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Keywords:

  • ABE fermentation;
  • Alternative hosts;
  • Clostridia;
  • Keto acid road;
  • n-Butanol

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References

In the context of the global objective of shifting from petroleum to a biomass-based economy, the research on fermentative strategies to produce alternative biofuels and chemicals has become a predominant field of study. Microorganisms, because of their substrate versatility and metabolic efficiency, are promising to partially support our increasing needs for materials and fuels, opening up scenarios for the use of alternative sources, including wastes. Butanol is a very attractive molecule since it can be seen both as a chemical platform and as a fuel. Today, it is principally derived from petroleum, but it also represents the final product of a microbial fermentation. Although Clostridia are the natural and traditional organisms employed in butanol production, systematic approaches to improve production and resistance traits are currently impeded by a lack of characterization and genetic tools. This is the main reason why, besides their optimizations, a significant and growing amount of research is centered on the engineering of alternative robust cell factories capable of elevated production, possibly combined with higher tolerance. Here, we review the most recent advances in n-butanol production in non-Clostridial microbial hosts, including not only other prokaryotic but also eukaryotic microorganisms, which might eventually be seen as second-generation hosts.

Abbreviations
ABE

acetone, butanol, ethanol

AdhE

alcohol dehydrogenase

Bcd/Etf

ferrodoxin-dependent butyryl-CoA dehydrogenase complex

Bdh

butanol dehydrogenase

Bldh

CoA-acylating butyraldehyde dehydrogenase

Crt

crotonase

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References

Petrol chemistry associated with crude oil refineries has profoundly changed our society: the vast majority of fuels utilized by combustion engines are derived from it, and the same can be stated for many of the materials present in our daily lives, such as plastics, fibers, solvents, fertilizers, as well as for specialties including some pharmaceutical preparations. While in ancient times fossil resources were only modestly used, resulting in a sustainable mode for their exploitation, the Industrial Revolution brought a profound change, characterized by a trend of increasing dependency on them. Even more, the new comforts and devices contributed to an increase in population and life expectancy and to a wider distribution of goods and services. This in turn has continuously generated new demands and markets for fossil fuels with no indications of a turnabout in sight.

As a consequence, we can speculate, argue, or even thoroughly discuss about the amount of residual fossil resources available on the planet, but this will not change the fact that they will be consumed at a rate infinitely greater than that at which they will be replenished by geological processes. This is how microbial metabolism came to the stage with a new light, after being the main engine of modern biochemistry at the end of 19th century—beginning of 20th century. Actually, the most efficient and brilliant activity that is pursued by the microbial world is the transformation of molecules, which can be used or transformed from organic to inorganic and from complex to simple state, and vice versa. Moreover, very often the product of one metabolism becomes the substrate for the other, ensuring the natural cycling of molecules and elements. Remarkably, very few substrates are recalcitrant for microorganisms, opening up a scenario of many possible transformations that can match our need for alternative sources with the increased generation of wastes.

At the moment, the possibility to completely substitute oil products with microbial metabolites is a dream, but due to some brilliant and successful examples (such as 1,3-propanediol and the derived Sorona® polymer [http://origin.dupont.com/Sorona̲Microsite]; lactic acid and the derived polylactic acid polymer [www.natureworksllc.com]; bioethanol as commercialized by many companies, see [www.task39.org] for a recent report on the current status of bioethanol plants; succinic acid [www.reverdia.com/home]), this appears as the most important and most stimulating challenge for industrial microbiologists. This implies looking at the problem with an open mind: in many cases, it will not be the case to substitute processes or molecules, but more radically to reimagine them, or improve their possible use. As an additional piece of the challenge, we should also learn from nature how to make the process sustainable.

In this context, the microbial production of butanol deserves consideration since it represents one of the most interesting biotechnological alternatives to petroleum derivatives to be offered on the future market, as announced and commented in recent commentaries (www.cobalttech.com; www.butamax.com; www.metabolic-explorer.com; [1]). It has to be specified that the term “butanol” is usually referred to the straight chain isomer of a 4-carbon alcohol with the functional group at the terminal carbon, also known as 1-butanol or n-butanol (in this review). The branched isomer with the alcohol at the terminal carbon is isobutanol. 2-Butanol or sec-butanol isomer presents the alcohol at an internal carbon of the straight chain, while the branched isomer with the alcohol at the internal carbon is tert-butanol. The first two isoforms are the most interesting as potential solvents in chemical industry and as biofuels, due to their limited solubility and closer similarity with gasoline if compared with ethanol.

The escalation in the number of papers and reviews on the subject underscores the large effort of the scientific and industrial communities toward the goal of n-butanol and isobutanol production from microbial fermentations. Here, we point out first the most important technical traits of the process of production in Clostridia and then we deeply discuss the recent advances in research on n-butanol production in non-Clostridial microbial hosts, currently under development to assess possible advantages over the natural producers.

2 Butanol and n-butanol production by Clostridia

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References

n-Butanol is an important product and intermediate for the chemical industry with several large-volume derivatives: it is used in plasticizers, amino resins, and butylamines, and can be additionally used as a solvent for many different preparations [1] (Fig. 1). Moreover, n-butanol has been suggested as a bio-based oxygenated fuel for blending in gasoline in high concentrations without vehicle modifications (85% or more with gasoline): with respect to ethanol, the energy content of n-butanol is closer to that of gasoline (27 versus 32 MJ/L, respectively), it has no miscibility problems, and is compatible with the current infrastructure [2, 3].

image

Figure 1. Uses of n-butanol. On the left, the main application areas are reported (dimension of the cartoons is just indicative, and not related to market volume). On the right, the global butanol applications are shown (demand by main regions: Europe 25%, China 34%, North America 24%, Asia except China 13%, other countries 4%, as reported by Cathay Industrial Biotech Ltd, 2010 report) (figure redrawn from www.icis.com/blogs/green-chemicals/2011/07/cathay-industrial-biotech-file.html).

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The world market for n-butanol is estimated to be more than 5 million tons per year, with an average cost for a gallon between 3 and 4 US$. The global market value is close to 6 billion US$, and is expected to reach 9 billion US$ in 2015, while the global market for butanol derivatives is about 50 billion US$ (www.nexant.com).

Butanol production from glucose (acetone, butanol, ethanol [ABE] fermentation) has been possible since the early 1900s. The process based on the fermentation of agricultural derived sugars was developed industrially for the production of acetone first and n-butanol later in Russia and China up to the 1980s, when the chemical process from oil becomes more economical. In the last decade, however, new plants are being built or revamped driven by the changing oil economics and interest in bioproducts. In China alone, a production capacity of 1 million tons/year of ABE is expected based on newly built and revamped plants [1].

All the known bacteria naturally capable of producing n-butanol belong to the group of the Clostridia, ubiquitous anaerobic species commonly found in the environment (soil, organic waste), whose several species are capable of fermenting different sugars into short-chain alcohols, acids, and a large number of diverse metabolites [4]. The Clostridial path for the synthesis of n-butanol from glucose brings to the parallel accumulation of lesser quantities of acetone and ethanol, acetic and butyric acids, together with CO2 and hydrogen where the ABE solvents are typically accumulated in relative amounts of about 3:6:1 in Clostridium acetobutylicum, with a total solvent production of around 20 g/L (reviewed in [5]).

Fermentations with natural strains of Clostridia suffer from some important limitations. Possibly the strongest limit of the system is the low tolerance of the cells to the products, among which n-butanol is the most toxic. An n-butanol concentration of 11–12 g/L is the upper limit of accumulation for natural strains, but mutants and engineered strains have been selected, which tolerate (and are able to grow in) the presence of up to 19 g/L of said alcohol [6]. This low value for product tolerance has a strong impact on process design and product purification, which constitute an important fraction of the total cost of production. Other limitations include the intricacies of a multiproduct fermentation lowering the yield to a theoretical 0.94 mol/mol of glucose, simultaneous “nonproductive” spore formation, low cell densities attainable with Clostridia during anaerobic fermentation, low productivities (0.35–0.4 g/L/h at best, but see below), and a poorly characterized phenomenon known as “strain degeneracy” that leads to production instability with longer times of fermentation (see for reviews [2, 7-9]). Much past and present research has focused on improving these aspects, both at the level of strain selection and process design [9].

2.1 Strain selection

Significantly improved strains have been isolated by classical mutagenesis and selection as far as product tolerance and resistance to phage infections is concerned (see e.g. [1, 2, 7]). However, further improvement is expected based on the development of new tools for Clostridium engineering. In the last decade, specific vector systems based on gene recombination have been useful for the development of laboratory strains and protocols to obtain new mutants. Based on these systems, and on extensive genome sequencing, targeted mutants have been produced on specific genes in the ABE pathway and on pathways influencing productivity [4, 10-12].

2.2 Process improvement

Batch ABE fermentations of solventogenic Clostridia are characterized by a primary phase of acid accumulation and a switch to a solvent production phase [13]. While continuous production would be economically convenient, semicontinuous variants are used in practice in existing plants, to avoid “strain degeneracy.” This type of design has been utilized both in Russia [14] and China [1, 15] in several thousand tons per year capacity industrial plants, with typical running times of around 4 wk. Product toxicity imposes continuous solvent removal and concentration and is at the basis of process designs that include gas stripping or pervaporation (recently reviewed in [9, 16]). The low cell densities could be circumvented by fermentation with immobilized cells (biofilm reactor systems), which have been reported to “stabilize” the culture for 200–700 h and attain high densities, or membrane cell recycling systems [9, 17].

Combined optimization of the fermentation process and product recovery mode have improved considerably the productivity, up to 1–1.5 g/L/h for n-butanol [18] or even to 11 g/L/h for ABE [15], but have not been tested yet on an industrial scale due to the high costs of product recovery.

2.3 Clostridium advantages

While natural strains and their mutant derivative have been used industrially and are well characterized as n-butanol producers, within the limits illustrated above the Clostridium genus still has potential as a low-cost producer based on its diversified capabilities of substrate utilization. In fact, several strains (not necessarily n-butanol producers) can use a large spectrum of simple and complex carbohydrates such as starch, cellulose, and hemicellulose (reviewed in [19]). Remarkably, some strains are able to utilize glucose and xylose simultaneously [4]. On these bases, industrial processes were and are based on the utilization of starchy or sugar-rich agro products such as byproducts from corn, cassava, potatoes, molasses in China, and molasses and hydrolyzates in Russia. Moreover, comparatively high-yield fermentation was demonstrated on barley straw hydrolyzate [20] and other lignocellulosic agro residues [21] with productivity and yield similar to, or better than, those based on glucose fermentation. Clostridia have generally shown to have good resistance to some of the compounds released from lignocellulosic biomass pretreatment, such as acids, while fermentation is inhibited by low levels of ferulic acids and phenolic compounds that would need to be removed prefermentation [7, 22]. Other “clever” Clostridia strains were shown to be naturally able to utilize CO [23], CO2, and H2 [24], although with efficiency limited by gas/liquids transfer rates. This combination of substrate utilization would facilitate the use of Clostridia as producers from syngas derived from diverse types of biomass. Finally, Clostridia utilization of a fraction of carbohydrates derived from microalgae is possible and could be improved, gaining accessibility on this desirable source of renewable biomass for the production of bioalcohols [25, 26].

The relatively recent developments of Clostridial engineering tools and protocols will possibly make feasible to evolve strains with a combination of desired traits for processes based on lignocellulosic biomass, where interesting product profile modifications were obtained by substituting acetone production with isopropanol (see e.g. [27]).

In conclusion, up to now most of the advantages of n-butanol come from its properties as a fuel, not from the current production technology. Traditionally, low yields—in the 15–25% range—plague n-butanol production and toxicity remains the major limitation, with n-butanol being toxic at about 20 g/L. With dilute product concentrations of 1–2%, recovery technologies have been quite costly. However, major strain improvements are considered to be possible through modeling and metabolic engineering approaches [19] (see also Section 'Concluding remarks').

3 Non-Clostridial microbial host

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References

As well known to any industrial microbiologist, the ideal host for a production process does not exist, or does not exist yet, in the prerevolution era for synthetic biotechnology. The expertise of the key players will be another piece of the puzzle: the background knowledge about a specific cell factory together with the characteristics of preexisting plants can also play an important role in the choice of microorganism. The development of non-Clostridial microbial host for butanol production has to be analyzed in this context.

Two main metabolic strategies were exploited to obtain n-butanol in microorganisms, which are not natural producers (Fig. 2). The first one takes in consideration the transfer of the Clostridial ABE fermentation pathway, which was realized in different microorganisms using each time different optimization strategies, mainly devoted to harmonizing the energy requirement and redox balance (see as an example [28] and further details in the next sections). In this path, butyryl-CoA is synthesized from acetyl-CoA and is then reduced to n-butanol. The second metabolic strategy takes advantage of keto acids as intermediates to produce fusel alcohols, among which n-butanol (see as an example [29] and further details in the next sections). Keto acids are present in all living microorganisms since they are intermediate in amino acids biosynthesis and degradation metabolism, through the Ehrlich pathway (reviewed in [30]).

image

Figure 2. (A) The Clostridial acetone-butanol-ethanol (ABE) fermentation pathway. (B) The Ehrlich pathway for amino acids degradation into alcohols.

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It can be generally stated that most of the efforts have been concentrated on alternative bacterial hosts, with a unique example for the utilization of a eukaryotic host, represented by the well-known and established baker's yeast. Other physiological and metabolic features characterize alternative cell factories for n-butanol, as listed in Table 1, where most of the differences turn out to have a very significant impact on the productive process. In the same table, the highest production value reported in the literature for every host is also given.

Table 1. Physiological and metabolic features characterizing the reviewed cell factories for n-butanol production. Readers are invited to refer to the text, where references reporting specific numbers are specified
 Clostridium acetobutylicumEscherichia coliLactobacillus brevisPseudomonas putidaBacillus subtilisSynechoccus elongatusSaccharomyces cerevisiae
  1. ND = not determined.

Cellular typeProkaryoteProkaryoteProkaryoteProkaryoteProkaryoteProkaryoteEukaryote
Oxygen toleranceObligate anaerobeFacultative anaerobeFacultative aerobeFacultative anaerobeObligate aerobeFacultative anaerobeFacultative anaerobe
Substrate rangeLargeGoodGoodLargeGoodOrganic C independencySmall
Phototrophy and autotrophyNoNoNoNoNoYesNo
Genetic tractabilityLowHighGoodHighGoodGoodHigh
n-Butanol tolerance (growth)1.5% w/v1.5% w/v2–3% w/v0.75% w/v1.25% w/vND2% w/v
n-Butanol tolerance (viability)∼ 2% w/v∼ 2% w/v3–6% w/v6% w/v5% w/vNDND
n-Butanol production (via acetyl-CoA)19 g/L30 g/L300 mg/L122 mg/L24 mg/L29.9 mg/L2.5 mg/L
n-Butanol production (via keto acid)ND1 g/LNDNDNDND92 mg/L

Escherichia coli, Saccharomyces cerevisiae, Lactobacillus brevis, Pseudomonas putida, Bacillus subtilis, and Cyanobacteria spp. are the protagonists of the following sections, with a particular attention to the “why and how” that led the scientists to prefer some systems than others.

4 The Clostridial ABE pathway road

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References

n-Butanol production in Clostridia starts with the condensation of two acetyl-CoA units to produce the C4 backbone: in three subsequent reactions, acetoacetyl-CoA is reduced and dehydrated to butyryl-CoA, further reduced to butyraldehyde, and finally to n-butanol (Fig. 2A). The genes encoding for each reactions are very well described and the corresponding enzymes are also characterized [13]. The expression of the entire pathway was thus pursued in the best-known, largely used microbial workhorses. Among bacteria, E. coli steal the scene given its simplicity, feasibility of manipulation, and genetic tractability.

However, the simple overexpression of genes is very often not enough to accomplish the desired metabolic switch: in this case, overexpressing the ABE genes led to the production of just a small amount of n-butanol, 13.9 mg/L in 40 h under anaerobic conditions [31]. This was possibly explained considering the great susceptibility of some of the corresponding enzymes to oxygen together with their need for specific cofactors, as demonstrated for the ferrodoxin-dependent butyryl-CoA dehydrogenase (Bcd/Etf) complex [31]. Different strategies were then applied to increase production. The best results were achieved by substituting the Bcd/Etf complex with a transenoyl-coenzyme A (CoA) reductase (Ter), blocking mixed acid fermentation and acetate formation (deletion of lactate dehydrogenase [Ldh], alcohol dehydrogenase [AdhE], fumarate reductase [Frd], and phosphotransacetylase [Pta] genes), and overexpressing the formate dehydrogenase gene to increase the acetyl-CoA and reduction equivalent pool. This last combination resulted in a new E. coli strain able to accumulate 15 g/L of n-butanol in 3 days [28].

It is important to remark that a fermentation process can efficiently occur only if the redox balance is addressed, as also stated in the paper cited above. Metabolic models and in silico analyses are increasingly helpful in supporting new strains design. A recent elementary mode analysis has shown that E. coli engineered to produce n-butanol would incur in NADH deficiency under anaerobic conditions, while isobutanol production would not produce this effect [32]. Results from the in silico analysis suggested that six deletions (glucose-6-phosphate dehydrogenase [zwf], malate dehydrogenase [mdh], frd, NADH:ubiquinone oxidoreductase [ndh], adhE, and ldhA) could lead to the production of n-butanol as a single product and couple anaerobic growth and n-butanol production during the growth phase [32]. The same paper also suggests to carefully consider the ways of avoiding acetate production: the deletion of both Pta and pyruvate oxidase B genes would benefit n-butanol production but would also generate a deficit of acetyl CoA, which is detrimental for anaerobic growth. In an even more recent paper, a flux-balanced analysis approach suggests an alternative way to increase the flow of NADH via a screening of triple knockouts [33]. Once again, the data confirmed that disruption of the ethanol (adhE) and acetate (pta) production pathways is crucial for increasing the production of n-butanol in engineered E. coli cells. Moreover, the authors suggest that when the n-butanol pathway is present, there is no need to inactivate the lactate production pathway since the first one is a better electron sink (4 mol—at least—versus 2 mol of NADH are oxidized in the production of 1 mol of n-butanol versus 2 mol of lactate, starting from 2 mol of pyruvate). The disruption of the acetate pathway would also play in the same direction, since the consequent lack of ATP is balanced by increasing the carbon flux in the TCA cycle, accompanied by an increase in NADH production. The authors finally suggest redirecting the flux through the pentose phosphate pathway: this should increase the NADPH pool, which in turn, because of transhydrogenase activities, could provide additional NADH for n-butanol production [33].

The research activities just described (wet and dry) underline the complexity of manipulating a native metabolism with the final aim of a complete redirection of the carbon flux to the desired product. An alternative way to overcome this limitation is to count on the cellular capability to reorganize when a central mechanism (also defined as nonpathway components, such as transcription or translation) is perturbed. This was well described by McKee and coauthors: by perturbing the carbon storage regulator system, previously described to cause a profound reorganization of E. coli central metabolism [34], they were able to obtain a twofold improvement of n-butanol production with a concomitant acetate decrease [35]. The basis for these results remains to be understood, since the prominent effect of the perturbation was an increase in amino acid levels. However, the approach appears intriguing as it could be applied to other prokaryotic cells, being carbon storage regulator a conserved system.

A similar approach, based on the perturbation of the transcriptional machinery, was applied for improving n-butanol tolerance [28, 36], which remains a key limitation to be overcome for any productive system. At the moment, E. coli sensitivity to the product is similar to that of the Clostridia strains, posing in this respect the same process constraints. In fact, the best production value of 30 g/L was reached due to gas stripping of the alcohol during the fermentation [28].

One of the principal reasons for developing other nonnatural producers is to achieve increased tolerance to n-butanol: obtaining a higher product percentage in the final beer would be significant to reduce the costs for recovery.

Heterologous hosts with known natural solvent tolerance and high industrial utility, such as P. putida [37, 38] and B. subtilis [39], were then explored for the reconstruction of the n-butanol biosynthetic pathway. Pseudomonas putida S12 solvent tolerance is based on an increased proportion of transunsaturated fatty acids in its cytoplasmic membrane and on the use of active efflux pump systems [37]. In the literature, a moderate n-butanol tolerance (around 0.75% w/v) was reported for this microorganism [37, 40]. Solvent-tolerant species of Bacillus have also been isolated that are still viable at n-butanol concentrations as high as 2.5–3.7% w/v. In this case, the tolerance mechanisms include adaptations to the cell-wall composition and the activity of stress response proteins [41, 42]. More generally, B. subtilis strains can still grow when n-butanol concentration is around 1.25% w/v [43]. Nielsen et al. successfully expressed the native n-butanol pathway in these two alternative hosts [43]. More in detail, the Gram-negative P. putida, when cultured in terrific broth medium, produced 50 and 122 mg/L of n-butanol using glucose and glycerol as carbon source, respectively, under aerobic conditions. Notably, in this microorganism, the Clostridial Bcd/Etf complex seems not to be inhibited or inactivated by oxygen. The Gram-positive B. subtilis was additionally modified by deleting three genes encoding for α-amylase, threonine synthase, and dihydroorotate dehydrogenase (amyE, threonine synthase [ThrC], and dihydroorotate dehydrogenase [pyrD]). The derived strain was cultivated in terrific broth medium and produced 23 and 24 mg/L of n-butanol using glucose and glycerol as carbon source, respectively, under anaerobic conditions [43]. While being the most solvent tolerant and despite sharing greater phylogenetic similarity with Clostridium compared to P. putida, B. subtilis turned out to be the poorest producing strain.

Berezina et al. [44] demonstrated that in L. brevis, the conversion of butyryl-CoA into n-butanol was performed by endogenous aldehyde and alcohol dehydrogenases activities acting as butyraldehyde (Bldh) and butanol dehydrogenase (Bdh), respectively. These results coupled with the high n-butanol tolerance of L. brevis (it can grow up to 2% and adapt up to 3% w/v of n-butanol) were the principal factors for choosing this bacterium as a host for the expression of the Clostridial butanol metabolic pathway [44]. The presence of Bldh and Bdh activities reduced the need of heterologous expression. In particular, two plasmids were constructed to express the Clostridial gene encoding for 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA-dehydrogenase complex (bcd), and two subunits of the electron transfer flavoprotein (etfAB), one with and one without the thiolase gene. Interestingly, the n-butanol titer was 250 mg/L when the strain was transformed with a plasmid containing the thiolase gene and 300 mg/L when this gene was not introduced. This also demonstrated the higher efficiency of an endogenous thiolase [44]. It can be agreed with the authors that the host system seems to offer interesting margin for optimization, going from the inactivation of competing endogenous fermentative pathways to the utilization of balanced expression systems.

Moving in the direction of alcohol tolerance, it is quite natural to think about S. cerevisiae. This budding yeast has a long history of industrial use for the production of ethanol, beer, and wine from sugar. It represents the key player in bioethanol production both in first- and in second-generation plants, based on its robustness not only in the presence of high-ethanol concentrations but also in the presence of the inhibitory compounds often present in cheap medium (for a recent review comprising metabolic, systemic, and synthetic approaches to engineer the baker's yeast, see [45]). The natural tolerance of S. cerevisiae to n-butanol is lower than its tolerance to ethanol, being around 2% w/v [46, 47], but still significant considering that very few optimization attempts have been pursued.

The Clostridial pathway was first expressed in this host and then different bacterial enzymes were substituted with isoenzymes from various other species to better match with the host metabolism [48]. Different thiolases, 3-hydroxybutyryl-CoA dehydrogenases, and butyryl-CoA dehydrogenases have been tested, including endogenous activities both for the first (thiolase) and the last (aldehyde and alcohol dehydrogenases) steps. The best strain overexpressed the native thiolase, the 3-hydroxybutyryl-CoA dehydrogenase (NADH dependent), crt, butyraldehyde, and Bdh from Clostridium beijerinckii and butyryl-CoA dehydrogenases from Streptomyces collinus (ccr), producing 2.5 mg/L of n-butanol from 2% w/v galactose as a carbon source [48].

Saccharomyces cerevisiae is the sole eukaryotic organism in which the n-butanol pathway was expressed: very likely the greatest limitation is represented by acetyl-CoA, the key compound of the pathway but poorly available in the cytoplasm [49], where the heterologous enzymes are active. The possibility to further develop S. cerevisiae through this pathway without a profound visitation of its metabolism remains dubious. Metabolic models and in silico analyses will be crucial for addressing this open question. An alternative way of making the process more viable would be to reduce the substrate cost, which presently dominates the overall cost, followed by product recovery.

Autotrophic organisms might offer the solution. In the very last years, the use of Cyanobacteria has received increasing attention due to their prokaryotic and phototrophic nature together with their ability to fix carbon through the Calvin–Benson–Bassham cycle. From an industrial perspective, they offer different advantages such as relatively fast cell growth, simple nutrient requirements, the potential to be genetically engineered, and the capacity to grow in a variety of locations, even those unfit for agriculture, as recently reviewed [50].

It was calculated that for producing one molecule of n-butanol using photosynthesis and the Calvin cycle, 48 photons are required, considering that energy has to be devoted both to glyceraldehyde-3-phosphate (2 mol required) and to reducing equivalent formation [51]. The Clostridia n-butanol pathway was introduced by recombination into the genome of Synechococcus elongatus PCC 7924 by first integrating atoB (E. coli) and adhE2 (C. acetobutylicum) and then integrating hbd, crt (C. acetobutylicum), and ter (Treponema denticola) genes. This strain was able to accumulate only about 2.2 mg/L of n-butanol when grown under light possibly due to a technical inefficient system for oxygen removal (a continuous bubbling of CO2/N2). n-Butanol accumulation reached 14.5 mg/L after 7 days only when the strain was cultivated in dark anoxic conditions [51].

The node of acetoacetyl-CoA formation seems to be the limiting step in this microorganism, being dependent on acetyl-CoA and NADH availability. For addressing cofactor availability, the S. elongatus was then further modified by converting enzymatic dependence to the higher available NADPH [52] (as also confirmed by the intracellular ratio of about 1:30 between NAD+ and NADP+ [53]), directly produced during photosynthesis. Under photosynthetic conditions, acetyl-CoA has low availability; however, it was considered that acetoacetyl-CoA can also be formed through decarboxylative condensation of malonyl-CoA, which derives from ATP-activated acetyl-CoA. Consequently, ATP consumption through this alternative route can shift the equilibrium versus the desired compound. In summary, the final strain comprised the acetyl-CoA carboxylase, the malonyl-CoA-acetyl-CoA condensing enzyme, the NADP+-depending alcohol dehydrogenase, and the CoA-acylating butyaldehyde dehydrogenase (Bldh). The major n-butanol production was almost 30 mg/L, as also reported in Table 1 [52, 54].

While the fermentative nature of many bacteria seems to offer a more natural solution to the heterologous expression of the butanol pathway, photoautotrophic bacteria offer their substrate “independence” as an invaluable plus. However, it remains difficult to combine a fermentative process with oxygenic photosynthesis. The possibility to inactivate photosystem II has been demonstrated [51], but this poses even more sharply another problem, which is the well-demonstrated low efficiency of photosynthesis, constituting at the moment the major limitation in the industrial exploitation of microbial photosynthesis.

5 The keto acids road

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References

The cost of feedstock for low-added value biotechnological process is an important issue. Until now, efforts to engineer microbes have mainly focused on fatty acids and sugars as substrates [55]. All these approaches, however, underestimate the value of the protein fraction that could derive from leftover biomass from fermentative processes, constituting a substantial waste product since proteins are the dominant fraction in fast-growing microorganisms. These protein-based byproducts are typically used as animal feed, but the corresponding market has a limited capacity [56]. Huo et al. recently proposed the use of proteins or, more precisely, amino acids derived from protein hydrolysis, as feedstock for biofuels and chemical productions [57].

The last two steps of the Ehrlich pathway consist of the conversion of 2-keto acids, as intermediate in amino acid biosynthesis pathway, to the corresponding aldehydes by a keto acid decarboxylase and then to the alcohols by alcohol dehydrogenase (ADH [30]; Fig. 2B). Using this approach, Atsumi et al. obtained the production of n-butanol from the corresponding 2-keto acid precursor, ketovalerate, in E. coli cells [58]. Keto acid decarboxylase gene was overexpressed from Lactococcus lactis or ARO10 (aromatic amino acid requiring gene encoding for phenylpyruvate decarboxylase) from S. cerevisiae as keto acid decarboxylase activity, coupled with the ADH2 from S. cerevisiae as ADH activity. The engineered E. coli strain was able to accumulate 16.3 mg/L of n-butanol using glucose as substrate, these low levels possibly being dependent mainly on the small amount of 2-ketovalerate synthesized. Since this intermediate can be generated from the l-threonine pathway through the 2-ketobutyrate formation, the authors then overexpressed ilvA (threonine deaminase) and leuABCD (isopropylmalate synthases) in E. coli strain and added 8 g/L of l-threonine as substrate, increasing n-butanol production up to 237.1 mg/L [58]. n-Butanol production was further improved by deleting the ilvD gene, a dihydroxy-acid dehydratase enzyme that produces both 2-ketoisovalerate (a precursor for leucine and valine) and 2-keto-3-methyl-valerate (a precursor for isoleucine). This deletion eliminated the formation of side intermediates as 2-ketoisovalerate, reducing the substrate competitive inhibition for keto acid decarboxylase gene, finally obtaining 667 mg/L of n-butanol. To further increment the keto acid pool through the deregulation of threonine degradation, Shen and Liao considered the allosteric feedback inhibition of ThrA (homoserine dehydrogenase) by threonine [29]. Overexpressing a feedback resistant ThrA mutant (ThrAfbr), the production of both propanol and n-butanol increased and together with the elimination of competing pathway the highest amount of n-butanol using the keto acid road was obtained. As reported, when the metA, threonine dehydrogenase (tdh), ilvB (acetolactate synthase I), ilvl, and adhE were deleted, the n-butanol amount coproduced with propanol at a 1:1 ratio was 1 g/L [29].

This demonstrates the possibility to obtain fusel alcohols via the keto acids route in E. coli. What is also known is that wine yeasts under glucose starvation and in the presence of amino acids can also accumulate fusel alcohols, among which is n-butanol. These metabolites are interpreted by the cells as a signal of nutritional limitation, evoking a pseudohyphal differentiation [59].

Differently from E. coli, ketovalerate was never experimentally measured in S. cerevisiae, while the pathway to isoketovalerate was better elucidated and recently successfully exploited for isobutanol formation [60, 61]. Even more recently, an in silico analysis suggesting a possible, but not characterized, connection between glycine and butanol [62] was utilized to experimentally demonstrate a novel pathway for butanol production in S. cerevisiae, with a possible isobutanol coproduction. During kinetic growth, 15 g/L of glycine, n-butanol and isobutanol accumulate in the medium up to 92 and 58 mg/L, respectively, was added to glucose medium [63, 64].

6 Concluding remarks

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References

In the scenario described, it seems more reasonable to draw perspectives and views rather than conclusions. This possibly renders the n-butanol production challenge even more fascinating.

It is highly possible that the first bio-butanol in the market will derive from engineered Clostridium spp., according to the latest announcements and consequent efforts about new or revamped plants for ABE fermentation [1] and considering the challenge taken by a number of key players in the market of fuels and chemicals (as cited in the Introduction section). It sounds also more feasible in the near future to imagine a chemical market instead of a fuel market for bio-butanol, according to the different volumes and selling prices. In this respect, it is important to remark that fossil oil is renewable, but in a time scale that is not compatible with its exploitation, as well emphasized in [65]. Very likely, in the longer term, the market demand for nonfossil-derived products will become higher.

Different strategies of process optimization for n-butanol production are emerging, here mainly analyzed in the perspective of alternative hosts: (i) increasing production and productivity, which can additionally have a direct reflection on recovery costs and (ii) considering alternative pathway and substrates, which can also lead to increasing the range of possible products.

6.1 Increasing production and productivity

A deeper knowledge of the metabolism and physiology of the cell factory can suggest what might be adapted and what should be changed for improving the process of production. Modeling can be of great help, facilitating scientists to calculate the distance between experimental and theoretical yields, eventually suggesting how to move from the first to the second (just to cite two recent examples, see [32, 66]). Genetic tractability of Clostridia remains a major limitation, even if quickly improving [19].

Another great limitation is n-butanol tolerance, this alcohol being much more toxic than ethanol and also more toxic than its branched isomer, isobutanol [67]. Together with the search for alternative hosts being naturally more tolerant, many efforts are invested in improving this trait in the already chosen hosts, starting from Clostridia and moving to other bacteria and yeasts (see as an example some recent papers reported here [41, 58, 68-72]). The development of n-butanol tolerant strains will open the possibility of having a fermented beer with higher alcoholic concentration, with positive consequences in terms of recovery. However, it has to be mentioned that most of the techniques aimed at selecting tolerant strains are not coupled with fermentation properties: this means that it should be not taken for granted that an improved tolerance will reflect in an improved production.

6.2 Alternative pathways and substrates

The difficulty in transferring the Clostridia ABE pathway into non-native hosts has been reported here, and becomes even more evident when considering that production of related compounds results in much higher titer in E. coli, as it was demonstrated for isobutanol (20–50 g/L) [58, 73] and isopropanol (40–143 g/L) [74]. As stated earlier, part of this difference relates with product tolerance, but the butanol fermentation poses per se some constraints, more related to the efficiency of some enzymatic complexes and to the need of strictly reducing conditions. This is how the keto acid road came into consideration and this is how the possibility to produce more and different fusel alcohols came under evaluation [57]. In the logic of an integrated biorefinery, the protein waste could form a still underestimated source of different chemicals, and different technologies for protein and amino acid isolation are under development [75] (Fig. 3). Alternatively, by even more sophisticated engineering approaches, it might be possible to synthesize a desired amino acid from lignocellulosic sugars, as very recently reported for isobutanol production from d-xylose through valine biosynthetic pathway in an S. cerevisiae strain overexpressing a bacterial xylose isomerase [76]. Finally, a well-established cell factory might be first engineered for n-butanol production and further manipulated for extending the carbon chain length, obtaining higher value products [77, 78].

image

Figure 3. Integrated biorefinery. Main stream of production from feedstock is accompanied by reutilization of protein-based waste accumulating at the end of the fermentation process. This waste can be utilized to feed the main stream process or, alternatively, as a source of materials for side stream productions.

Download figure to PowerPoint

In conclusion, different bacteria and the yeast S. cerevisiae were differently engineered for n-butanol production, as alternative hosts to natural Clostridium spp. All these studies have the invaluable merit of adding pieces of knowledge for an ancient, but still not completely solved, metabolism. These pieces will be possibly valorized by systems and synthetic biology approaches, not forgetting that the final host has to be robust enough to also address process constraints.

Practical application

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References

Microbial fermentations are promising to partially support our increasing need for materials and fuels, most of which are currently derived from fossil resources. Butanol is a very attractive molecule since it can be seen both as a chemical platform and as a fuel. The natural butanol producers, bacteria of the Clostridium genus, can be exploited, but their usage imposes process limitations. This is the main reason why, besides working on their optimization, microbiologists are also evaluating the pros and cons of alternative microorganisms, reviewed here, which might eventually emerge as second-generation hosts.

7 References

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Butanol and n-butanol production by Clostridia
  5. 3 Non-Clostridial microbial host
  6. 4 The Clostridial ABE pathway road
  7. 5 The keto acids road
  8. 6 Concluding remarks
  9. Practical application
  10. Acknowledgements
  11. 7 References
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