- Open Access
Renewable synthesis of n-butyraldehyde from glucose by engineered Escherichia coli
© The Author(s) 2017
Received: 18 August 2017
Accepted: 26 November 2017
Published: 4 December 2017
n-Butyraldehyde is a high-production volume chemical produced exclusively from hydroformylation of propylene. It is a versatile chemical used in the synthesis of diverse C4–C8 alcohols, carboxylic acids, esters, and amines. Its high demand and broad applications make it an ideal chemical to be produced from biomass.
An Escherichia coli strain was engineered to produce n-butyraldehyde directly from glucose by expressing a modified Clostridium CoA-dependent n-butanol production pathway with mono-functional Coenzyme A-acylating aldehyde dehydrogenase (Aldh) instead of the natural bifunctional aldehyde/alcohol dehydrogenase. Aldh from Clostridium beijerinckii outperformed the other tested homologues. However, the presence of native alcohol dehydrogenase led to spontaneous conversion of n-butyraldehyde to n-butanol. This problem was addressed by knocking out native E. coli alcohol dehydrogenases, significantly improving the butyraldehyde-to-butanol ratio. This ratio was further increased reducing media complexity from Terrific broth to M9 media containing 2% yeast extract. To increase production titer, in situ liquid–liquid extraction using dodecane and oleyl alcohol was investigated. Results showed oleyl alcohol as a better extractant, increasing the titer of n-butyraldehyde produced to 630 mg/L.
This study demonstrated n-butyraldehyde production from glucose. Through sequential strain and condition optimizations, butyraldehyde-to-butanol ratio was improved significantly compared to the parent strain. Results from this work may serve as a basis for further development of renewable n-butyraldehyde production.
Strains and plasmids
rrnB T14 ΔlacZ WJ16 hsdR514 ΔaraBAD AH33 ΔrhaBAD LD78
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacI q ZΔM15 Tn10 (Tetr)]
BW25113/F’ [traD36 proAB + lacIqZΔM15 (Tetr)]
JCL16 ΔadhE ΔldhA ΔfrdBC Δpta
JCL299 ΔyqhD ΔyjgB
JCL299 ΔyqhD ΔyjgB ΔfucO
JCL299 ΔyqhD ΔyjgB ΔfucO ΔeutG
JCL299 ΔyqhD ΔyjgB ΔfucO ΔeutG ΔybbO
JCL299 ΔyqhD ΔyjgB ΔfucO ΔeutG ΔybbO ΔadhP
JCL299 ΔyqhD ΔyjgB ΔfucO ΔeutG ΔybbO ΔadhP ΔgldA
JCL299 ΔyqhD ΔyjgB ΔfucO ΔeutG ΔybbO ΔadhP ΔgldA ΔyahK
JCL299 ΔyqhD ΔyjgB ΔfucO ΔeutG ΔybbO ΔadhP ΔgldA ΔyahK ΔyghA
Pack::atoB, adhE2, crt, hbd; ColE1 ori; Ampr
PadhE::fdh; pSC101 ori; Cmr
PadhE::ter; Cola ori; Kanr
Pack::atoB, aldh (Clostridium beijerinckii), crt, hbd; ColE1 ori; Ampr
Pack::atoB, aldh (mutant gene; Clostridium beijerinckii), crt, hbd; ColE1 ori; Ampr
Pack::atoB, aldh (Clostridium saccharobutylicum), crt, hbd; ColE1 ori; Ampr
Pack::atoB, aldh (Clostridium saccharoperbutylacetonicum N1-4), crt, hbd; ColE1 ori; Ampr
Biological production of aldehydes is limited due to toxicity and reactivity. While few aldehyde products have been produced by engineered microbes [3–6], the biochemical repertoire for aldehydes needs to be expanded to support the effort in sustainability. n-Butyraldehyde has been previously reported in a mutant strain of Clostridium acetobutylicum  lacking alcohol dehydrogenase, capable of secreting up to 1.6 g/L of n-butyraldehyde. However, Clostridia are more difficult to work with than other well-characterized microorganism such as Escherichia coli and Saccharomyces cerevisiae due to their complex physiology and metabolism, as well as having less developed genetic manipulation tools. In addition, facultative anaerobes such as E. coli are often preferred for bio-based chemical productions because they grow rapidly during aerobic cultivation and conserves carbon for production under anaerobic conditions, increasing product yield due to elimination of respiration. Therefore, commercial interests are in engineering E. coli for n-butyraldehyde production . However, due to the presence of numerous native alcohol dehydrogenases (Adh) in E. coli, n-butyraldehyde is spontaneously converted to n-butanol, thereby lowering the yield of aldehyde. This same behavior was observed in isobutyraldehyde production in E. coli . Through knocking out several endogenous genes coding for Adh in E. coli, isobutyraldehyde production was significantly improved to roughly 2.5 g/L in test tubes and up to 35 g/L with gas stripping as in situ product removal. Inspired by the E. coli isobutyraldehyde production, here we also deleted adh genes and showed significant improvement in n-butyraldehyde production. Furthermore, in the process of constructing a n-butyraldehyde production pathway, we identified an alternative and better CoA-acylating aldehyde dehydrogenase than what has been previously reported. Lastly, instead of using gas stripping for in situ product removal as has been demonstrated for isobutyraldehyde, we tested in situ removal through organic overlay for liquid–liquid extraction and showed that oleyl alcohol is a suitable extractant for n-butyraldehyde production. The results obtained from this study provided a method for renewable synthesis of n-butyraldehyde with significantly reduced butanol co-production.
Strains and plasmids construction
Strains and plasmids used in this study are listed in the Additional file 1: Table S1. Primer sequences used are listed in Additional file 1: Table S1. Strains ELeco1 to KS8 were constructed from JCL299  by sequential deletion of aldehyde reductase genes. All gene deletions were carried out using P1 transduction  with Keio collection  as donor strains. Kanamycin resistance marker was removed via FLP-mediated recombination. The successful gene deletions were subsequently verified by PCR (Additional file 1: Figure S1). All plasmids in this study were constructed using Gibson assembly . Plasmids pKU48, pKU49, pKU50, and pKU51 were constructed by replacing adhE2 in pRW13 with aldh (CB), aldh (CB(mut)), aldh (CS), and aldh (CS(N1-4)), respectively. Briefly, a fragment containing plasmid vector, atoB, crt, and hbd was amplified using primers KU115 and KU116 using pRW13 as a template. This fragment was assembled with individual aldh fragments amplified by the specified primers in Additional file 1: Table S1 using the genomic DNA of the corresponding source organism. aldh (CB(mut)) gene was cloned from a cloning vector containing the gene in our lab collection. For its sequence, see Additional file 1. Plasmids were then verified through sequencing.
Culture media and growth conditions
All chemicals were purchased from Sigma-Aldrich or JTBaker. Media were purchased from BD-biosciences. All E. coli strains were cultured at 37 °C in a rotatory shaker (250 rpm). Luria broth (LB) and LB plates (1.5% w/v, agar) were routinely used for E. coli cultivation unless otherwise specified. Terrific broth (TB; 12 g tryptone, 24 g yeast extract, 2.31 g KH2PO4, 12.54 g K2HPO4, 4 mL glycerol per liter of water) supplemented with 20 g/L glucose was used as complex medium for n-butyraldehyde production. For medium analysis, M9 medium (12.8 g Na2HPO4·7H2O, 3 g KH2PO4, 0.5 g NaCl, 0.5 g NH4Cl, 1 mM MgSO4, 1 mg vitamin B1 and 0.3 mM CaCl2 per liter of water) supplemented with 20 g/L glucose and various concentrations (0.125–2.0%) of yeast extract (YE) and tryptone were used. When required, antibiotics were added into culture medium for selection at the following concentrations: kanamycin (Kan), 50 μg/mL; chloramphenicol (Cm), 50 μg/mL; ampicillin (Amp), 100 μg/mL; tetracycline (Tet), 15 μg/mL. Cell growth was routinely determined by measuring optical density at wavelength of 600 nm (OD600) of cultures using a Biotek epoch 2 microplate spectrophotometer. Path length was adjusted to 1 cm.
1% (v/v) of overnight cultures in LB was used to inoculate 3 mL of production media (TB or M9 with varying concentration of yeast extract) containing 20 g/L glucose with appropriate antibiotics in test tubes. When the cultures reached OD600 of 0.4–0.6, they were switched to anaerobic by transferring them into a 10-mL BD vacutainer tube. Head space was then purged with anaerobic gas (95% N2, 5% H2). Cultures were then sampled at specified times for optical density measurement and product quantification.
In situ removal of n-butyraldehyde by liquid–liquid extraction
1% (v/v) of overnight cultures were inoculated into 20 mL TB supplemented with 20 g/L glucose in 250-mL baffle flasks. When the culture OD600 reached 0.4–0.6, and 10 mL or 20 mL of either dodecane or oleyl alcohol was added as extractant to the culture. Subsequently, the cultures were switched to anaerobic by placing them in BD GasPak Anaerobe Gas Generating Pouch System with Indicator. When taking samples, anaerobic pouch was opened inside an anaerobic chamber to maintain the anaerobic environment.
Culture samples (1 mL) were centrifuged at 10,000×g for 5 min. The supernatants were then collected for product analysis. When analyzing product content in extractant, extractant was diluted using ethyl acetate. Aldehyde and alcohol concentrations in both medium and extractant were quantified by a Shimadzu GC-2010 gas chromatography (GC) equipped with a barrier ionization discharge (BID) detector. The separation of compounds was performed by SH-Rtx-wax GC column (30 m, 0.32 mm i.d., 0.50-μm-thick film). GC oven temperature was initially held at 40 °C for 2 min and increased with a gradient of 5 °C/min until 80 °C followed by a gradient of 12 °C/min until 120 °C. Then the temperature continues to rise with a gradient of 20 °C/min until 230 °C and held for 2 min. Helium was used as the carrier gas. The injector was maintained at 220 °C, and the detector was maintained at 230 °C. 1 μL of samples was injected in split injection mode (1:15 split ratio) using 2-methyl-1-pentanol or 1-pentanol as the internal standard. Glucose consumption was determined by subtracting the glucose concentration in samples from the concentration in original medium. Glucose concentration was measured using Agilent 1260 HPLC equipped with a refractive index detector. The injection volume used was 20 μL. The mobile phase consisted of 5 mM H2SO4 with a linear flow rate of 0.6 mL/min. Separation of metabolites was done by Agilent HiPlex-H (700 × 7.7 mm) organic acid analysis column maintained at 65 °C. A Bio-Rad Micro-Guard Cation H guard column (30 × 4.6 mm) was connected in front of the analysis column. Glucose was monitored by refractive index detector. Concentration of glucose in the collected samples was determined by standard curve constructed from HPLC analysis of standard glucose solutions.
Partition coefficient determination for n-butyraldehyde in dodecane and oleyl alcohol
Results and discussion
Selection of CoA-acylating aldehyde dehydrogenase for n-butyraldehyde production
Improving aldehyde-to-alcohol ratio by knocking out native alcohol dehydrogenases
Improving n-butyraldehyde titer by in situ product removal
Effect of reducing media complexity on n-butyraldehyde production
n-Butyraldehyde production was more sensitive to tryptone concentration than that of yeast extract as cultures containing 0.125 and 0.25% tryptone showed lower n-butyraldehyde titer compared to the corresponding concentrations of yeast extract (Fig. 5c, d). Increasing tryptone concentration led to increased n-butanol production, indicating that tryptone contributed towards the lowered aldehyde-to-alcohol ratio for using TB as production media. By comparing the components of yeast extract and tryptone from the manufacturers’ manual, we noticed that tryptone has higher percentage of larger molecules with molecular weight in the range of than 500–2000 Da, indicating a larger amount of oligopeptides. On the other hand, yeast extract contains mostly smaller molecules with molecular weight less than 250 Da. It is possible that this discrepancy led to different expression patterns which may include non-specific native alcohol dehydrogenases capable of reducing n-butyraldehyde. Nonetheless, the exact mechanism to why tryptone causes increase in n-butanol production is unclear.
This study demonstrated n-butyraldehyde production from glucose using engineered E. coli. We showed that aldh gene from C. beijerinckii outperformed the other aldh genes tested in achieving highest butyraldehyde-to-butanol ratio. Subsequent knockouts of endogenous adh genes including yqhD, yjgB, fucO, adhP, gldA, and yahK, in situ product removal by oleyl alcohol, and medium optimization using M9 2% glucose with 1–2% yeast extract significantly improved both the n-butyraldehyde titer (from 10 mg/L to 630 mg/L) and butyraldehyde-to-butanol ratio. Compared to E. coli glucose-based n-butanol production (with titer up to 15 g/L in test tubes), n-butyraldehyde production using similar strain and pathway resulted in significantly lower titer. It is possible to achieve renewable n-butyraldehyde production via bio-butanol followed by chemical conversion. The chemical conversion of n-butanol to n-butyraldehyde is possible using Cu [27, 28]- or Pt -based catalysis. However, the Cu-based catalysis requires high temperature of 500 to 800 K. While the Pt-based catalysis can produce n-butyraldehyde from n-butanol at lower temperatures, leaching of the expensive Pt-based catalyst increases cost of the overall process. Therefore, sugar-based direct production of n-butyraldehyde remains an attractive potential direction. In order for it to become industrially viable in the future, further optimization of genetic expression, media, and product removal techniques is necessary.
JTK, WS, and EIL designed the experiments. JTK and WS performed the experiments. JTK, WS, and EIL analyzed the data. EIL supervised the experiments. JTK and EIL wrote the manuscript. All authors read and approved the final manuscript.
This work was funded by the Ministry of Science and Technology (MOST), R.O.C. Taiwan through Grant 105-2221-E-009-164, partially through MOST 106-3113-E-007-002, and the laboratory start-up fund from the National Chiao Tung University. The authors would like to thank Dr. Claire R. Shen for providing plasmids used in this study.
The authors declare that they have no competing interests.
Availability of data and materials
Data and material supporting the findings can be found at National Chiao Tung University Department of Biological Science and Technology in Hsinchu, Taiwan.
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