, the fatty acid β-oxidation reversal was successfully utilized to generate the intermediates of mcl-PHA from renewable feedstocks in this study. By integrating two parallel precursor-supplying modules, the E. coli strain was confirmed to produce mcl-PHA containing both odd- and even-chain monomers efficiently. After optimization of the odd-numbered monomer module and the chassis, E. coli was found to synthesize mcl-PHA up to 6.23 wt% harboring odd-numbered monomers about 20.03 mol% from glucose and propionate. To the best of our knowledge, this is by far the first report on the novel mcl-PHA production both with even- and odd-numbered monomers with the highest yield. When grown on glucose and other related fatty acids, the recombinant E. coli was capable of producing other molar ratios of the monomers. This allows for generation of more and more PHA smart materials with diverse properties. Therefore, the engineered E. coli will be recruited as potential valuable and intelligent cell factories for industrial production to meet various applications.
Microbial strains and media
Strains and plasmids used in this study
Strains and plasmids
Source or references
E. coli DH5a
F−, endA1, hsdR17, (rk−, mk+), supE44, thi-1, λ−, recA1, gyrA96, ΔlacU169 (Φ80 lacZ ΔM15)
E. coli LS5218
F+, fadR601, atoC512 (Const)
E. coli LS5218 ΔptsG::FRT ΔtesB::FRT ΔyciA::FRT
Zhuang et al. 
E. coli LS5218 ΔptsG::FRT ΔtesA::FRT ΔpflB::FRT ΔpoxB::FRT
lacPOZ mobRP4, low-copy-no. cloning vector; KmR
Kovach et al. 
pTrc99a derivative, yqeF and fadB from E. coli MG1655, phaJ1 Pa and phaC2 Pa from P. aeruginosa PAO1, ter from Treponema denticola
Zhuang et al. 
pBBR1MCS2-prpP; pBBR1MCS-2 derivative, prpP from R. eutropha H16
pBBR1MCS2-acs; pBBR1MCS-2 derivative, acs from E. coli MG1655
pBBR1MCS2-prpE; pBBR1MCS2-derivative, prpE from R. eutropha H16
pBBR1MCS2-pct; pBBR1MCS-2 derivative, pct from R. eutropha H16
pBBR1MCS2-prpP-acs; pBBR1MCS-2 derivative, prpP from R. eutropha H16 and acs from E. coli MG1655
pBBR1MCS2-prpP-prpE; pBBR1MCS2-derivative, prpP and prpE from R. eutropha H16
pBBR1MCS2-prpP-pct; pBBR1MCS2-derivative, prpP and pct from R. eutropha H16
For the even-chain monomer supply, the construction of plasmid pQQ05 has been previously described . Briefly, the genes yqeF, fadB, phaJ1Pa, ter and phaC2Pa were all cloned and ligated into the corresponding sites of pTrc99a which were cut with the same restriction enzymes stepwise to generate plasmid pQQ05.
The construction of odd-chain monomer generation pathway was as follows. The codon-optimized prpP gene was cloned into the pBBR1MCS2 vector between the KpnI and BamHI sites to construct the plasmid of pZQ01. Later, in order to form the plasmid pBBR1MCS2-acs, namely pZQ02, the acs gene amplified via polymerase chain reaction (PCR) using E. coli MG1655 genomic DNA (gDNA) as template was also inserted into the pBBR1MCS2. The prpE and pct fragments amplified from R. eutropha H16 gDNA with primers prpE-F/prpE-R and pct-F/pct-R were separately ligated into the pBBR1MCS2 to yield the plasmids pZQ03 and pZQ04. Subsequently, co-expression of two genes prpP and acs, prpP and prpE, prpP and pct in the pBBR1MCS2 was utilized to form the plasmids pZQ05, pZQ06 and pZQ07, respectively. All of the genes were under the control of the lac promoter with separated ribosomal binding site located upstream of each gene to facilitate the translation. The R. eutropha H16 template used for these PCR reactions was isolated using the TIANamp Bacterial DNA Kit (TIANGEN BIOTECH, China). The primers used to amplify different fragments for cloning reactions are listed in Additional file 1: Table S1.
In all cases, PCR was performed using an S1000 Thermal Cycler (Bio-Rad, USA). PrimeSTAR HS DNA polymerase was purchased from Takara (Tokyo, Japan), restriction endonucleases were from Fermentas/Thermo Scientific (Pittsburgh, USA), and T4 DNA ligase was from New England Biolabs (Ipswich, USA). Propagated plasmids were prepared by TIANGEN Plasmid Mini Extraction Kit (TIANGEN BIOTECH, China), and restriction enzyme-digested products were purified using an E.Z.N.A.™ Gel Extraction Kit (Omega, USA). DNA sequencing of all constructed plasmids were performed by Liuhe BGI Tech Co. Ltd (Beijing, China). All of the constructed plasmids were transformed into the strain LZ05 and the optimum double plasmids were then transformed into the strain LZ08 according to standard procedures .
The gene pflB which encodes pyruvate formate lyase was knocked out by the one-step inactivation method as described previously  and poxB encoding pyruvate oxidase was knocked out by linearized DNA fragments with extending homologous sequence . First, the linerized DNA fragments with the FLP recognition target sites and 39 bp homologous sequences were obtained via PCR using pKD4 (KmR) as a template and pflB-F/pflB-R as primers. After the DNA gel extraction, the purified PCR product was electroporated into the host cells which carried the plasmid pKD46, and then E. coli LZ05 was induced by 0.3% (w/v) l-arabinose to express the λ Red system. The positive transformants were selected and identified by colony PCR using the primers pflB-test-F/pflB-test-R. Regarding the poxB deletion, primers poxB-F/poxB-R and chromosomal DNA of the strain QZ1111 were applied to amplify the linearized DNA fragments for poxB. The deletion procedure of poxB gene was as follows. After DpnI digestion, the PCR products were then purified and electroporated into the competent strain E. coli LZ05 containing the plasmid pKD46. Transformant cells were selected in solid LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, and 1.5% agar powder) containing chloramphenicol (CmR). Candidate clones were screened by PCR employing primers poxB-F/poxB-R. The PCR products were ultimately sequenced in Liuhe BGI Tech Co. Ltd (Beijing, China) if necessary. After removing pKD46, the corresponding KmR or CmR cassette was removed with the helper plasmid pCP20. The plasmids pKD46 and pCP20 were eliminated by overnight cultivation at 42 °C. Finally, the strain LZ08 with the above two gene inactivation was generated.
The medium of shake flask study contains 10 g/L tryptone, 5 g/L yeast extract, 30 mM NH4Cl, 5 mM (NH4)2SO4, 1.48 mM Na2HPO4, and 100 μM FeSO4 supplemented with 125 mM MOPS.
For all shake flask experiments, single colony was inoculated into 5 mL LB broth and grown at 37 °C overnight. 0.5 mL pre-culture was inoculated to 300 mL Erlenmeyer flask containing 50 mL LB and cultivated for 8 to 10 h and then 1% (v/v) seed inoculum for shake flask cultivation was incubated in 50 mL fermentation medium. When all liquid fermentation medium (50 mL) was incubated in 300 mL conical flasks at 37 °C with an agitation of 250 rpm to an optical density at 600 nm (OD600) of 0.6–0.8, 1 mM IPTG was added to the culture broth as an inducer. After induction, 30 g/L glucose was supplied as the sole carbon source at the appropriate time and then fermented for 72 h at 30 °C with shaking at 250 rpm. When necessary, ampicillin (100 μg/mL), kanamycin (50 μg/mL) or chloramphenicol (25 μg/mL) was added to the medium to maintain the stability of the plasmids. After cultivation, cells were gathered by centrifugation at 12,000 rpm for 15 min, washed with water twice and treated with ethanol once and then lyophilized.
PHA production analysis
The content and monomer compositions of intracellular accumulated PHA were analyzed by gas chromatography (GC) as described previously . PHA content was defined as the percent ratio of PHA concentration to CDW. Liquid culture was centrifuged to obtain the supernatant and cellular biomass. 15 mg lyophilized cells were subjected to methanolysis in the presence of 1 mL of chloroform and 1 mL of 3% (v/v) sulfuric acid in methanol for 1 h at 100 °C. The samples were cooled to room temperature and then 1 mL of distilled water was added in order to extract the cell debris that is soluble in the aqueous phase. 10 mg/mL pentadecanoic acid in ethanol was added as an internal standard. The mixture was vortexed and centrifuged at 12,000 rpm for 10 min. After the layer separation, the organic (chloroform) phase (500 μL) was transferred to another new vial and analyzed using a Shimadzu GC2010 gas chromatograph (Kyoto, Japan) equipped with an AOC-20i auto-injector and a RestekRxi®-5 column. PHA standard samples were dissolved in chloroform and also analyzed according to the method above by GC. The temperature program used was as follows: 80 °C hold for 1 min, ramp from 60 to 230 °C at 10 °C per min and a final hold at 230 °C for 10 min .
Cell growth, glucose consumption and acetate assimilation analyses
Cell growth was monitored by measuring OD600 utilizing a spectrophotometer (Shimazu, Japan). Glucose and acetate were quantitatively analyzed by high-performance liquid chromatography (HPLC) (Shimazu, Japan) which equipped with a refractive index detector (RID-10A) and an Ion Exclusion column (Bio-Rad, HPX-87H). The samples were first centrifuged at 12,000 rpm for 10 min, and then the supernatant was filtrated with a 0.22 μm filter membrane. 5 mM sulfuric acid was utilized as the mobile phase of HPLC with the flow rate of 0.6 mL/min and the utilized column temperature was 65 °C.
All data examined were expressed as mean ± SD. Statistical analyses of the data were carried out using two-tailed Student’s t-test between two groups, and one-way ANOVA followed by the post hoc Tukey’s test for multiple groups. P < 0.05 was considered significant. The * denotes P < 0.05, the *** denotes P < 0.001.
QQZ designed and carried out all the experiments, acquired the data and wrote the manuscript. QSQ supervised this study and revised the manuscript for important intellectual content. Both authors read and approved the final manuscript.
This research was financially supported by the grants from the National Natural Science Foundation of China (21808114), the Key Research and Development Program of Shandong Province (2019GSF107044), the Natural Science Foundation of Shandong Province (ZR2016CB03), and the Young Doctoral Cooperation Fund Project of Qilu University of Technology (Shandong Academy of Sciences) (2017BSHZ007).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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