Biofuel production in engineered microorganisms - advances and research needs
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(2) IAMB 20121028. candidate for replacing gasoline is butanol [1,2,4,6,13,17,20]. FAEE (fatty acid ethyl esters) could replace diesel as well as fatty alcohols [6,9,11,15]. Jet fuel could be substituted by biologically synthesized alkanes [6,9]. Some of these molecules are produced naturally by microorganisms. For example, Clostridium species produce butanol [1,2,21,1,13]. Cyanobacteria produce alkanes [5]. Acinetobacter calcoaceticu have especial enzymes that catalyze the production of fatty alcohols [15]. Some of the metabolic pathways for the production of these molecules have been biochemically and genetically characterized. These pathways have something in common; the intermediate that initiates the production of these molecules is acetylCoA[6,8]. There are four main groups of pathways that start after the production of acetyl-CoA from pyruvate. The fermentative pathways, amino acid biosynthesis, fatty acid synthesis pathway and the isoprenoid pathways. However the physiology of these pathways in native producers is very complex. There’s no broad knowledge of the genome of the native producers and no available tools for their manipulation [1,2,6,13,21]. Moreover, their growth rate is usually low [6,13]. Many strains produce these molecules in very low concentrations and yields. Furthermore, it is known that they only use a very low percent of biomass components. Xylose, who represents 30% of the lignocellulose biomass, is nonmetabolizable by the majority of the microorganisms [6,8]. Thus, the transformation of biomass to glucose, which is the main substrate of these metabolic pathways, by microorganisms is not efficient. Therefore, the utilization of native strains for biofuel production is not an economically viable solution to supply the current demand for transportation fuels. This is why metabolic engineering has been focusing on designing biosynthetic pathways for the production of advanced biofuels in microorganisms with a broad metabolic potential. To accomplish the increasing demand user friendly hosts are used, which are. organisms with fast growth rates and that are already extensively genetically characterized [2]. These hosts have various genetic tools available for their modification. The most popular user friendly hosts are Escherichia coli, Saccharomyces cerevisiae and Synechococcus elongatus [1,2,4,13,15,20]. The objective is to produce molecule fuels in high titers at high yields and with high specificity. To do so, it is necessary to express the native pathways of the native producers in user friendly hosts. Introduction of heterologous genes, gene overexpression, gene knockouts and deletion of competitive pathways are some of the techniques used for the optimization of heterologous pathways [8]. However, metabolic engineering still faces some challenges. Heterologous genes can cause metabolic imbalance in the user friendly host [4]. The accumulation of certain metabolites can cause cytotoxicity [4,8]. All the pathways are no fully understood so they face some problems due to the difficulty of redox control balance [21]. In this review we present the most recent advances in metabolic engineering of microorganisms for the production of advanced fuels. Although important advances in ethanol production exist our focus is limited to the production of the most important advanced and drop in fuels. These are: Isopropanol, 1propanol, isobutanol, Butanol, FAEE, FAME and alkenes. We also identify future research needs for the conversion of simple sugars to biofuels.. ISOPROPANOL Isopropanol is a secondary alcohol used to complete the esterification of fats and oils for the production of a unique diesel. This biodiesel has a special characteristic that is ideal to fuel airplanes; it’s capable of supporting low temperatures without crystallizing, a situation that cannot be avoided when methanol is used to fulfill this function [2,7,20]. As fuel itself, Isopropanol has a high energy density and low hygroscopicity [6]. 2.
(3) IAMB 20121028. These characteristics allow mixing with gasoline at a high proportion [20]. With further investigation it is believe that isopropanol could replace gasoline at a 100%. If isopropanol is dehydrated, propylene can be obtained, another fuel that is part of synthetic gasoline [7]. At an industrial level, the secondary alcohol is being produced at high titers due to its potential to produce other chemical products. This makes isopropanol ideal for commercial production [7]. Biologically, Isopropanol can be produced using the CoA-dependent pathway of acetone followed by an alcohol dehydrogenase. The by-products that result from this pathway include acetone and ethanol. Clostridium species like Clostridium acetobutylicum and Clostridium beijerinckii are natural producers of acetone [3,7,19]. Researchers have already expressed the acetone pathway in the user friendly host Escherichia coli [3]. In an initial experiment 5.4 g/L of acetone were produced by an E. coli strain that expressed clostridial genes [3]. The production level was as high as the one found naturally in Clostridium acetobutylicum strains [2, 3]. Based on this result, E. coli was selected in the following experiments as a host for isopropanol production. The native clostridium pathway expressed in E. coli for the production of isopropanol consisted of four enzymes: Acetyl-CoA acetyltransferase, acetoacetyl-coA transferase, acetoacetate decarboxylase, and a secondary alcohol dehydrogenase [2,3,6,7,19]. The clostridium acetobutylicum genes that encode for the first three enzymes were thl, ctfAB and adc, respectively [2,3,6,7,19]. For the secondary alcohol dehydrogenase enzyme, that reduce acetone to isopropanol, two effective genes that encode for this enzyme were identified: adh(cb) from C. Beijrinckii [2,6] and adh(tb) from Thermoanaerobacter brockii HTD4[19]. Two genes that encode the enzymes that catalyze the first two steps in the biosynthesis of acetone in E.coli have also been identified. These are atoB and atoAD [2,6,19].. Research conducted by Hanai and coworkers investigated which one was the best combination of the aforementioned genes to produce isopropanol in E. coli under aerobic conditions at 37°C. They maintained the growth medium conditions and cultivation techniques in each essay and quantified the production of isopropanol that resulted after 12 h, by the time glucose was depleted [19]. In the presence of thl, ctfAB, adc and adh(cb) a titer of 2.16 g/L of isopropanol was obtained [19]. Acetone and Ethanol sub production was very low. In the second gene combination atoB from E. coli replaced C. acetobutylicum acetylCoA acetyl-transferase (thl). This pathway produced 2.28 g/L of isopropanol [19]. The production improved, but in a low proportion when compared to the first combination. Subproduction of ethanol also increased, but only traces of acetone were detected. As this combination resulted in a higher titer, researchers maintain the expression of atoB and replaced ctfAB with E. coli’s native gene atoAD. The results showed an increase in isopropanol production of approximately 0.6 g/L[19]. Here ethanol production was the lowest while acetone production increased. Although isopropanol production was higher, acetone production is undesirable. Therefore the next step was to express thl instead of atoB and maintain atoAD. This isopropanol titer was the highest, making this selection of genes the best combination for the coA-dependent pathway. Nonetheless, ethanol production increased and acetone remained at the same level. The final step was to test the efficiency of SADHs. The expression of Thermoanaerobacter adh resulted in the lowest production of isopropanol; below 0.3 g/L [19]. Acetone production was even higher; it reached a concentration of 2.32g/L [19]. This shows that SADH from T. brockii has a very low activity. Therefore, it is not appropriate for this pathway and it is highly recommended to keep using adh from C. beijerinckii.. In brief, the E. coli engineered strain that produced the highest titer of isopropanol included adh from de C. beijerinckii NRRL B593, thl, adc from C. acetobutylicum and 3.
(4) IAMB 20121028. atoAD from E. coli. It is possible to obtain a concentration of 4.91 g/l after 30.5 hours, with a maximum production rate of 0.41g/L/h [19]. The yield reached 43.5% (mol isopropanol/mol glucose) of the theoretical maximum [19]. This pathway was proven to be stable. When glucose was added, the rate of production was similar in both periods. Even in the period when glucose was exhausted, between 12 and 14 hours, researchers were able to register Isopropanol production [19] Inokuma et al. took the pathway proposed by Hanai et al. to improve the yield of isopropanol production by E. coli, aiming to achieve industrial or large scale production. To accomplish this, the process had to be efficient and come at a low cost. Researchers began by improving growth conditions and by lowering the cytotoxicity caused by the heterologous molecules in E. coli TA76 [7]. Inokuma et al. determined that the initial step to increase isopropanol concentrations would to improve the medium and growth conditions. The first problem researchers detected was that when the pH decreased and the medium became more acidic, an inhibitory effect was produced and isopropanol production began to decrease. To solve this problem, they proposed to maintain pH above 5 by adding 10 M of KOH when necessary [7]. When there was no pH regulation, and the final pH was 4, Inokuma et al. obtained an isopropanol titer of 14.7 g/L after 48h of Fed-Batch fermentation. In the following essay with pH regulation the titer was much higher; 40,1± 0.5 g/L of isopropanol after 60 hours, and a yield of 73.2% (mol isopropanol/mol glucose) [7]. Later on, taking a look at the time course of pH-controlled fed-batch fermentation Inokuma et al. perceived that after 48 hours, the production rate decreased substantially. This situation showed that isopropanol accumulation in the medium produced an inhibitory effect. In order to solve this problem, researchers proposed to remove and gather isopropanol and other volatile gases by gas stripping. Using the pH-controlled FedBatch fermentation with a gas stripping-based recovery system, an isopropanol production of. 79,6 g/L was obtained [7]. This means almost twice the previous concentration obtained; thus inhibition was avoided. However, the production rate did decrease with time. To solve this problem, Inokuma et al. recommended adding a condenser to increase the recovery rate of isopropanol. An improvement in the humidification system was also necessary to prevent evaporation of water from the culture media [7]. Even with the gas stripping-based recovery system the production rate decreased with time. Inokuma et al. detected that nutrient depletion occurred when there were low oxygen levels. In order to improve these growth conditions, 2ML of SD-8 medium were added. This time, the production rate did improve. The total amount of Isopropanol produced was 143g/L in 240 h with a yield of 67.4% (mol isopropanol/mol glucose) [7]. After this period no isopropanol concentration was registered. This means that other inhibition factors may exist, remaining in the process, even with the gas stripping-based recovery system. One hypothesis strongly supported by Inokuma et al. was that acetone was coming out of the culture media even with the stripping gas recovery system. This means that an important quantity of acetone was volatilizing and so, it wasn’t transforming to isopropanol. To fix this, it is required to increase the activity of the secondary alcohol dehydrogenase. Hence, adc from C. beijerinckii NRRL B593, should be overexpressed [7]. Inokuma et al. achieved to improve isopropanol production. Compared with the highest titer reached by Hanai et al. there was a 29-fold increase. It remains the highest production of isopropanol achieved by a metabolic engineered E. coli strain [6]. There were other attempts to replicate this experiment, where similar concentrations of isopropanol were obtained [7]. This means that this is a highly replicable process and there is potential for further industrial and commercial production.. 4.
(5) IAMB 20121028. 1-PROPANOL 1-propanol is a branched chain alcohol. It comes as an attractive fuel due to its high energy density and low hygroscopicity [4]. These properties allow the mixture of 1propanol with gasoline [20]. In nature, 1propanol can be derived from threonine catabolism, which is found in clostridium and as a product of beer fermentation by yeast [4, 20]. So far, a microorganism that produces 1propanol from glucose in high titers naturally hasn't been found [4,20]. Nor the renewable sources needed for a large-scale production. However, there are successful metabolic engineering approaches for 1-propanol production in E. coli. It is essential to use the threonine biosynthesis. This strategy avoids using heterologous routes since amino acid synthesis is present in all organisms. Therefore, metabolic imbalance and cytotoxicity should not cause a significant problem [4,20] The pathway consists principally of 2ketobutyrate production. This 2-ketoacid is an intermediate in threonine biosynthesis. Once this intermediary is produced, it can be converted to an aldehyde by a 2-ketoacid decarboxylase (KDC), and then to 1-propanol by an alcohol dehydrogenase (ADH) [4,20]. In an early study, Atsumi S et al. investigated which was the best KDC for the production of branched chain alcohols as biofuels, one of these being 1-propanol.This kind of enzymes are not common in bacteria [4] ; hence the importance of this study. Five KDC’s from different microorganisms were proposed for exploration in an E. coli strain. The natural hosts from which the genes were obtained were; Saccaromyces cerevisiae, Lactococcus lactis and C. acetobutylicum. Thereby, five E. coli clones were grown in a minimal media with 0.2% of Glucose [4]. Each one overexpressed one of this KDCs and an alcohol dehydrogenase (AdhE2) from C. acetobutylicum. Atsumi S et al. demonstrated that the best KDC for this purpose due to its high activity and adaptability is encoded by KivD from Lactococcus lactis lactis. The total amount of 1-propanol produced by E. coli. when KivD was overexpressed was 520 mM [4]. The second best KCD was encoded by ARO10 from S. cerevisiae. It only achieved 290 mM of 1-propanol [4]. This means less than half the production of KivD. Later, Liao JC et al. intended to optimize this pathway. It should be mentioned that in this study authors intended to produce 1-propanol and 1-butanol at the same time in an E. coli strain. They detected all the pathways that were competing for pyruvate and other substrates to determine which genes were needed to be deleted to improve the specificity of the pathway, thus the production rate of 1propanol [20]. The native pathway of theronine in E. coli starts with phosphoenolpyruvate (PEP), which is transformed to oxaloacetate by Phosphoenolpyruvate carboxylase (ppc). Then oxaloacetate is coverted to aspartate and to homoserine by aspartate aminotransferase (aspC) and homoserine dehydrogenase (thrA), respectively. Homoserine is then condensed by thrBC (homoserine kinase and threonine synthase) to generate Threonine. ilvA encoding threonine deaminase catalyzes the conversion of homoserine into 2-ketobutyrate. Finally, 1-propanol is generated from this 2ketoacid by KivD and Adh2, which are the only non-native steps. [4,20] In the initial trial, ilvA, KivD and ADH2 were overexpressed in E.coli. This clone produced 60 mg/L of 1-propanol in 24 h [20]. When these genes weren’t overexpressed, 1-propanol production was below the detection limit. After a 24 hour period, only isobutanol and ethanol production were detected [20]. Ethanol is produced from acetyl-coA by aldehyde-alcohol dehydrogenase (adhE), meaning that the process occurred before 2-ketobutyrate transformation. Researchers acknowledge that theonine production was causing a bottleneck scenario. This could be due to feedback inhibition of threonine [20]. When there is a big production of an amino acid in the cell, the final product binds to the allosteric side of the first enzyme involved in the biochemical pathway [28]. This causes a transformation of the active site of the enzyme and so the whole 5.
(6) IAMB 20121028. pathway stops because it cannot longer transform the substrate [28]. In order to solve the conformational change, it is necessary to add a Thra feedback resistant mutant .When this resistant mutant was overexpressed in E. coli the production achieved was 4 times higher, rising approximately 200 mg/L of 1propanol [20]. The next step was to eliminate the routes that compete for substrates of the 2-ketoacid pathway used by 2-ketobutyrayte. Researchers detected four of these mechanisms [20]. (i) Methionine pathway, where metA (homoserine O-succinyltransferase) starts the synthesis of homoserine; (ii) 2-amino-3-ketobutyrate pathway where tdh (threonine dehydrogenase) catalyzes threonine dehydration. (iii) The pathway for ethanol biosynthesis, coming from the reduction of acetyl-coA by adhE (aldehyde-alcohol dehydrogenase). (iv) The superpathway of Valine, leucine and isoleucine leaded by the enzymatic activity of the acetolactate synthase isozymes (ilvI, ilvH,ilvN, ilvB). Specifically, valine and leucine biosynthesis competes for pyruvate. Isoleucine biosynthesis competes for 2-ketoburyrate and led to the by-product formation of isobutanol. This study was able to register how deletions increased the specificity of the pathway. As byproducts formation decreased, 1-propanol production increased [20]. In the initial experiment the methionine pathway derived from homoserine was deleted. The engineered E. coli strain produced 600 mg/L [20]. Isobutanol and ethanol concentrations had a marked decrease (nearly 200 mg/L each one when the pathway increased its specificity). In the following clone tdh was deleted also and so by-product level decreased again, with a major effect in ethanol concentration. The obtained 1-propanol titer was 800 mg/L [20]. In the succeeding clone ilvB and ilvl genes were deleted. As expected Isobutanol production diminished to very low levels, below 50mg/L [20]. 1-butanol production benefited from this deletion, with a marked increase in production to 800mg/L [20]. 1-propanol titer didn’t have any improvement. The Final strain contained the. combined effect of these manipulations plus the deletion of adhE. This deletion decreased ethanol production to 100mg/L [20]. As a result, the best 1-propanol titer achieved in 72 hours was 1g/L [20]. Liao JC et al. achieved to reduce by-products to very low levels and at the same time enhance 1-propanol and 1butanol titers in the same E. coli strain.. ISOBUTANOL Isobutanol is an important, interesting fuel, due to its similarities to gasoline, which makes it a suitable replacement [2,4,6,22,24,25]. Its chemical and physical properties include high octane number and energy density (26.25 MJ/L), low vapor pressure and no hygroscopicity [2,22]. Isobutanol is not naturally produced in high titers in microorganisms [4]. However, this branched chain alcohol can be derived from 2ketovalerate, a 2-keto acid. It is an intermediate in valine biosynthesis, one of the twenty most common amino acids [4]. As reviewed for the 1-propanol production, 2-ketovalerate is converted to isobutyraldehyde by KivD from L. lactis chosen as explained before, for its highbroad specificity [4]. Then this aldehyde is reduced to produce isobutanol by an alcohol dehydrogenase. It is necessary to overexpress these heterologous genes to achieve high titers [4,22,24]. Several groups have reported the expression of this synthetic pathway [4,22,24]. One of this studies expressed the pathway in cyanobacterium Synechococcus elongatus, with light and CO2 as substrate instead of glucose, since it is a heterotrophic organism. This strain obtained an isopropanol production of 450 mg/L [24]. Atsumi S et al. and Connor MR et al. engineered E. coli strains for the production of isobutanol, using also the 2ketoacids pathway. Despite the toxicity to isobutanol, E. coli strains achieved to grow and accumulate high titers. In Atsumi S et al. study, isobutanol production was performed under micro aerobic conditions at 30°C in an E. coli strain. The strain overexpressed KivD from L. lactis, ADH2 from 6.
(7) IAMB 20121028. S. cerevisiae and ilvIHCD from E. coli. This strain produced 1.83 g/L of isobutanol [4]. In a succeeding experiment, five genes were eliminated to increase available pyruvate: adhE (pyruvate formate-lyase deactivase), ldhA(lactate dehydrogenase), frdAB(fumarate reductase), fnr (ferredoxin-NADP+ reductase )and pta (phosphate acetyltransferase). This manipulation increased Isobutanol production to 2.2 g/L [4]. This means that the specificity of the pathway did improve. To further increase the isobutanol production titer E. coli native gene from valine biosynthesis, ilvIH gene was deleted and replaced by acetolactate synthase (alsS) from Bacillus subtilis. This strain led to a final titer of 3, 6 g/L of isobutanol [4]. Finally, another pyruvate competitor, encoded by plfB (pyruvate formate-lyase) gene was inactivated, in order to enhance even more isobutanol production titer. The combined effects of these manipulations led to 22 g/L of isobutanol in 112 hours and yielded 86% of the theoretical yield [4]. Later Wu. T et al. studied the enzymatic activity of ADHs in the isobutanol pathway via 2-ketovalerate. There are other candidates different from ADH2 from S. cerevisiae that catalyze the reduction of isobutyraldehyde to isobutanol in a NADH-dependent reaction. AdhA from L. lactis is one of them. Actually E. coli itself has 6 different ADHs, one of them, adhE. The gene encoding this enzyme was already deleted from the synthetic pathway to remove Ethanol production [22, 26]. The ADH that appears more suitable for this reaction is YqhD, a NADP-dependent dehydrogenase. It is known that YqhD has higher affinity to higher alcohol and undetectable activity with short-chain alcohols [22]. To evaluate if E. coli ADHs had a significant contribution to isobutanol production, two strains were constructed. One overexpressed ADH2 from S.cerevisiae and the other didn’t. Both E.coli clones produced 7.4 g/l in 24 hours [22]. Therefore ADHs from E. coli play a big role in reducing isobutyraldehyde to isobutanol [22].. Next, to evaluate if YqhD was the ADH responsible for this role, two strains were used, both with overexpression of ADH2, and one without YqhD. When YqhD was deleted isobutanol production decreases substantially, the strain only produced 3.8 g/l in 24 hours [22]. When neither ADH2 nor YqhD were expressed, production lowered down to 1.4 g/L in 24 hours [22]. The results indicated that Adh2 from S. Cervisiae is taking part in the pathway but does not have high activity towards isobutyriladehyde as ADHs from E. coli, especially YdhD [22]. Therefore ADH2 overexpression isn’t necessary to improve isobutanol production [22]. Yqhd overexpression was also tested; however it didn’t make a difference in isobutanol production titer. Other alcohol dehydrogenases were tested to enhance isobutanol production. L. lactis genes showed high broad range substrate specificity for alcohol production from aldehydes [4, 22] Therefore, adhA from L. lactis, was suggested for overexpression. As a result of the adhA overexpression the strain produced ≈ 8.5 g/L in 24 hours[22]. This isobutanol production was even higher than the one produced with YqhD only. Actually the second best production was achieved by the strain in which YqhD was deleted and adhA overexpressed. Therefore the alcohol dehydrogenase more appropriate for the production of isobutanol from 2ketovalerate in E. coli is, adhA from L. lactis [22].. BUTANOL Ethanol is the most popular transportation biofuel because it is already produced at an industrial scale [16]. However ethanol doesn’t have the appropriate chemical and physics properties to fully replace any existing fuel, or to blend at high rates [2,4,6,8,9,13,17]. Actually it is not compatible with the existing infrastructure [2,6,8,9]. Ethanol transportation has to be done in special pipelines because it is corrosive to the existing ones [6,8,9]. Butanol is by far the best potential fuel substitute. It has approximately 84% of 7.
(8) IAMB 20121028. gasoline energy density, being 27 Mj/L vs 32 Mj/L [1,2,4,613,21]. It lacks hygroscopic features [1,2,4, 6,13,21]. It has very low vapor pressure [2,13] and therefore low volatility [4]. All of these characteristics allow butanol to completely replace gasoline or blend at any ratio [1,2,4,13,21]. Especially since it is 100% compatible with the existent infrastructure [1,2,4,13] and no corrosive to the transportation pipelines [2,6,21]. Microbial production of this alcohol usually happens naturally in clostridium species in a homofermentative CoA-dependent pathway from butyrate and acetone under anaerobic conditions; due to the nature of clostridium species [1, 2, 21, 1, 13]. It is generally found in Clostridium acetobutylicum and clostridium beijerinckii strains [13]. 1-butanol can also be derivated from 2-ketovalerate, an intermediate in the unnatural synthesis of norvaline, which is at the same time is derived from 2ketobutyrate from leucine synthesis [2, 4, 20, 21]. Several groups have a reconstructed clostridium pathway in different hosts such as E. coli [13,17,14,21], Synechococcus elongatus PCC 7942 [1], Saccharomyces Cerevisiae[1] , Lactobaccilus brevis [1], Pseudonoma putida [1], Bacillus subtilis [1]. However, only E. coli has been used as a host to produce butanol from the 2-ketoacid pathway [4, 20]. The best titer for both pathways has been achieved by E. coli metabolically engineered strains. Using the 2ketoacid pathway the highest butanol production obtained was 0.9 g/L [2]. With the CoA-dependent synthetic pathway the E. coli strains achieved a superior production level; 30 g/L in 7 days and 70% (mol butanol/ mol glucose) of the theoretical yield [21]. This is a very good result given that 1-butanol production in E. coli is considered a high titer when it exceeds its toxic level of 10mg/l [21]. However, this high titer was not achieved in the first study. The first engineered synthetic pathway expressed in an E. coli strain was designed from C. acetobutylycum ATCC 824. Initially it involved the expression of 6 C. acetobutylycum. genes thl , hbd, crt, bcd, etfAB, thl, hbd and Adh2. This experiment was conducted under anaerobic conditions by Atsumi S et al. which led to a low production of 13.9 mg/L of 1butanol. Specifically speaking, AcetoacetylCoA thiolase (Thl) catalyzed the transformation of acetyl-CoA to acetoacetylCoA in the first step of the synthetic pathway. Then acetoacetyl-CoA was converted to 3hydroxybutyryl-CoA and to crotonyl-CoA by 3-Hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt). Crotonyl-CoA was then reduced to butyryl-CoA by butyryl-CoA dehydrogenase complex (bcd-etfAB). Finally, butyryl-CoA was transformed to butyraldehyde and then reduced by alcohol dehydrogenase (Adh2) to obtain 1-butanol [13,21]. Every step of this pathway is NADH dependent except for the step mediated by butyryl-CoA dehydrogenase, which also required an electron transfer from flavoproteins A and B, as a reducing co-factor. In total, for the production of one molecule of 1-butanol, 4 NADH coenzymes are needed [13,21]. To further enhance the production of butanol in E. coli using this synthetic pathway, Atsumi S et al. replaced the enzymatic activity of thl for the overexpression of a native gene in E. coli, atoB (acetyl-CoA acetyltransferase). The overexpression of this gene led to a higher butanol production. It was assumed that the pathway was oxygen sensitive due to the strict nature of clostridium. However when growth conditions were changed to semi-aerobic, the final production of 1-butanol increased even more. From 41.7 mg/L in 40 h obtained under anaerobic conditions to 70 mg/L under semiaerobic conditions, both expressing atoB from E. coli [13] Another native gene in E. coli that was thought to be the replacement of one heterologous gene in the pathway was AdhE (aldehyde-alcohol dehydrogenase). Nevertheless, its enzymatic activity is higher toward acetyl-CoA rather than to butyryl-CoA [13]. It means that the overexpression of this gene could lead to a higher production of ethanol, a fermentation by-product, instead of an increase in the 1– butanol titer. AdhE2 from C. acetobutylycum 8.
(9) IAMB 20121028. showed higher activity to butyryl-CoA than to acetyl-CoA. Therefore, alcohol dehydrogenase remains in the pathway to catalyze the reaction of butyryl-CoA to butyrylaldehide [13,21] As seen in previous studies, [2,4,6,7,14,15,21] deletion of by-product pathways leads to a higher titer of the objective product. This manipulation causes an increase in the availability of important substrates involved in the pathway that are consumed to form the byproduct. This enforces the specificity of the pathway. Later, the deletion of by-products pathways, specifically ethanol succinate and lactate that were competing for NADH and acetyl-CoA was achieved through the elimination of the E. coli genes adhE, frdBC (fumarate reductase proteins) and ldhA (lactate dehydrogenase) [13]. These deletions increased 1-propanol production up to 270 mg/L in 24h in the same semi-aerobic conditions mentioned before [13]. Lactate, ethanol and succinate concentration decreased considerably. Out of all the byproduct concentrations, the succinate one diminished the most. It declined from 41mM to 5.4mM [13]. However, this knockout technique didn’t work out to inhibit E. coli acetate pathway that competes for acetyl-CoA. When pta (phosphate acetyltransferase), the principal gene involved in acetate production was deleted at the same time with adhE, frdBC and ldhA, the E. coli strain decreased acetate and butanol production at the same time. The final concentration in 24h for 1-butanol was 200 mg/L [13]. These results made clear that the pathway wasn’t fully understood. The first problem that Atsumi S et al. found during this study was that the bcd-trfAB enzimatic activitiy was the only one that remained unidentifiable. Homologous and isoenzymes were tested but this situation didn’t change [13]. The second problem found was that contraire to the nature of the pathway, higher butanol titers were achieved in semi-aerobic conditions. The main reason was that the acetyl-CoA production was being catalyzed by Pfl (pyruvate formate lyase) and not from pyruvate, as it was designed [13].. This is also one of the reasons why after the deletion of the competing pathways for NADH and acetyl-CoA the pyruvate final concentration increased. This means that the NADH supply is insufficient to satisfy the pathway under anaerobic conditions because Pyruvate dehydrogenase is inactive [13]. They established then, that in order to improve 1butanol production in E. coli, a balance of each step of the pathway needs to be done [13]. In a subsequent study Shen C.R et al. made a complete analysis of each step in the pathway. First they observed that in this synthetic CoAdependent pathway lacked an irreversible reaction that could direct the carbon flux to 1butanol construction. To accomplish this, a NADH driving force strategy was proposed as a solution. | To create the NADH driving force, bcd-etfAB complex was replaced by Ter from Treponema denticolla [21]. This is a novel trans-2-enoylCoA reductase with high activity for crotonylCoA [21]. In fact NADH is the only reducing power that its enzymatic product needed. Therefore, the reduced ferredoxin is no longer an inconvenient additional requirement. Shen C.R et al found during the investigation of the activity of trans-2-enoyl-CoA that crotonylCoA reduction was developed in an irreversible reaction. This means that the carbon flux is successfully directed to 1butanol production, and so the expression of Ter and the deletion of the competing pathways for NADH mentioned above, coupled the NADH driving force to the pathway [21]. The deletion of adhE, frd ldhA and the overexpression of atoB, Hbd, Crt, adhE2 and Ter in a E. coli engineered strain produced 1.8 g/L of butanol in 24 hours under anaerobic conditions [21]. Higher titers were obtained also under these conditions [21]. However this production level is not yet considered as a high titer. For further optimization of the pathway Shen C.R et al. deleted pta to decrease acetate byproduction and overexpressed the formate dehydrogenase from candida boidinii, fdh. 9.
(10) IAMB 20121028. This overexpression was aimed at avoiding pyruvate and formate accumulation. Fdh transforms formate to CO2 and NADH. This reinforces even more the NADH driving forces and effectively increases 1-butanol production [21]. The new NADH source solved the redox imbalance of the pathway. Without fdh overexpression 2 molecules of glucose were needed to produce one of butanol. Now with the overexpression of fdh, formate resulting from pyruvate can be transformed to yield an additional NADH [21]. From one molecule of glucose 2 molecules of pyruvate and 2 NADH’s can be formed. Then each pyruvate molecule is converted to one molecule of formate and one of acetyl-CoA. Until this point the pathway produced 2 moles of NADH, 2 moles of acetyl-CoA and 2 moles of formate. The last two molecules of formate are then transformed into 2 moles of NADH and 2 moles of CO2. The final result is 4 mol of NADH and 2 moles of acetyl-CoA. This fulfills the demand to produce exactly one molecule of 1-butanol. As a result, from 1 mol of glucose, the pathway is able to produce 1 mol of 1-butanol. This means a theoretical maximum yield of 100% (1mol of butanol / 1mol of glucose). The combined effects of these manipulations led to a butanol production of 15g/L in 3 days representing 88% of the theoretical maximum yield [21]. Shen C.R et al. finished the experiment by optimizing the medium conditions. As in the isopropanol engineered strain proposed by Jan Y-S et al. they implemented the gas stripping system to gather and recover the butanol production from the medium in a pH batch fermentor. The expression of this synthetic pathway produced 30 g/L in 7 days of butanol under anaerobic conditions with the gas stripping system [21].. FATTY ACIDS Biodiesel is nowadays produced from plants and animal fatty acids [8]. Its demand is so big that it competes with food production. In 2010, the growth in demand for biodiesel was 3 times higher than for gasoline [15]. Only in the USA the production of biodiesel in 2011 was of 802. million gallons1. This causes biodiesel prices to be prone to increasing. Moreover, the cultivation of oil extraction plants requires intensive land use and water supply which could lead to environmental damage. Therefore, a better option would be to produce fatty acids from fermentative pathways. [15] Fatty Acids have high energy density [6] as they are used by cells for energy storage and other chemical functions [15]. Therefore they were chosen as components of biodiesel, specifically fatty acid methyl esters (FAME) and fatty acid ethyl esters (FAEE) [15]. These are currently derived after a process of transesterification of animal and plant oils [2, 15]. This Biodiesel is fully compatible with the existing infrastructure [8, 15]. Other compounds derived from free fatty acids such as fatty alcohols, alkanes and alkenes are also potential biofuels [6,15] Fatty acid synthesis occurs in the cell membrane of all the microorganisms [6]. The objective is to transform them to phospholipids. This mechanism is continuous and efficient [6,15] and has been studied extensively. Then, there’s a lot of genetic information and tools to manipulate fatty acid biosynthesis [6,15]. It is desirable to produce FAME, FAEE and fatty alcohols from metabolic engineered microorganism. To do so, in an initial study an E. coli strain was engineered to express its native thioesterase, TesA in the cytosol [15]. This thioesterase is capable of transforming fatty acyl-ACP to free fatty acids. As the objective was to produce FAEE, FAME or fatty aldehydes, the fatty acid degradation was disrupted in the first step of the fatty acid degradation. This is when free fatty acids are converted to acyl-ACP COA by fadD. To accomplish this, the E. coli fadD (fatty acylCoA synthetase) gene was overexpressed and adhE (acyl-CoA dehydrogenase) was deleted. In summary to produce acyl-ACP-CoA from fatty acyl-ACP the E. coli strain overexpressed TesA and fadD and knocked out fadE [15].. 1. National biodiesel board, nov 29 , 2011. 10.
(11) IAMB 20121028. When the objective was to produce FAEE, atfA from (wax-ester synthase) from Acinetobacter sp was expressed in E. coli. 2% of ethanol was added to the medium in order to induce the esterification of fatty acids and ethanol [15]. This way, Acyl-ACP CoA was synthetized to produce FAEE by atfA. This E. coli engineered strain produced 400 mg/L of FAEE in 48 hours [15]. For the production of fatty alcohols, atfA is replaced by Acr1 from Acinetobacter calcoaceticus BD413. Acr1 transforms acylAPC to fatty alcohols in a NADPH dependent reaction. The other overexpressed and knockout genes for the FAEE production did not change for this essay. As a result the strain produced 60mg/L of fatty alcohols [15]. However, the production wasn’t as high as in FAEEs case. Eric J. et al. proposed to search for another acyl-CoA reductase with higher activity for acyl-ACP CoA reduction. On the other hand, FadD was essential in the process: without it there’s was no detectable production of fatty alcohols and very low production of FAEE [15]. Since diesel quality and performance is directly affected by the carbon chains length and its saturation, it is important to maintain diesel quality by controlling the FAEE composition [15]. To modify the length of fatty acid chains, there were different plant thioesterases used in another study [15]. Here different thioesterases produced FAEEs or fatty alcohols with different frequencies for every range. It seems like each thioesterase has its own preference for a carbon chain length. For example, the thioesterase AtfatA3 produced FAEE where 80% were in the range from C2 to C16. This way depending on the cetane number it could be chosen the appropriate thioesterases[15]. Metabolic engineering aims to produce advanced biofuels directly from cellulose in future investigations. In the last case the objective was to synthetize glycosyl hydrolases. Which enzymes are capable of hydrolyzing hemicelluloses to produce simple sugars.Specifically the objective was to produce xylose which contains 5 carbon atoms. The hemicelluloses were expressed in an E.. coli protein. This E. coli strain expressed also the engineered pathway for FAEE. The production media contained 0.2% of Glucose and 2% of xylan added to a xylose composed molecule. The obtained titer was 11.6 mg/L of FAEE [15]. This is a strategy for lowering production costs [15]. To obtain a low cost process that is also replicable at large scale further research needs to be conducted. This research must be addressed to investigate the transformation of biomass to hemicellulose and the production of xylose without the use of expensive enzymes.. ALKANES Alkanes are present in the main transportation fuels; gasoline jet fuel and diesel. They can be derived microbiologically from many organisms. Specifically, they result from the decarboxylation of unsaturated fatty aldehydes. However, the lack of information and understanding of its biochemical ground makes it difficult to engineer a synthetic pathway [5]. In an initial approximation Schirmer A et al. tested 11 cyanobacteria strains. This was done in order to identify which genes from cyanobacteria could be related to the native production of alkanes. The strains were identically grown under photoautotrophically conditions. The results showed that out of 11, only one lacked production of alkanes [5]. Using genetic tools two unreported genes were identified in all the alkane producing cyanobacteria strains. Researchers choose Synechococcus elongatus PCC 7942 genes as the representative sample for further investigation. The assigned names were PCC7942_orfl1593 and PCC7942_orf1594. After a biochemical and genetic analysis of these genes they determined that PCC7942_orf1594was able to reduce fatty-acp to an unsaturated fatty aldehyde [5]. This is the role of an acyl carrier protein in the fatty acid biosynthesis. On the other hand, PCC7942_orf1593 was capable of catalyzing the decarboxylation of fatty aldehyde to form alkanes. This could be compared to the function of a ribonucleotide reductase R2 [5]. 11.
(12) IAMB 20121028. To validate these results, three E. coli strains were constructed and grown in a mineral medium. The first expressed both genes, PCC7942_orfl1593 and PCC7942_orf1594. A second one expressed only PCC7942_orf1594. The last one expressed PCC7942_orfl1593 [5]. E. coli was selected as the host due to its incapability to produce neither alkenes nor fatty aldehydes naturally [5]. Results revealed that the strain expressing only PCC7942_orf1594 acquire the capability of producing fatty aldehydes [5]. This reaffirms that PCC7942_orf1594 has reductive enzyme activity. Still, the alone expression of PCC7942_orf1593 didn’t led to alkene production as expected. Finally, when the E. coli strain expressed both genes, alkanes, fatty alcohols and fatty aldehydes were produced in detectable levels. The production titers of alkenes weren’t as high the titers obtained in E. coli for alcohol production. Nevertheless, they obtained a final production 300mg/l of alkanes [5]. As for hydrocarbons the E. coli strain produce in higher concentrations heptadecanol, with 60 mg/L when both genes were expressed and 90 mg/L when only PCC7942_orf1594 was expressed in the E. coli strain. This experiment validates the selected pathway for alkane’s production in a non-natural host.. CONCLUSIONS Recent studies have shown the feasibility of a large scale production of advanced biofuels using metabolic engineering. Genetically modified strains with the capability to produce potential fuel molecules from renewable sources are being developed. Furthermore, these engineered strains have achieved to produce potential substitutes of petroleum based-fuels in high titers and high yields. The most popular user friendly host until now is Escherichia coli. However, there has been increasing attention to the Cyanobacteria, Synechococcus elongatus, due to its heterotrophic nature. The production of isobutanol and butanol by S.elongatus using. carbon dioxide as carbon source avoids the transformation of biomass to simple sugars. Further experiments that could lead to high titers as those produced by E. coli, are required. Butanol is the most suitable biofuel to replace gasoline. The metabolic engineering of pathways for its production in E. coli strains has been complex. Hitherto, strains expressing the fermentative pathway have achieved higher titers than with the 2-ketoacid pathway for 2ketovalrerate. The main challenge was the understating of the whole balance of the redox reactions in each step. Driving forces to direct the flux to selected product should be implemented in the production of other molecules to obtain higher titers. It is very important that the pathway has an irreversible reaction that could direct the carbon flux to the production of the target molecule. Because of the complexity on chemical a genetic properties of alkanes it has only been proved the feasibility to produce them in E. coli strains. Further investigation should lead to an increase in the specificity of the pathway. Alkanes are the most important candidates to replace Jet Fuel. Thanks to the study case of propanol production through the 2-ketoacid pathway [21], a bottleneck in the pathway was solved through the implementation of a mutant feedback resistant. This highlights the importance of considering in cell signaling pathways in the host. They could be limiting the production of the desired molecule. Some of the most important steps to optimize an engineered pathway is the deletion of competitive pathways. This intends to decrease by-product concentration increasing the availability of the substrates involved in main metabolic pathway. This technique directs the flux to the production of the desired molecule. 12.
(13) IAMB 20121028. When this doesn’t occur it is recommendable to reevaluate the balance of each step of the pathway.. production from Escherichia Coli. Current Opinion in Biotechnology 2008, 19:414-419. Continuous removal of the product is important to improve production and to decrease the cytotoxicity caused by the accumulation of heterologous metabolites. The most representative study related to this was in the production of isopropanol [7]. They achieved a continuous production for 240 h. The final concentration obtained was 143g/l. This is the highest concentration obtained in all the study cases. The product removal technique used was Gas stripping. So far it has been the most selected process because of its efficiency and simple installation.. 3. Bermejo LL, Welker NE, Papoutsakis ET: Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification. Appl Environ Microbiol 1998, 64:1079-1085.. Research to find other homologous genes and isoenzymes is very important. The reviewed studies showed that native genes are not always the most efficient, neither the heterologous genes. Pathway optimization should include always experiments to determinate which is the best combination of genes that generates the highest titer. Optimization of the conditions of growth and production is also an essential step in pathway engineering. For example, pH control increased isopropanol production in 3 fold. It is still important to keep investigating about the transformation of biomass to simple sugars. Without a lower cost process to accomplish this bioconversion the feasibility of introducing these pathways in a higher scale are low.. References 1. Lan EI, Liao JC. Metabolic engineering of cyanobacteria for 1butanol production from carbon dioxide Metab. Eng. 2011, 13:353–363 2. Atsumi S, Liao JC. Metabolic engineering for advanced biofuels. 4. Atsumi S, Hanai T, Liao JC: Non Fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 2008, 451:86-89. 5. Schirmer A, Rude M, Li M, Popova E, B del cardayre. S Microbial Biosynthesis of Alkanes. Science 2010, 329: 559-562 6.. Zhang F, Rodriguez S, Keasling J. Metabolic engineering of microbial pathways for advanced biofuels production. Current Opinion in Biotechnology 2011, 22:775-783.. 7. Inokuma K, Liao J, Okamoto M, Hanai T. Improvement of isopropanol production by metabolically engineered Escherichia coli using gas stripping. Journal of Bioscience and Bioengineering 2010, 110(6): 699-710. 8. Fischer C, Klein-Marcuschamer D, Stephanopoulos G. Selection and optimization of microbial hosts for biofuels production. Metabolic Engineering 2008, 10:295-304. 9. Sung KL, Chou H, Ham T.S, Taek SL, Keasling JD. Metabolic engineering of microorganism for biofuels production: from bugs to synthetic biology to fuels. Current Opinion in Biotechnology 2008, 19: 556-563. 10. Jan Y-S, Park JM, Choi S, Choi YJ, Seung DY, Cho JH, Sang YL. Engineering of microorganisms for 13.
(14) IAMB 20121028. the production of biofuels and perspectives based on systems metabolic engineering approaches. Biotechnology Advances 2011, 8(15). 11. Wackett LP. Biomass to fuels via microbial transformation. Current Opinion in chemical biology 2008 , 12: 187-193 12. Wackett LP. Engineering microbes to produce biofuels. Current Opinion in Biotechnology 2011, 22: 388-393. 13. Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJ, Hanai T, Liao JC. Metabolic engineering of E.coli for 1-butanol production. Metabolic engineering 2008. 10: 305-311. 14. Dürre P. Fermentative production of butanol--the academic perspective. Curr Opin Biotechnol. 2011; 22(3):3316. 15. Eric J. Steen, Yisheng Kang, Gregory Bokinsky, Zhihao Hu, Andreas Schirmer, Amy McClure, Stephen B. del Cardayre & Jay D. Keasling. Microbial production of fatty-acidderived fuels and chemicals from plant biomass. Nature 2010, 463:559562. 16. Mussatto SI , Dragone G, Guimarães P MR, Silva JPA, Carneiro LM, Roberto IC, Vicente A, Domingues L, Teixeira JA. Technological trends, globalmarket, and challenges of bioethanol production Biotechnology Advances Volume 2010, 28:817–830. 17. Huang H, Liu H, Gan YR. Genetic modification of critical enzymes and involved genes in butanol biosynthesis from biomass. Biotechnol Adv. 2010, 28(5):651-657.. 18. Peter H. Pfromm, Vincent AmanorBoadu, Richard Nelson. Sustainability of algae derived biodiesel: A mass balance approach. Bioresource Technology, 2011; 102 (2): 1185-1193. 19. Hanai T, Atsumi. S, Liao J.C. Engineered synthetic pathway for isopropanol production in Escherichia Coli. Applied and environmental Microbiology. 2007; 73(24):7814-7818. 20. Liao JC, Shen C.R. Metabolic engineering of Escherichia coli for 1butanol and 1-propanol production via the keto-acid pathways. Metabolic Engineering 2008; 10: 312-320. 21. Shen C.R, Lan E.I, Dekishima Y, Baez A, Cho Myung K, Liao J.C. Driving Forces Enable High-Titer Anaerobic 1-Butanol synthesis in Escherichia Coli. Applied and Environmental Microbiology 2011; 77(9): 2905-2915. 22. Atsumi. S,Wu. T, Eckl E.M, Hawkins S.D, Buelter T, Liao JC. Engineering the isobutanol biosynthetic pathway in Esherichia coli by comparison of three aldehyde reductas/alcohol dehydrogenase genes. Applied Genetics and Molecular Biotechnology. 2010; 85: 651-657 23. Rodriguez FC, Roldán MD. Biotecnología ambiental. Editorial Tebar, SI. Madrid (2005) 24. Atsumi Shota, Higashide Wendy and Liao JC. Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde Nature Biotechnology. 2009; 27: 1177 – 1180. 25. United States Environmental Protection Agency. Final Report: SecondGeneration Isobutanol Producing Biocatalyst. 2009. Available url: http://cfpub.epa.gov/ncer_abstracts/inde 14.
(15) IAMB 20121028. x.cfm/fuseaction/display.abstractDetail/ abstract/8958/report/F 26. Connor MR and Liao JC. Engineering of an Escherichia coli Strain for the Production of 3-Methyl-1-Butanol. Appl. Environ. Microbiol. 2008; 74(18):5769-5775 27. Tucci S and Martin W. A novel prokaryotic trans-2-enoyl-CoA reductase from the spirochete Treponema denticola. FEBS Letter 58. 28. Mc Graw Hill. Feedback Inhibition of Biochemical Pathways. Available url: http://highered.mcgrawhill.com/olcweb/ cgi/pluginpop.cgi?it=swf::535::535::/sit es/dl/free/0072437316/120070/bio10.s wf::Feedback%20Inhibition%20of%20 Biochemical%20Pathway. 15.
(16) IAMB 20121028. List of annex A. B. C. D. E. F.. Engineered Synthetic Pathway Engineered Synthetic Pathway Engineered Synthetic Pathway Engineered Synthetic Pathway Engineered Synthetic Pathway Engineered Synthetic Pathway. diagram for Isopropanol Production diagram for propanol Production diagram for isobutanol Production diagram for butanol Production diagram for fatty acids Production diagram for alkanes Production. 16.
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