Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase
© Zheng et al.; licensee BioMed Central Ltd. 2012
Received: 8 September 2012
Accepted: 5 October 2012
Published: 11 October 2012
Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production.
We cloned and expressed a cytosolic Acinetobacter baylyi thioesterase (‘AcTesA) in E. coli by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser10, Gly48, Asn77, Asp158 and His161 residues composed the active centre of ‘AcTesA. The engineered strain that overexpressed ‘AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type E. coli, demonstrating that the expression of ‘AcTesA indeed boosted the synthesis of FFAs. The ‘AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of ‘AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12–16 h post induction.
For the first time, an A. baylyi thioesterase was cloned and solubly expressed in the cytosol of E. coli. This leaderless thioesterase (‘AcTesA) was found to be capable of enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In addition, ‘AcTesA exhibited different substrate specificity from other thioesterases previously reported, and can be used to supply the fatty acid-based biofuels with high quality of FFAs. Altogether, this study provides a promising thioesterase for FFAs production, and is of great importance in enriching the library of useful thioesterases.
KeywordsThioesterase Acinetobacter baylyi Escherichia coli Free fatty acid Substrate specificity Active-site residues
Acyl carrier protein
Free fatty acids
Polymerase chain reaction
Flame ionization detector.
Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs). They play an essential role in chain termination during de novo fatty acid synthesis and have been proven to be important in fatty acid bioengineering . Therefore, thioesterases are widely used for the microbial production of FFAs, which can be further applied to produce fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes [2–5].
In addition, thioesterases from different organisms have varied substrate specificities, and can be used to tailor the composition of the FFAs. For examples, Cp FatB1 from Cuphea palustris has a substrate preference towards C8- and C10-ACPs, Uc FatB1 from Umbellularia californica prefers the C12-ACPs, and thioesterases from Ricinus communis and Jatropha curcas accumulated three major products, including C14, C16:1 and C16 straight chain FFAs [6–10].
In wild-type E. coli, the fatty acid biosynthesis was inhibited by fatty acyl-ACPs in the absence of phospholipids synthesis. Though not a few thioesterases have been reported to be capable of releasing the feedback inhibition of fatty acyl-ACPs, no extensive examination was carried out to test their abilities to produce FFAs in microbial cells. Only a few thioesterases were applied to overproduce FFAs [11–15]. Zhang et al. examined the effect of the overexpression of four different plant thioesterases on FFAs production of E. coli. Some of the thioesterases they examined were able to produce over 2.0 g/L FFAs, representing a strong ability of accumulating FFAs . In addition, they also found that the level of FFAs production mainly depended on the acyl-ACP thioesterase employed . Therefore, it is of great significance to find a promising thioesterase that has a strong ability of FFAs accumulation or a novel substrate specificity.
Above-mentioned thioesterases are all from plant sources. Little attention has been paid to bacterial thioesterases except the ‘TesA of E. coli. Many bacterial enzymes are superior in chemical production to their eukaryotic counterparts. For example, the mevalonate (MVA) production increased 50 folds by replacing the MVA upper pathway genes from Saccharomyces cerevisiae with those from Enterococcus faecalis. In addition, their substrate specificities are probably quite different from the plant thioesterases so far reported. Therefore, more types of FFAs, which may contribute to improving the performance of fatty acid-derived biofuels, can be expected by expressing bacterial thioesterases. Though a bacterial thioesterase from Streptococcus pyogenes was employed for improving the fatty acid synthesis, expression of this thioesterase in E. coli only obtained 1.3-fold more total fatty acids than the wild-type E. coli, still with C16 and C18 fatty acids as its major components . So this S. pyogenes thioesterase did not obviously enhance the production of FFAs. It again demonstrates that the thioesterase plays the key role in determining the amount and composition of FFAs. So it prompts us to discover some promising bacterial thioesterase genes for further improving the FFAs production.
The Acinetobacter baylyi thioesterase is expected to be functional in hydrolyzing fatty acyl-ACPs to FFAs, as A. baylyi naturally accumulates wax ester, whose formation requires the participation of FFAs [18, 19]. But unfortunately, no investigation has been carried out to study the A. baylyi thioesterase thus far.
In this study, a thioesterase gene was cloned from A. baylyi, and was heterologously expressed in E. coli BL21(DE3). To investigate the enzymatic activity and substrate specificity of this thioesterase, its catalytic product was determined by gas chromatography. In addition, protein sequence alignment and structure analysis were carried out to elucidate its possible active centre, which was further determined by site-directed mutagenesis. The expression level of A. baylyi thioesterase and the fermentation medium were also optimized to further improve the production of FFAs. Finally, a fed-batch fermentation was performed to evaluate the FFAs production in a scaleable process.
Results and discussion
Ser10-Asp154-His157 and Ser10-Gly44-Asn73 compose the catalytic triad and the oxyanion hole of ‘TesA, respectively [22–24]. The ‘AcTesA shares the same catalytic-triad residues as ‘TesA (Figure 1), suggesting that ‘AcTesA may function as a SGNH-hydrolase. The ‘AcTesA has a similar but not the same catalytic triad as the plant thioesterases , which use cysteine to compose their catalytic triad instead of serine. The plant thioesterases were still highly active when their cysteines were mutated to serines, while the thioesterases from rat and chicken livers retained up to 90% of the activities when their serines were substituted with cysteines [26–28]. These results demonstrated that the sulfhydryl group of cysteine and the hydroxyl group of serine were both able to nucleophilically attack of the substrates’ carbonyl carbon atom.
Expression of recombinant ‘AcTesA protein in E. coli BL21(DE3)
Changes of the FFAs production by expressing ‘AcTesA
To check if the expression of ‘AcTesA is capable of boosting the FFAs biosynthesis of E. coli, we determined the FFAs production in wild-type and engineered E. coli strains. In shake-flask experiments, the wild-type E. coli BL21(DE3) accumulated 20.5 mg/L FFAs, while LL8 produced ~25-fold more FFAs (501.2 mg/L) than BL21(DE3). It demonstrated that the activity of the endogenous thioesterase of wild-type E. coli was strictly regulated, which resulted in the tiny production of FFAs, and overexpression of ‘AcTesA could enhance the production of FFAs by releasing the feedback inhibition caused by fatty acyl-ACPs. The titer of 501.2 mg/L represents a high FFAs production achieved in shake flask.
Three-dimensional structure analysis of thioesterase ‘AcTesA
The ‘AcTesA from A. baylyi has the same catalytic-triad residues and oxyanion-related residues as the ‘TesA from E. coli (Figure 1). The mechanism for ‘TesA catalysis is that the hydroxyl group from the catalytic serine nucleophilically attack of the thioester bond of the substrate fatty acyl-ACPs, assisted by a histidine, which functions as an acid–base catalyst .
The Ser and His residues located at the active centre play a direct role in the catalysis. This also applies to plant thioesterases, given the enzyme will be completely inactivated if the cysteine or histidine in the catalytic triad is mutated to alanine . Therefore, it is reasonable to believe that the bacterial and plant thioesterases utilize the uniform catalytic mechanism to hydrolyze the fatty acyl-ACPs.
In addition, all the strains had almost equivalent cell masses, except the strains expressing native ‘AcTesA and N77A mutant got slightly higher cell masses (Figure 5b). It demonstrated that the expression of recombinant thioesterases did not obviously affect the cell growth. The strains that accumulated more FFAs (‘AcTesA and N77A) even obtained the higher cell masses (Figure 5), suggesting the synthesized FFAs were not harmful to the E. coli cell.
Optimization of the expression level of thioesterase ‘AcTesA
Lennen et al. achieved a high production of fatty acids by using a medium-strength araBAD promoter . But in this study, a higher FFAs production was obtained by using a strong T7 promoter. It is worth mentioning that the T7 strains (LL8, LL18 and LL28) obtained higher cell masses than the araBAD strains (LL38, LL48 and LL58) in the same sampling time (Figure 6b). It is probable that the lower cell masses resulted in the decreased FFAs production, and prolonged culture time may increase the FFAs production of the araBAD strains to some extent. But generally speaking, the T7 strains achieved the higher productivities than the araBAD strains. The high productivity is of great importance to decreasing the cost in mass production.
Effect of carbon and nitrogen sources on FFAs accumulation
Glucose and glycerol are the most commonly used carbon sources. So the effect of glucose and glycerol on the FFAs production was firstly compared. Without addition of any organic nitrogen, LL18 produced 236.5 mg/L FFAs using glucose as the carbon source, while 222.9 mg/L using glycerol as the carbon source. This result demonstrates that both glucose and glycerol can be efficiently converted to FFAs by E. coli. This may be the reason why both the glucose and glycerol were widely used in the FFAs production [4, 10, 15, 34].
Using tryptone as the organic nitrogen, the levels of organic nitrogen addition on the FFAs production were further tested. The LL18 was cultured in the media containing 0, 1, 2.5, 5, 10, 20 and 30 g/L tryptone, respectively, and its FFAs production in corresponding medium was determined. With elevated levels of tryptone, the obtained cell densities increased accordingly. The 5 g/L tryptone culture got a medium cell mass, but it achieved the highest FFAs production. The reduced tryptone addition led to decreased cell masses and FFAs production. The higher levels of tryptone contributed to the obviously increased cell masses, but resulted in decreased FFAs production (Figure 7c, Figure 7d).
The results demonstrate that the source and levels of organic nitrogen can greatly affect the FFAs production. The improved FFAs production in the medium containing 5 g/L tryptone mainly benefits from the modest cell growth and recombinant thioesterase (‘AcTesA) expression. The higher levels of organic nitrogen led to decreased FFAs production. The possible explanation is that the elevated levels of organic nitrogen resulted in the excessive quantities of functional thioesterase and thus initially rapider rates of FFAs accumulation, disturbing the cell viability and normal FFAs production of E. coli. The lower levels of organic nitrogen lead to reduced cell masses and insufficient recombinant thioesterase, so the FFAs production also decreased.
Lu et al. optimized the FFAs-producing E. coli strain by detailed metabolic engineering, and they finally obtained 2.5 g/L FFAs in fed-batch fermentation . In this study, without detailed optimization of the strain and fermentation, we achieved a FFAs titer of 3.6 g/L, ~1.5 fold higher than that obtained by Lu et al.. It demonstrates that the ‘AcTesA is a quite promising thioesterase for the production of FFAs. The FFAs production can be further greatly improved by co-overexpressing the acetyl-CoA carboxylase to increase the supply of malonyl-CoA, an important precursor for fatty acid synthesis, and deleting the endogenous acyl-CoA synthetase to block the degradation of FFAs in a single host [15, 36].
The FFAs yield of 6.1% is higher than the maximal yield of 4.8% obtained by Lu et al., but is still much lower than the theoretical maximal yield of ~30% (purely stoichiometric yield) . A further improvement of FFAs production can be expected by efficient genetic reconstruction of the strain and detailed optimization of the fermentation conditions.
For the first time, an A. baylyi thioesterase was solubly expressed in the cytosol of E. coli by deleting its leader sequence. This leaderless thioesterase (‘AcTesA) was discovered to be capable of greatly enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, a encouraging titer of 3.6 g/L was finally achieved. In addition, it exhibited different substrate specificities from other thioesterases previously reported. The ‘AcTesA can be used to supply the biodiesel and chemical production with high quality of FFAs, and is of great importance in enriching the library of useful thioesterases. In the summary, our study provides a promising thioesterase for FFAs production.
A. baylyi ATCC 33305 was purchased from ATCC. E. coli BL21(DE3) were purchased from Invitrogen (Carlsbad, CA, USA). The expression vectors pCOLADuet-1, pACYCDuet-1 and pET-28a(+) were purchased from Novagen (Darmstadt, Germany). The expression vectors pBAD18, pBAD30 and pBAD33 were originally constructed by Guzman et al. . All restriction enzymes and T4 DNA ligatase were purchased from Fermentas (Vilnius, Lithuania). The PrimeSTAR HS DNA polymerase was supplied by Takara Biotechnology (Dalian, China). Oligonucleotides were ordered from Generay Biotechnology (Shanghai, China). The arachidic acid was ordered from Alfa Aesar (Ward Hill, MA).
Bacterial strains and plasmids used in this study
Plasmid or strain
Reference or source
pBR322 ori lacI T7lac Kanr
P15A ori lacI T7lac Cmr
ColA ori lacI T7lac Kanr
pBR322 ori araBAD Ampr
pACYC184 ori araBAD Ampr
pACYC184 ori araBAD Cmr
pCOLADuet-1 harboring ‘AcTesA from A. baylyi
pACYCDuet-1 harboring ‘AcTesA from A. baylyi
pET-28a(+) harboring ‘AcTesA from A. baylyi
pBAD18 harboring ‘AcTesA from A. baylyi
pBAD30 harboring ‘AcTesA from A. baylyi
pBAD33 harboring ‘AcTesA from A. baylyi
pCOLADuet-1 harboring ‘AcTesA with a mutation to Ala at Ser10 residue
pCOLADuet-1 harboring ‘AcTesA with a mutation to Ala at Gly48 residue
pCOLADuet-1 harboring ‘AcTesA with a mutation to Ala at Asn77 residue
pCOLADuet-1 harboring ‘AcTesA with a mutation to Ala at Asp158 residue
pCOLADuet-1 harboring ‘AcTesA with a mutation to Ala at His161 residue
F- ompT gal dcm lon hsdSB(r B - m B - ) λ(DE3)
E. coli BL21 (DE3) bearing pLL8
E. coli BL21 (DE3) bearing pLL18
E. coli BL21 (DE3) bearing pLL28
E. coli BL21 (DE3) bearing pLL38
E. coli BL21 (DE3) bearing pLL48
E. coli BL21 (DE3) bearing pLL58
E. coli BL21 (DE3) bearing pS10A
E. coli BL21 (DE3) bearing pG48A
E. coli BL21 (DE3) bearing pN77A
E. coli BL21 (DE3) bearing pD158A
E. coli BL21 (DE3) bearing pH161A
Primers used in this study
GCCAGTGTAAGTG C GGAAACCACCAGTGGTGC
SDS-PAGE analysis of recombinant protein
E. coli BL21(DE3) harboring pLL8, namely LL8 strain, was cultivated in LB medium supplemented with kanamycin at 37°C until its OD600nm reached 0.6~0.8. Then 0.5 mM isopropyl β-D-thiogalactoside (IPTG) was added and the culture was switched to grow at 30°C for 6 h. The cells were first harvested by centrifugation at 10,000 g for 2 min, resuspended with 0.05 M sodium phosphate buffer (pH 7.4) after washed twice with the same buffer, and finally disrupted by sonication. The resultant supernatant by centrifuging for 30 min at 13000 g was collected for SDS-PAGE analysis.
The models of the theoretical ‘AcTesA structures were built on a public website Swiss-Model (http://swissmodel.expasy.org), using the thioesterase I of Escherichia coli (PDB ID: 1IVN) as a template.
A method based on the amplification of the entire plasmid using primers that include the desired changes was employed for the site-directed mutagenesis . The PrimeSTAR HS DNA polymerase (Takara) was used for the PCR. The Ser-10, Gly-48, Asn-77, Asp-158 and His-161 of the thioesterase ‘AcTesA were separately mutated to Ala by using the mutant primers S10A-F/S10A-R, G48A-F/G48A-R, N77A-F/N77A-R, D158A-F/D158A-R and H161A-F/H161A-R, respectively (Table 2).
Bacterial strains, media, and growth conditions
The bacterial strains used in this study are listed in Table 1. E. coli BL21(DE3) (Invitrogen, Carlsbad, CA) was used as the host to overproduce proteins. During strain construction, cultures were grown aerobically at 37°C in Luria Broth (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract). Kanamycin (50 mg/L), Ampicillin (100 mg/L) or chloramphenicol (34 mg/L) was added if necessary. For initial production experiments in shake flasks, strains were grown in a M9 medium (37.8 g/L Na2HPO4·12H2O, 7.5 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 4 mM MgSO4) containing 20 g/L of glucose or 20 g/L of glycerol. The engineered strains were fed with glycerol as carbon source if they carried the recombinant plasmids using araBAD promoter, otherwise they were fed with glucose. In addition, beef extract powder, beef extract, yeast extract or tryptone was added into the M9 medium if necessary. The media contained 5 g/L beef extract powder if there was no specific explanation. Protein production was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 30°C, and the cultures were harvested after 40 h post induction.
The fed-batch fermentation was carried out in a 5 L BIOSTAT® B plus fermentor (Sartorius Stedim Biotech GmbH, Goettingen, Germany). The strain was grown in the M9 medium supplemented with 5 g/L tryptone. The fermentation temperature was controlled at 30°C and the pH at 7.0. The pH was maintained using NH3·H2O. Cells were induced at an OD600nm of ~18 using 0.5 mM IPTG. The glucose feed solution was continuously added into the cultures at the rates of 2~3 g/L/h.
Detection of FFAs
The harvested cultures were firstly adjusted to pH 2.0 with 1 M HCl. The arachidic acid, which was dissolved in chloroform, was then added into the acidified cultures and used as the internal standard. An equal volume of chloroform–methanol (v/v, 2:1) with the culture was next added to extract the lipids . The resultant blends were vortexed for a few minutes and then left overnight. The FFAs were directly quantified without derivatization by a gas chromatograph (GC) equipped with a flame ionization detector (FID) . The system consisted of model 450-GC (Varian, Walnut Creek, CA) and a model 8400 AutoSampler (Varian). The separation of FFAs was performed using a CP-FFAP CB capillary column (25 m×0.25 mm; 0.2 μm film thickness) purchased from Agilent Technologies. The oven temperature was initially held at 100°C for 1 min, then raised with a gradient of 10°C/min until reaching 250°C, and finally held for 10 min. Nitrogen was used as the carrier gas. The injector and detector were held at 270°C and 300°C, respectively.
This work was financially supported by Guangdong Province-Chinese Academy of Sciences Joint Project (2009B091300146), National Natural Science Foundation of China (21106170), National Defense Foundation of the Chinese Academy of Sciences (CXJJ-11-M56) and Main Direction Program of Knowledge Innovation of Chinese Academy of Sciences (KSCX2-EW-G-13).
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