Due to the increasing concerns about limited fossil resources and environmental problems, there has been much interest in developing biofuels from renewable biomass. Ethanol is currently used as a major biofuel, as it can be easily produced by existing fermentation technology, but it is not the best biofuel due to its low energy density, high vapor pressure, hygroscopy, and incompatibility with current infrastructure. Higher alcohols, including 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol, which possess fuel properties more similar to those of petroleum-based fuel, have attracted particular interest as alternatives to ethanol. Since microorganisms isolated from nature do not allow production of these alcohols at high enough efficiencies, metabolic engineering has been employed to enhance their production. Here, we review recent advances in metabolic engineering of microorganisms for the production of higher alcohols.
In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen.
The fermentation carried out by the solvent-producing bacterium, Clostridium acetobutylicum, is characterized by two distinct phases: acidogenic and solventogenic phases. Understanding the cellular physiological changes occurring during the phase transition in clostridial fermentation is important for the enhanced production of solvents. To identify protein changes upon entry to stationary phase where solvents are typically produced, we herein analyzed the proteomic profiles of the parental wild type C. acetobutylicum strains, ATCC 824, the non-solventogenic strain, M5 that has lost the solventogenic megaplasmid pSOL1, and the synthetic simplified alcohol forming strain, M5 (pIMP1E1AB) expressing plasmid-based CoA-transferase (CtfAB) and aldehyde/alcohol dehydrogenase (AdhE1). A total of 68 protein spots, corresponding to 56 unique proteins, were unambiguously identified as being differentially present after the phase transitions in the three C. acetobutylicum strains. In addition to changes in proteins known to be involved in solventogenesis (AdhE1 and CtfB), we identified significant alterations in enzymes involved in sugar transport and metabolism, fermentative pathway, heat shock proteins, translation, and amino acid biosynthesis upon entry into the stationary phase. Of these, four increased proteins (AdhE1, CAC0233, CtfB and phosphocarrier protein HPr) and six decreased proteins (butyrate kinase, ferredoxin:pyruvate oxidoreductase, phenylalanyl-tRNA synthetase, adenylosuccinate synthase, pyruvate kinase and valyl-tRNA synthetase) showed similar patterns in the two strains capable of butanol formation. Interestingly, significant changes of several proteins by post-translational modifications were observed in the solventogenic phase. The proteomic data from this study will improve our understanding on how cell physiology is affected through protein levels patterns in clostridia.
Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone-butanol-ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol-butanol-ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab-scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot-scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab-scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production.
Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.
Biofuel from renewable biomass is one of the answers to help solve the problems associated with limited fossil resources and climate change. Butanol has superior liquid-fuel characteristics, with similar properties to gasoline, and thus, has the potential to be used as a substitute for gasoline. Clostridia are recognized as a good butanol producers and are employed in the industrial-scale production of solvents. Due to the difficulty of performing genetic manipulations on clostridia, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered the development of engineered clostridia strains with highly efficient and selective butanol-production capabilities. In recent years, the butanol-producing characteristics in clostridia have been further characterized and alternative pathways discovered. More recently, systems-level metabolic engineering approaches were taken to develop superior strains. Herein, we review recent discoveries of metabolic pathways for butanol production and the metabolic engineering strategies being developed.
To improve butanol selectivity, Clostridium acetobutylicum M5(pIMP1E1AB) was constructed by adhE1-ctfAB complementation of C. acetobutylicum M5, a derivative strain of C. acetobutylicum ATCC 824, which does not produce solvents due to the lack of megaplasmid pSOL1. The gene products of adhE1-ctfAB catalyze the formation of acetoacetate and ethanol/butanol with acid re-assimilation in solventogenesis. Effects of the adhE1-ctfAB complementation of M5 were studied by batch fermentations under various pH and glucose concentrations, and by flux balance analysis using a genome-scale metabolic model for this organism. The metabolically engineered M5(pIMP1E1AB) strain was able to produce 154 mM butanol with 9.9 mM acetone at pH 5.5, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.84, which is much higher than that (0.57 at pH 5.0 or 0.61 at pH 5.5) of the wild-type strain ATCC 824. Unlike for C. acetobutylicum ATCC 824, a higher level of acetate accumulation was observed during fermentation of the M5 strain complemented with adhE1 and/or ctfAB. A plausible reason for this phenomenon is that the cellular metabolism was shifted towards acetate production to compensate reduced ATP production during the largely growth-associated butanol formation by the M5(pIMP1E1AB) strain.
Clostridium acetobutylicum is an industrially important organism that produces acetone-butanol-ethanol (ABE). The main objective of this study was to characterize the effects of increased cell density on the production of ABE during the phase transition from acidogenesis to solventogenesis in C. acetobutylicum. The increased ABE productivity of C. acetobutylicum was obtained by increasing the cell density using a newly designed medium (designated C. a cetobutylicum medium 1; CAM1). The maximum OD(600) value of C. acetobutylicum ATCC 824 strain obtained with CAM1 was 19.7, which is 1.8 times higher than that obtained with clostridial growth medium (CGM). The overall ABE productivity obtained in the CAM1-fermetation of the ATCC 824 strain was 0.83 g/L/h, which is 1.5 times higher than that (0.55 g/L/h) obtained with CGM. However, the increased productivity obtained with CAM1 did not result in an increase in the final ABE titer, because phase transition occurred at a high titer of acids.
Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while the adhE1(D485G) gene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD(**)) strain. By reinforcing the direct butanol-forming flux in C. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain with in situ recovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production in C. acetobutylicum described here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer. IMPORTANCE Renewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered development of engineered clostridia having highly efficient and selective butanol production capability. Here we report for the first time the results of systems metabolic engineering studies of two butanol-forming routes and their relative importances in butanol production. Based on these findings, a metabolically engineered Clostridium acetobutylicum strain capable of producing butanol to a high titer with high yield and selectivity could be developed by reinforcing the direct butanol-forming flux.
Global energy crisis and limited supply of petroleum fuels have rekindled the worldwide focus towards development of a sustainable technology for alternative fuel production. Utilization of abundant renewable biomass offers an excellent opportunity for the development of an economical biofuel production process at a scale sufficiently large to have an impact on sustainability and security objectives. Additionally, several environmental benefits have also been linked with the utilization of renewable biomass. Butanol is considered to be superior to ethanol due to its higher energy content and less hygroscopy. This has led to an increased research interest in butanol production from renewable biomass in recent years. In this paper, we review the various aspects of utilizing renewable biomass for clostridial butanol production. Focus is given on various alternative substrates that have been used for butanol production and on fermentation strategies recently reported to improve butanol production.
Platform chemicals composed of 2-6 carbons derived from fossil resources are used as important precursors for making a variety of chemicals and materials, including solvents, fuels, polymers, pharmaceuticals, perfumes, and foods. Due to concerns regarding our environment and the limited nature of fossil resources, however, increasing interest has focused on the development of sustainable technologies for producing these platform chemicals from renewable resources. The techniques and strategies for developing microbial strains for chemicals production have advanced rapidly, and it is becoming feasible to develop microbes for producing additional types of chemicals, including non-natural molecules. In this study, we review the current status of the bio-based production of major C2-C6 platform chemicals, focusing on the microbial production of platform chemicals that have been used for the production of chemical intermediates, building block compounds, and polymers.
Growing concerns over limited fossil resources and associated environmental problems are motivating the development of sustainable processes for the production of chemicals, fuels and materials from renewable resources. Metabolic engineering is a key enabling technology for transforming microorganisms into efficient cell factories for these compounds. Systems metabolic engineering, which incorporates the concepts and techniques of systems biology, synthetic biology and evolutionary engineering at the systems level, offers a conceptual and technological framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal production of desired products. Here we discuss the general strategies of systems metabolic engineering and examples of its application and offer insights as to when and how each of the different strategies should be used. Finally, we highlight the limitations and challenges to be overcome for the systems metabolic engineering of microorganisms at more advanced levels.
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