Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3-hydroxybutyrate) production in Escherichia coli
- Yan Zhang†1, 2, 3, 5,
- Zhenquan Lin†1, 2, 3, 5,
- Qiaojie Liu1, 2, 3, 5,
- Yifan Li1, 2, 3, 5,
- Zhiwen Wang1, 2, 3, 5,
- Hongwu Ma4,
- Tao Chen1, 2, 3, 5Email author and
- Xueming Zhao1, 2, 3, 5
© Zhang et al.; licensee BioMed Central. 2014
Received: 22 September 2014
Accepted: 23 November 2014
Published: 16 December 2014
Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources.
In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP+] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source.
Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.
KeywordsEscherichia coli poly(3-hydroxybutyrate) L-serine deaminate Entner-Doudoroff pathway Pyruvate dehydrogenase complex
Polyhydroxyalkanoates (PHAs) are diverse polyesters synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses . Since PHAs possess thermoplastic or elastomeric properties and are completely biodegradable, PHA bioplastics offer an exciting alternative to petrochemical-derived plastics . Poly(3-hydroxybutyrate) (PHB) is the most wide spread and best-characterized member of PHAs and many different fermentation strategies and recovery methods have been developed for its production as a model polymer .
There has been a range of studies to evaluate PHB production in E. coli. Enhancing the availability of the precursor acetyl-CoA or/and cofactor NADPH increased the production of PHB. By overexpressing the fructose-bisphosphate aldolase (encoded by fbaA) or/and triosephosphate isomerase (encoded by tpi) , the recombinant E. coli accumulated more PHB than the reference strains due to the increase in acetyl-CoA concentration. By inactivating the phosphoglucose isomerase (encoded by pgi) gene, more NADPH was produced from pentose phosphate (PP) pathway, and eventually the PHB production was enhanced . Transketolase (encoded by tktA) or transaldolase (encoded by talA) was used to improve the metabolism in non-oxidative PP pathway to enhance the production of PHB [6,7]. Overexpressing glucose-6-phosphate dehydrogenase (encoded by zwf) and 6-phosphogluconate dehydrogenase (encoded by gnd) in oxidative pentose phosphate pathway increased the PHB production in E. coli by increasing the NADPH availability . In addition, to increase product yields, NADPH levels have been manipulated in the past by overexpressing the NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans .
In E. coli, acetyl-CoA was derived mostly from pyruvate which is a key intermediate in catabolic and biosynthetic reactions. Most pyruvate was synthesized through the coupled mechanism of glucose transport by the phosphotransferase transport system or the glycolytic pathways including Embden-Meyerhof-Parnas (EMP), Entner-Doudoroff (ED), and PP pathway . E. coli only produced two mole of NADH per mole of glucose through the EMP pathway, and caused the carbon loss through the PP pathway. Compared with these two pathways, ED pathway enables the strain to produce one molecular NADPH, a direct cofactor for the PHB production, without the carbon loss. Moreover, previous research based on proteome analysis revealed that ED pathway plays an important role during PHB production from glucose [11,12]. In E. coli, pyruvate also can be synthesized from L-serine by L-serine deaminase . L-serine deaminases (encoded by sdaA, sdaB and tdcG)  catalyze the conversion of L-serine to pyruvate and ammonia, which may be employed to improve the PHB production in E.coli.
In this research, combined engineering of the Serine-Deamination Pathway (SD pathway, shown in Figure 1) with ED pathway resulted in enhanced PHB production, but accompanying with pyruvate accumulation. Overexpressing PDH complex eliminated the pyruvate accumulation and led to further improvement of the intracellular acetyl-CoA concentration and PHB production. Finally, the metabolically engineered E. coli strain was able to synthesize significant amount of PHB from different carbon sources such as glucose, xylose and glycerol.
Results and discussion
Overexpressing L-serine deaminase for improved PHB production
Influence of gene overexpression in L-serine biosynthetic pathway on PHB production
It is reasonable that efficient conversion of L-serine to pyruvate requires sufficient availability of L-serine. To enhance the biosynthesis of L-serine, we overexpressed the genes of de novo L-serine biosynthetic pathway. L-serine is synthesized from D-3-phosphoglycerate by three reactions catalyzed by D-3-phosphoglycerate dehydrogenase, D-3-phosphoserine aminotransferase and phosphoserine phosphatase, which are encoded by serA, serC and serB, respectively (Figure 1). D-3-phosphoglycerate dehydrogenase is regulated by allosteric end-product inhibition. Moreover, a published report has showed that a truncated D-3-phosphoglycerate dehydrogenase (PGDH) serA Δ197 was no longer inhibited by L-serine in C. glutamicum . As such, we combined serA Δ197 together with serB and serC into an artificial operon driven by the constitutive promoter trc, creating plasmid pTSer. Strain SD01 was transformed with plasmid pTSer for activating the Serine-Deamination (SD) pathway. After 48h cultivation, 3.96 g/L PHB was accumulated by SD01 (pBHR68/pTSer), which was only slightly higher than that of the reference strain SD01 (pBHR68/pTrc101) (Figure 2).
However, the strategy of overexpressing serABC mainly led to improvement of cell growth, rather than specific PHB accumulation (Figure 2). This phenomenon might be due to the difference between the kinetic parameters of the two enzymes, SHMT and SdaA. The Km value for L-serine of SdaA is higher than that of SHMT (2.67 vs 0.3 mM [19,20]). The overexpression of serABC improved the availability of L-serine, which contributed much more to the reaction catalyzed by SHMT than the reaction catalyzed by SdaA. SHMT is the main source of C1 carbon and glycine for cell growth in E.coli, so residual cell dry weight was increased under this condition [21,22].
On the other hand, the kcat value of SdaA is much higher than that of SHMT (436 vs 5 s−1 [19,20]), and the corresponding kcat/Km values are 163.3 and 16.7 mM−1 · s−1, respectively. This means only when the intracellular concentration of L-serine is further improved to some extent, the serine deamination reaction can obviously overwhelm the competing reaction catalyzed by SHMT. Previous researches have shown that pgk (encodes phosphoglycerate kinase) overexpression could divert carbon flux into the D-3-phosphoglycerate pool and further promote the biosynthesis of L-serine . To push more flux into the SD pathway, pgk gene was ligated into pTSer, creating plasmid pTPSer. The PHB production of SD01 (pBHR68/pTPSer) reached 4.92 g/L, increasing by 30.2% compared to that of SD01 (pBHR68/pTrc101) harboring the empty vector (Figure 2). However, when pgk was overexpressed alone, the PHB production of SD01 (pBHR68/pTPgk) showed no significant change, compared with that of SD01 (pBHR68/pTrc101). These results suggested that activating the SD pathway by co-overexpressing the L-serine deaminase, enzymes in L-serine biosynthesis pathway and phosphoglycerate kinase had a significant effect on PHB production.
Effect of combining gene overexpression in ED pathway with the SD pathway on PHB production
Redirecting the carbon metabolism into the ED pathway in the engineered strain is beneficial to PHB production, since it avoids the carbon loss and balance the NADPH need [12,26]. Moreover, enhancing ED pathway might increase the availability of pyruvate and further enrich the in vivo pool of acetyl-CoA. As such, we tried to test the effect of enhancing the ED pathway on PHB accumulation.
Cell growth, PHB production, and by-products formation by strains study at the end of the cultivation
CDW (g/L) c
PHB (g/L) c
PHB content (%CDW) c
Pyruvate (g/L) c
Acetate (g/L) c
Specific glucose uptake rate (g/g rCDW·h) b, c
Comparison of the intracellular NADP + , NADPH concentrations and [NADPH]/[NADP+] ratios of the recombinant strains without the PHB synthesis genes
NADP + (μmol/gCDW) b,c
NADPH (μmol/gCDW) c
[NADPH]/[NADP + ] ratio c
Overexpression of the pyruvate dehydrogenase complex for enhanced PHB production
In E. coli, pyruvate is metabolized by the pyruvate dehydrogenase (PDH) complex and pyruvate oxidase during aerobic growth conditions [15,29]. To enhance the expression of PDH complex, fragment Trc-162 was inserted into the upstream of the PDH operon in SD02 and SD04 as mentioned in the methods section, resulting in strains SD06 (SD02, PTrc-162-aceEF) and SD07 (SD04, PTrc-162-aceEF). The transcript levels of genes encoding the PDH complex were presented in Figure 3C, which indicated that the genes of the PDH complex were successfully overexpressed. The PHB production of SD06 (pBHR68) was 4.63 g/L (69.1% CDW), no significant change compared with the reference strain SD02 (pBHR68) (Table 1), as well as the intracellular concentration of acetyl-CoA (Figure 4).
In contrast, the PHB concentration of the strain SD07 (pBHR68) was 5.54 g/L and PHB content was 81.1% CDW, significantly increased from 2.90 g/L of the parent strain SD04 (pBHR68). As expected, no trace of pyruvate and acetate was detected in the medium of SD07 (pBHR68) (Table 1), and the intracellular acetyl-CoA concentration of SD07 was further increased compared with that of SD04 (Figure 4). These indicated that the diversion of excess pyruvate to acetyl-CoA enhanced the availability of acetyl-CoA for PHB synthesis, and thus rebalanced the flux distribution at the pyruvate node.
PHB fermentation using other unrelated carbon sources
PHB accumulation of the strains in xylose or glycerol
CDW (g/L) b
PHB (g/L) b
PHB content (%CDW) b
Through combined engineering of SD pathway, ED pathway and PDH, a recombinant E. coli strain was obtained which led to significantly enhanced PHB accumulation. The final strain produced 5.52 g/L PHB from glucose with a content of 81.11% CDW. A similar phenomenon was observed when xylose or glycerol was served as carbon sources. In conclusion, enhancing the availability of acetyl-CoA via engineering the SD pathway, ED pathway and the PDH complex offered an effective way for improving the PHB production in E. coli from different carbon sources.
Bacterial strains, primers, and plasmids construction
Strains and plasmids used in this study
Strain or plasmid
Relevant genotype b
Source or reference
E. coli DH5α
Coli Genetic Stock Center strain (CGSC) No. 12384
E. coli JM109
recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, Δ(lac proAB)/F’ [traD36, proAB + , lac q lacZΔM15]
TaKaRa (Dalian, China)
SD01, P sdaA ::Ptrc
SD02, SerC:: Ptrc-pgk-SerABC
SD02, P edd ::P J23119
SD02, P edd-eda ::P J23119 , P zwf ::PTrc-162
SD02, P aceE ::PTrc-162
SD04, P aceE ::PTrc-162
Expression vector, pSC101 replication, constitutive trc promoter, Cmr
pSC101 replication, Cmr, Ptrc -serA-serB-serC
pSC101 replication, Cmr, P trc - pgk-serA-serB-serC
pSC101 replication, Cmr, Ptrc-pgk
pBluscript SK(-) derivative, phbA Re , phbB Re , phbC Re cloned from R. eutropha
pSC101 replication, temperature sensitive replication origin, Spc r , P araBAD -driven I-SceI gene, Red recombinase expression plasmid, lac-inducible expression
p15A replication, Cm r , Tet r , I-SceI restriction sites
Genome replacement manipulation
Primer sequence used in this study
Primer sequence (5’→3’)
Primers for genetic manipulation
Primers for RT-PCR
For swapping the promoter of zwf, PDH complex operon and pgk-serAB fragment insertion, the tetA fragment was amplified from strain Tet-Trc-162, and fused with the up and low homologous flanks (Figure 5C). The low flank of pgk-serABC fragment was amplified from the plasmid pTPSer (Figure 5D). Fragment Trc-162 consisted of the trc promoter core sequence and M1-162 in tandem.
During strains and plasmids construction, cultures were grown at 30°C or 37°C, in Luria broth (per liter: 10 g tryptone, 5 g yeast extract, and 10 g NaCl) with or without agar (2%) as indicated. Minimal sodium medium (MS medium) with 1 g/L yeast extract was used as seed culture and shake flask medium which contained (in grams per liter): glucose/xylose/glycerol 20.0, (NH4)2SO4 2.0, MgSO4 · 7H2O 0.4, Na2HPO4 3.83, KH2PO4 1.5, Fe(III)-NH4-citrate 0.05, CaCl2 0.02, and 1 mL/L trace element . When necessary, a final concentration of 10 μg/mL chloromycetin and/or 100 μg/mL ampicillin were added. Colonies were inoculated into 5-ml LB culture medium and grown at 37°C with shaking overnight. Then the culture was inoculated with 1% into 250-ml flask with 50 ml culture medium as seed culture grown at 37°C in MS medium for 12 h at 220 rpm on a rotary shaker. Seed culture was then inoculated into 500-mL flask with 100 ml culture medium (with the initial OD600 of 0.04) and grown at 37°C and 220 rpm on a rotary shaker for 48 h. Three biological replicates were performed to detect the accumulation of PHB. To assess the significant difference of the PHB accumulation, data was subject to Student’s t test analysis with p < 0.05 being significantly different.
The growth of cell was monitored by measuring the OD600 with an ultraviolet spectrophotometer (Beijing Puxi Universal Co Ltd). Glucose in the fermentation broth was determined utilizing a SBA sensor machine (Institute of Microbiology, Shangdong, China). Bacteria were harvested by centrifugation at 8,000 × g for 10 min and then washed with distilled water. Cell dry weight (CDW) was measured after lyophilization and vacuum drying. PHB content was analyzed by gas chromatography (Persee, China) with an Agilent J&W Capillary GC column after methanolysis of lyophilized cells in chloroform. To determine the concentration of pyruvate, acetate, glycerol and xylose, culture samples were centrifuged at 12,000 × g for 5 min and the aqueous supernatant used for HPLC analysis on an Agilent 1100 Series HPLC system equipped with an Aminex HPX-87H anion exchange column (Bio-Rad Laboratories, Richmond, CA, USA) and refractive index detector. A mobile phase of 5 mM H2SO4 at a 0.4 mL/min flow rate was used.
For the determination of intracellular acetyl-CoA, 40 mL mid-exponential phase cell culture was taken into precooled centrifuge tubes and centrifuged at 8000 g and 4°C for 10 min. The cell pellets were washed with 40 mL 100 mM Tris-HCl buffer (pH 8.0). Acetyl-CoA was analyzed by HPLC as previous reported [39,40]. For determination of intracellular NADP+ and NADPH, 10 mL mid-exponential phase cell culture was taken into precooled centrifuge tubes and centrifuged at 8000 g and 4°C for 10 min. The intracellular NADP+ and NADPH were analyzed by HPLC as previous reported .
Quantitative real-time reverse transcription (RT)-PCR analysis
The recombinant strains harboring pBHR68 plasmid were cultured with the same fermentation media and culture condition with 1% (w/v) glucose. Cells were harvested when OD600 reached 1. Total mRNA were extracted using the RNAprep pure Cell/Bacteria Kit (Tiangen, Beijing, China) as described by the manufacturer. The cDNA was amplified using FastQuant RT Kit (Tiangen, Beijing, China) with the total mRNA as the templates. Samples were then analyzed using a Light Cycler® 480 II (Roche, Basel, Switzerland) with RealMasterMix (SYBR Green I) (Tiangen, Beijing, China). Quantity real-time PCR (qPCR) amplification primers were designed and were listed in Table 5. The rrsA gene was selected as internal standard for normalization and three biological replicates were performed. The obtained data were analyzed by using the 2-ΔΔCt method described previously .
We thank Prof. Guo-Qiang Chen (Tsinghua University) for kindly providing the plasmid pBHR68. We thank Dr. Thomas E. Kuhlman for kindly providing requested plasmids. This work was supported by National 973 Project [2012CB725203, 2011CBA00804]; National Natural Science Foundation of China [NSFC-21176182, NSFC-21206112, NSFC-21390201]; and National High-tech R&D Program of China [2012AA02A702, 2012AA022103].
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