A microbial platform for renewable propane synthesis based on a fermentative butanol pathway
© Menon et al.; licensee BioMed Central. 2015
Received: 10 November 2014
Accepted: 23 February 2015
Published: 10 April 2015
Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli.
We report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L).
This study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde.
KeywordsPropane Butanol Microbial pathway engineering Cyanobacteria Aldehyde deformylating oxygenase Escherchia coli
Propane, a major component of autogas or liquefied petroleum gas (LPG), is an emerging fuel for future energy supply and transportation . Propane is the third most widely used motor fuel and about 20 million tonnes of propane gas are used per year to fuel motor vehicles . It is estimated that propane provides heat and energy for more than 14 million homes worldwide annually [3,4]. Propane also has an existing global market for a wide number of other stationary and mobile applications, such as low emission vehicles, gas burners and refrigeration systems . Easy separation from liquid biotechnological processes as a gas and less energy requirements for liquefaction and storage offers potential advantages to propane over other gaseous fuels .
The butanol pathway in Clostridium proceeds either via a keto acid route (Ehrlich pathway) or a CoA-dependent route. Higher yields of branched chain alcohols and aldehyde precursors (for example, isobutyraldehyde) from the decarboxylation of keto acids make the Ehrlich pathway less attractive because ADO has a strong preference for straight chain aldehyde substrates [11-13]. By contrast, butanol production by the CoA-dependent route initiates with the condensation of two molecules of acetyl CoA. Reduction in subsequent steps produces the end-product 1-butanol via a butyraldehyde intermediate. There are several reports of engineered CoA-dependent butanol pathways in Escherichia coli and other host organisms [14-20]. In the following work, we constructed and evaluated a series of CoA-dependent butyraldehyde pathways that eliminate the dependency on AdhE2, thereby allowing butyraldehyde to be re-routed towards propane instead of butanol.
The use of ADO (from Prochlorococcus marinus MIT9313) as a terminal decarbonylase has been used for the production of medium/long chain (C9-C17) as well as short chain-length alkanes (C3, C7) [8,10]. Variant forms of ADO have demonstrated improved activity with the shorter chain aldehydes that are not encountered in native cyanobacteria [7,12]. These variant forms of ADO are therefore attractive enzyme components for building new synthetic pathways with a greater productivity, as addressed in the current study.
Here, we report on the construction and evaluation of novel pathways for propane production in E. coli that are independent of the FAS pathway in a recent study, thereby opening up possibilities for further optimization of short chain-length alkane biosynthesis in future studies .
Synthesis of butyraldehyde based on the CoA-dependent 1-butanol pathway
Modification of the butyraldehyde pathway by replacement of AdhE2
In the clostridial pathway, butyryl-CoA is converted to butanol in two successive catalytic steps by the bifunctional aldehyde/alcohol dehydrogenase AdhE2 (Figure 1; AdhE2 route) . From an engineering perspective, this is highly undesirable since the local presence of an aldehyde reductase component of AdhE2 is likely to compete for the ADO substrate. Given this possibility and to avoid creating internal metabolic competition, we therefore replaced AdhE2 with a i) thioesterase (YciA) from Haemophilus influenzae to cleave butyryl-CoA to butyric acid and ii) ATP/NADPH-dependent carboxylic acid reductase (CAR) from Mycobacterium marinum to convert butyric acid to butyraldehyde [8,23]. Two variant routes were thus created based on this modification, namely atoB-TPC7, atoB-TPC7-ahr, nphT7-TPC7 and nphT7-TPC7-ahr (Figure 1; TPC7 route). As before, 1-butanol was monitored to verify the functionality of the parts for butyraldehyde synthesis. In both cases, 1-butanol production was observed signifying yet again that butyraldehyde synthesis was achievable with both routes. 1-Butanol production was increased with the atoB-TPC7 and nphT7-TPC7 routes when Ahr was overexpressed (1.5 and 1.3 times for atoB-TPC7 and nphT7-TPC7 routes, respectively), indicating that the conversion of butraldehyde to butanol by the endogenous aldehyde reductases was limiting total pathway flux.
Evaluation of pathway routes for propane synthesis
Increasing electron supply to ADO via ferredoxin
It has previously been shown that the supply of electrons to ADO via endogenous enzymes was inadequate for effective alkane biosynthesis in E. coli . To alleviate this constraint, the heterologous ferredoxin PetF (ssl0020), the presumed natural electron acceptor/donor to ADO in Synechocystis sp. PCC 6803, was overexpressed as part of the engineered pathways. Consequently, co-expression of PetF improved propane production by roughly twofold for both the atoB-TPC7 and nphT7-TPC7 pathway in combination with both ADO and ADOA134F (Figure 4). In order to verify the impact of the optimization efforts, residual butanol was also measured in selected strains (Additional file 1: Figures S4 and S5). If ADO was able to compete for butyraldehyde, the common precursor for both propane and butanol, one would expect to observe a lower butanol titre in strains with enhanced propane production. Indeed, residual butanol levels were decreased when ADO and ferredoxin were overexpressed. Similarly, when the ADOA134F variant replaced wild-type ADO, the accumulation of butanol in the media was also lower. This is consistent with the terminal ADO (and the ADOA134F variant) acting on butyraldehyde produced by the engineered synthetic pathway (Additional file 1: Figures S4 and S5).
Removing competing pathways for propane synthesis
Potential for large-scale production of propane
Recent progress in exploring various heterologous microbial platforms towards metabolites such as fatty acids, alcohols and alkanes has improved the prospect of advanced biofuel production. Propane (C3H8) was proposed as a new microbial biofuel target as it would be able to act as a direct drop-in replacement for the corresponding non-renewable products currently in use (for example, autogas, LPG). Propane also has very good physicochemical properties which allow it to be stored and transported in a compressed liquid form, while under ambient conditions, it is a clean-burning gas used in various applications ranging from heating to utilization as a transport fuel. The very first microbial biosynthetic pathway for producing propane was recently engineered in E. coli . In that study, it was found that the biosynthesis of propane was restricted by the poor activity of the enzyme ADO towards short-chain substrates in combination with competing pathways that limits the availability of the ADO substrate, butyraldehyde. The study reported here therefore explored alternative biosynthetic approaches to provide a more comprehensive understanding of the limiting factors in microbial propane biosynthesis and to find possible ways to overcome metabolic bottlenecks.
The pathways assembled in the present study are not dependent on type II fatty acid biosynthesis unlike in the previous study and instead use CoA- rather than ACP-based intermediates. Importantly, this strategy allowed us to bypass the strict regulatory control on native fatty acid biosynthesis flux [10,25,26]. The pathways engineered in this study comprise four parallel variations of the Clostridial butanol pathway, differing from one another in the conversion of (1) acetyl-CoA to acetoacetyl-CoA (atoB vs. nphT7 routes) and (2) butyryl-CoA to butyraldehyde (adhE2 vs. TPC7 routes). In the first stage of pathway evaluation, production of butanol was used as a measure of metabolic flux towards butyraldehyde, the immediate substrate for propane production by ADO. The TPC7 variant routes were developed in order to avoid the bifunctional activity of AdhE2 which includes the reduction of butyraldehyde to 1-butanol. Such a reaction would be expected to compete with ADO for the butyraldehyde intermediate and lower propane production. Therefore, in order to separate these functional activities and allow greater pathway flexibility, AdhE2 was replaced with a ‘TPC7’ module in combination with Ahr.
For the first two variant pathways, the (co)substrate requirements for AtoB and NphT7 are quite distinct; AtoB requires two molecules of acetyl CoA whereas nphT7 requires acetyl-CoA along with malonyl-CoA. Both reactions result in the release of CoA, though in the latter case, there is also release of CO2. The greater 1-butanol levels observed with the AtoB pathway suggest that it is a preferred route for acetoacetyl CoA production in E. coli. However, it is also important to note that AtoB has a higher protein expression level in E. coli compared to nphT7 (Additional file 1: Figure S1), and this may have limited 1-butanol production via the nphT7 route. When coupled with ADO, butyraldehyde is converted to propane instead of butanol, and propane production was observed in the majority of the alternative strains. Consequently, the most productive propane pathway in the wild-type E. coli cells was observed with the atoB-TPC7-ADO route (220 ± 3 μg/L). The flux towards propane through these pathways was compromised by competing native aldehyde reductase activites, as demonstrated by a significant increase in propane production and a concomitant reduction in butanol production following the deletion of two native genes encoding for such activities. In contrast to the two TPC7 pathways, the adhE2-dependent routes produced significantly less propane. This observation reinforced the view that AdhE2 converts butyraldehyde to butanol in successive steps without release of the butyraldehyde, thus preventing efficient interception of the pathway by ADO towards propane.
Quantitative comparisons revealed a difference of almost 3 orders of magnitude between the titres of butanol and propane (173 vs. 0.2 mg/L) in the wild-type strains of AtoB-TPC7-Ahr and AtoB-TPC7-ADO. This difference can be attributed partly to the poor efficiency of the last biosynthetic step (conversion of butyraldehyde to propane). This is consistent with the earlier reports that have demonstrated in vitro and in vivo in E. coli that ADO has very low activity even towards the most preferred native substrates (C10-C15) and especially towards C4 substrates [10,12]. To partially alleviate this constraint, the ADOA143F variant was introduced into the engineered pathways to replace native ADO. This resulted in a 1.8-fold improvement in propane, most likely reflecting more efficient utilization of the intracellular pool of available butyraldehyde. However, the remaining large difference between 1-butanol and propane levels suggests that the optimization efforts employed in the present study are still not sufficient and/or that other metabolic constraints are limiting. One obvious example, as confirmed in this study as well as others, is the reduction of ADO in the presence of a 2Fe-2S ferredoxin . Ferredoxin systems are known to improve the metabolic reactions by mediating electron transfer in partner enzymes as observed here [27-29]. Such an approach could be further aided by additional reducing components such as NAD(P)H:flavodoxin/ferredoxin oxidoreductase (for example, fpr) as demonstrated in the previous work .
The present work introduces an alternative and conceptually different pathway for propane production in comparison to the previous work  despite the fact that the propane titre remained slightly lower. However, a direct comparison between the FAS and CoA pathways is not suitable, particularly since the analysed experimental parameters differed between the two studies. Both the current, and previously reported, pathways will benefit from further optimisation strategies to improve carbon flux, involving detailed targeted metabolomics and mass balance calculation, pathway modelling and pathway refactoring along with modifications to the known limitations in the turnover rate constant of ADO.
We have reported here new pathways for propane production based on a fermentative butanol pathway. These are distinct from previously reported pathways that were based on fatty acid synthesis, where propane production is limited by the availability of butyraldehyde precursors and the poor activity of ADO with butyraldehyde. We have demonstrated that the new pathways are plausible alternatives for the construction of next-generation microbial propane production platforms. While the majority of the metabolic flux is still directed towards other products (that is, butanol), our work also demonstrates that there is strong potential for further optimization aimed at increasing propane production.
Materials and methods
All chemicals, solvents and standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific (Waltham, MA, USA) and were of analytical grade. Media components were obtained from Formedium (Norfolk, UK). Gene sequencing and oligonucleotide synthesis were performed by Eurofins MWG (Ebersberg, Germany). D-Glucose (GOPOD Format) assay kit was from Megazyme (Wicklow, Ireland).
Strains and plasmids
The pET-TPC7 plasmid was constructed in a pETDuetT-1 vector (ColE1 replicon, AmpicillinR, from Novagen) by replacing TE4 acyl-ACP thioesterase with a synthesized gene encoding YciA (Haemophilus influenza, GenBank ID: AAC22485.1) in pET-TPC4 backbone plasmid described in the previous study using restriction sites HindIII and NcoI . The final plasmid contained synthesized genes encoding Sfp (maturation factor phosphopantetheinyl transferase from Bacillus subtilis, GenBank ID: X65610.1) and CAR (carboxylic acid reductase from M. marinum, GenBank ID: ACC40567.1) upstream to yciA.
The pACYC-NHCT plasmid was constructed in a pACYCDuet-1 vector (P15A replicon, ChloramphenicolR, from Novagen) by subcloning a synthesized NcoI-BamHI gene fragment (GenScript, Piscataway, NJ, USA) carrying genes encoding NphT7 (acetoacetyl-CoA synthase from Streptomyces sp. CL190, GenBank ID: D7URV0.1) and Hbd (3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824, GenBank ID: P52041.2) into a synthesized pACYC construct harbouring crt (3-hydroxybutyryl-CoA dehydratase from C. acetobutylicum ATCC 824, GenBank ID: P52046.1) and ter (trans-2-enoyl-CoA reductase from T. denticola ATCC 35405, GenBank ID: Q73Q47.1).
pACYC-AHCT is a pACYCDuet-1 vector (P15A replicon, ChloramphenicolR, from Novagen) with atoB (Acetyl-CoA acetyltransferase from E. coli, GenBank ID: P76461.1), hbd (3-hydroxybutyryl-CoA dehydrogenase from C. acetobutylicum ATCC 824, GenBank ID: P52041.2), crt (3-hydroxybutyryl-CoA dehydratase from C. acetobutylicum ATCC 824, GenBank ID: P52046.1) and ter (trans-2-enoyl-CoA reductase from T. denticola ATCC 35405, GenBank ID: Q73Q47.1) genes inserted in the order. Gene atoB (acetyl-CoA acetyltransferase from E. coli, GenBank ID: P76461.1) was PCR-amplified from E. coli K-12 purified genome, using primers 5′-attaggtaccAAAAATTGTGTCATCGTCAGTGCGGTAC and 5′-attaaagcTTAATTCAACCGTTCAATCACCATCGCAAT, showing complementary regions in capital letters. The atoB PCR fragment was then subcloned into pACYC-NHCT, thus replacing the KpnI-HindIII fragment carrying NphT7, resulting pACYC-AHCT plasmid.
The pET-AdhE2 plasmid was constructed in a pET-Duet vector (f1 origin, AmpicillinR, from Novagen) with adhE2 by subcloning synthesized gene encoding AdhE2 (aldehyde-alcohol dehydrogenase, from C. acetobutylicum ATCC 824, GenBank ID: Q9ANR5) from a pUC57 parent vector provided by GenScript into pET-Duet vector, using restriction sites NcoI and AvrII.
pCDF-ADO and pCDF-ADOA134F
The pCDF-ADO plasmid was constructed in a pCDFDuet-1 vector (CDF replicon, streptomycin/spectinomycinR, from Novagen) with ADO (aldehyde deformylating oxygenase from Prochlorococcus marinus MIT9313, GenBank ID: Q7V6D4.1) cloned into the vector using NcoI and EcoRI restriction sites. The pCDF-ADOA134F vector was created by mutating the ADO insert, using A134F_forward (5′-GCA TTT GCG ATT TCT TTT TAT CAT ACG TAC-3′) and A134F_reverse primers (5′-GTA CGT ATG ATA AAA AGA AAT CGC AAA TGC-3′). The correct mutations were confirmed by complete plasmid DNA sequencing. Gene encoding ADO was originally provided by E. Neil G. Marsh (Department of Biological Chemistry, University of Michigan, USA) in a pET28b-cAD vector which was used for the previous study .
The pCDF-Ahr plasmid was constructed in a pCDFDuet-1 vector (CDF replicon, streptomycin/spectinomycinR, from Novagen). Gene encoding Ahr (aldehyde reductase from E. coli GenBank ID: P27250.2) was PCR-amplified from isolated E. coli K-12 genomic DNA, using primers 5′ATTAATCCATGGTCTAGATAATTAATGGATCCAGGAGGAAACATATGTCGATGATAAAAAGCTATGCCGCAAAAG-3′ and 5′-ATTAATCCTAGGAAGCTTCTCGAGTCAAAAATCGGCTTTCAACACCACGCGG-3′, and cloned into using restriction sites NcoI and AvrII .
The pRSF-PetF plasmid was constructed in a pRSF-Duet1 vector (RSF replicon, KanamycinR, from Novagen) with fdx (ferredoxin from Synechocystis sp. PCC 6803, GenBank ID: WP_010873424.1) by subcloning synthesized gene from a pUC57 parent vector provided by GenScript into a pRSF-Duet1 vector using NcoI and AvrII restriction sites.
Co-expression and introduction of the pathway in E. coli
The atoB-adhE2 route was introduced into E. coli or the knockout cells by co-expressing pACYC-AHCT and pET-AdhE2 vectors. The atoB-TPC7 route was introduced into E. coli or the knockout cells by co-expressing pACYC-AHCT and pET-TPC7 vectors. The nphT7-adhE2 route was introduced into E. coli or the knockout cells by co-expressing pACYC-NHCT and pET-AdhE2 vectors. The nphT7-TPC7 route was introduced into E. coli or the knockout cells by co-expressing pACYC-NHCT and pET-TPC7 vectors in the cells.
E. coli cells containing engineered pathways were further engineered by co-transforming either pCDF-ADO or pCDF-ADOA134F vectors in order to introduce ADO or the ADOA134F variant. The pRSF-PetF vector was co-expressed for Fdx, while pCDF-Ahr was used to introduce Ahr enzyme in the pathway. The presence of all the proteins in each individual plasmid was confirmed by SDS-PAGE and mass spectrometry analysis of the respective SDS-PAGE bands. In the case of proteins with a hexahistidine tag, Western blotting (using WesternBreeze Chemiluminescent Immunodetection kit from Invitrogen, Carlsbad, CA, USA) was also used to analyse the expression of his-tagged proteins.
SDS-PAGE analysis of protein expression in cells containing plasmids encoding pathway components
Protein expression levels in cells containing plasmids that encode the pathway enzymes were examined by SDS-PAGE. T5 media (20 mL; 12 g tryptone, 24 g yeast extract, 4 mL glycerol, 12.5 g K2HPO4, 2.3 g KH2PO4, 20 g glucose per litre) was inoculated with 1% (v/v) transformed E. coli cells and incubated at 37°C (180 rpm) until the optical density at 600 nm (OD600nm) reached 0.5. Cultures were then induced with isopropyl β-D-1-thiogalactopyranoside (final concentration of 0.5 mM). Cultures were grown for a further 24 h at 30°C (180 rpm). Samples (200 μL) from the cultures were taken for SDS PAGE analysis. Samples were taken before isopropyl β-D-1-thiogalactopyranoside (IPTG) induction and after 4 or 24 h of IPTG induction and cells harvested by centrifugation. Samples were electrophoresed in 12% RunBlue precast SDS-PAGE gels (Expedeon, Cambridge, UK). Protein bands were visualized by staining with Instant Blue protein stain (Expedeon).
Media, cultivation and detection of propane and butanol
Lysogeny broth (LB) liquid media (10 mL) was inoculated using E. coli glycerol stocks (from −80°C) and incubated at 37°C overnight at 180 rpm. Of T5 media, 50 mL (12 g tryptone, 24 g yeast extract, 4 mL glycerol, 12.5 g K2HPO4, 2.3 g KH2PO4, 20 g glucose per litre) was inoculated with 1% (v/v) of the inoculum and kept for incubation at 37°C (180 rpm) until the optical density at 600 nm (OD600) reached 0.5. The cultures were then induced with IPTG to a final concentration of 0.5 mM. The cell cultures were further grown for 4 h at 30°C and 180 rpm to prepare the samples for propane detection. In the case of total butanol produced in the butanol pathway, the culture was left at 30°C (180 rpm) for 72 h, while for residual butanol detection, samples were taken from this culture when OD600 reached 1.5. Control cultures were made using untransformed E. coli strain BL21 DE3 cells.
For propane formation analysis, 50 mL cell culture was centrifuged at 4,000 rpm, and the supernatant was discarded. The cell pellets were resuspended in a 6.25-mL T5 media with 0.5 mM IPTG, and 500 μL of the resuspended culture was transferred into 2-mL crimp sealed GC vial and used for propane analysis. The vials were incubated at 30°C, with shaking at 180 rpm for 3 h. Headspace of 1.0 mL from the cultures grown in the GC vial was manually removed and injected into the GC with a gas tight syringe. Propane detection was carried out using a Varian 3800 GC (Palo Alto, CA, USA) equipped with a DB-WAX column (30 m × 0.32 mm × 0.25 μM film thickness, J&W Scientific, Santa Clara, MA, USA). The injector temperature was 250°C with a split ratio of 10:1. The column temperature was set from 40°C hold for 2 min to 100°C at 20°C/min with helium flow at 1 mL/min and FID temperature at 250°C. Propane peak was identified by comparing it with the analytical propane standard, and quantification was done using a propane calibration curve.
For residual butanol detection, 50 mL liquid culture was spun down at 4,000 rpm for 10 min. Of the supernatant, 500 μL was extracted with 500 μL of ethyl acetate containing 0.2% hexane as internal standard and dried over MgSO4. Of the sample, 1 μL was analysed in GC using a Varian 3800 GC equipped with a HP-5 column (30 m × 0.32 mm × 0.25 μM film thickness, JW Scientific). The injector temperature was 250°C with a split ratio of 20:1. The column temperature was set from 40°C hold for 1 min to 280°C at 20°C/min with helium flow at 1 mL/min and FID temperature at 250°C. Butanol peak was identified by comparing with the analytical butanol standard, and quantification was done using a butanol calibration curve.
aldehyde deformylating oxygenase
carboxylic acid reductase
fatty acid synthesis
- OD600 :
optical density at 600 nm
The work was supported by a grant from the European Union FP-7 256808 to NSS and PRJ. NSS is an Engineering and Physical Sciences Research Council Established Career Fellow.
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