Application of new metabolic engineering tools for Clostridium acetobutylicum

The renewed interests in clostridial acetone-butanol-ethanol (ABE) fermentation as a next-generation biofuel source led to significantly intensified research in the past few years. This mini-review focuses on the current status of metabolic engineering techniques available for the model organism of ABE fermentation, Clostridium acetobutylicum. A comprehensive survey of various application examples covers two general issues related to both basic and applied research questions: (i) how to improve biofuel production and (ii) what information can be deduced from respective genotype/phenotype manipulations. Recently developed strategies to engineer C. acetobutylicum are summarized including the current portfolio of altered gene expression methodologies, as well as systematic (rational) and explorative (combinatorial) metabolic engineering approaches.     

Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum

Biosynthetic thiolases catalyze the condensation of two molecules acetyl‐CoA to acetoacetyl‐CoA and represent key enzymes for carbon–carbon bond forming metabolic pathways. An important biotechnological example of such a pathway is the clostridial n‐butanol production, comprising various natural constraints that limit titer, yield, and productivity. In this study, the thiolase of Clostridium acetobutylicum, the model organism for solventogenic clostridia, was specifically engineered for reduced sensitivity towards its physiological inhibitor coenzyme A (CoA‐SH). A high‐throughput screening assay in 96‐well microtiter plates was developed employing Escherichia coli as host cells for expression of a mutant thiolase gene library. Screening of this library resulted in the identification of a thiolase derivative with significantly increased activity in the presence of free CoA‐SH. This optimized thiolase comprised three amino acid substitutions (R133G, H156N, G222V) and its gene was expressed in C. acetobutylicum ATCC 824 to assess the effect of reduced CoA‐SH sensitivity on solvent production. In addition to a clearly delayed ethanol and acetone formation, the ethanol and butanol titers were increased by 46% and 18%, respectively, while the final acetone concentrations were similar to the vector control strain. These results demonstrate that thiolase engineering constitutes a suitable methodology applicable to improve clostridial butanol production, but other biosynthetic pathways involving thiolase‐mediated carbon flux limitations might also be subjected to this new metabolic engineering approach. Biotechnol. Bioeng. 2013; 110: 887–897. © 2012 Wiley Periodicals, Inc.     

Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum

Butanol as an advanced biofuel has gained great attention due to its environmental benefits and superior properties compared to ethanol. However, the cost of biobutanol production via conventional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is not economically competitive, which has hampered its industrial application. The strain performance and downstream process greatly impact the economics of biobutanol production. Although various engineered strains with carefully orchestrated metabolic and sporulation-specific pathways have been developed, none of them is ideal for industrial biobutanol production. For further strain improvement, it is necessary to develop advanced genome editing tools and a deep understanding of cellular functioning of genes in metabolic and regulatory pathways. Processes with integrated product recovery can increase fermentation productivity by continuously removing inhibitory products while generating butanol (ABE) in a concentrated solution. In this review, we provide an overview of recent advances in C. acetobutylicum strain engineering and process development focusing on in situ product recovery. With deep understanding of systematic cellular bioinformatics, the exploration of state-of-the-art genome editing tools such as CRISPR-Cas for targeted gene knock-out and knock-in would play a vital role in Clostridium cell engineering for biobutanol production. Developing advanced hybrid separation processes for in situ butanol recovery, which will be discussed with a detailed comparison of advantages and disadvantages of various recovery techniques, is also imperative to the economical development of biobutanol.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation

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.     

Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid

Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.     

Reaction engineering studies of acetone-butanol-ethanol fermentation with Clostridium acetobutylicum

Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum has been extensively studied in recent years because the organism is recognized as an excellent butanol producer. A parallel bioreactor system with 48 stirred-tank bioreactors on a 12 mL scale was evaluated for batch cultivations of the strictly anaerobic, butanol-producing C. acetobutylicum ATCC 824. Continuous gassing with nitrogen gas was applied to control anaerobic conditions. Process performances of ABE batch fermentations on a milliliter scale were identical to the liter-scale stirred-tank reactor if reaction conditions were identical on the different scales (e.g., initial medium, pH, temperature, specific evaporation rates, specific power input by the stirrers). The effects of varying initial ammonia concentrations (0.1-4.4 g L(-1) ) were studied in parallel with respect to glucose consumption and butanol production of C. acetobutylicum ATCC 824 as a first application example. The highest butanol yield of 33% (mol mol(-1) ) was observed at initial ammonia concentrations of 0.5 and 1.1 g L(-1) . This is the first report on the successful application of a 48 parallel stirred-tank bioreactor system for reaction engineering studies of strictly anaerobic microorganisms at the milliliter scale.     

Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production

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.     

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for 13C metabolic flux analysis

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA). Here, cells were grown in parallel cultures with [1-¹³C]glucose and [U-¹³C]glucose as tracers and ¹³C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of ¹³C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for ¹³C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased ¹³C-flux measurements in C. acetobutylicum.     

Nutrient modulation based process engineering strategy for improved butanol production from Clostridium acetobutylicum

The present study demonstrates a process engineering strategy to achieve high butanol titer and productivity from wild type Clostridium acetobutylicum MTCC 11274. In the first step, two different media were optimized with the objectives of maximizing the biomass and butanol productivity, respectively. In the next step, attributes of these two media compositions were integrated to design a two-stage fed-batch process which resulted in maximal butanol productivity of 0.55 g L⁻¹ h⁻¹ with titer of 13.1 g L⁻¹. Further, two-stage fed-batch process along with combinatorial use of magnesium limitation and calcium supplementation resulted in the highest butanol titer and productivity of 16.5 g L⁻¹ and 0.59 g L⁻¹ h⁻¹, respectively. Finally, integration of the process with gas stripping and modulation of feeding duration resulted in a cumulative butanol titer of 54.3 g L⁻¹ and productivity of 0.58 g L⁻¹ h⁻¹. The strategy opens up possibility of developing a viable butanol bioprocess. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2771, 2019.     

Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N -methyl- N '-nitro- N -nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N-methyl-N'-nitro-N-nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

The central metabolic pathway from acetyl-CoA to butyryl-CoA in Clostridium acetobutylicum

Abstract: The pathway from acetyl-CoA to butyryl-CoA serves as a major carbon metabolism channel in Clostridium acetobutylicum and other butyrate-forming clostridia, and the steps are similar to those involved in fatty acid metabolism. Recent findings are discussed, reviewing the isolation and characterization of the enzymes of the pathway, and the analyses of metabolic intermediate levels and possible points of regulation of enzyme activity by CoA compounds. DNA analyses have identified the genes for two thiolase proteins, and an apparent operon encoding five proteins involved in the conversion of acetoacetyl-CoA to butyryl-CoA. These five proteins are β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase and the α and β subunits of an electron transfer flavoprotein.     

Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1,3-propanediol from glycerol

Clostridium butyricum is to our knowledge the best natural 1,3-propanediol producer from glycerol and the only microorganism identified so far to use a coenzyme B12-independent glycerol dehydratase. However, to develop an economical process of 1,3-propanediol production, it would be necessary to improve the strain by a metabolic engineering approach. Unfortunately, no genetic tools are currently available for C. butyricum and all our efforts to develop them have been so far unsuccessful. To obtain a better "vitamin B12-free" biological process, we developed a metabolic engineering strategy with Clostridium acetobutylicum. The 1,3-propanediol pathway from C. butyricum was introduced on a plasmid in several mutants of C. acetobutylicum altered in product formation. The DG1(pSPD5) recombinant strain was the most efficient strain and was further characterized from a physiological and biotechnological point of view. Chemostat cultures of this strain grown on glucose alone produced only acids (acetate, butyrate and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized in chemostat culture, 1,3-propanediol became the major product, the specific rate of acid formation decreased and a very low level of hydrogen was observed. In a fed-batch culture, the DG1(pSPD5) strain was able to produce 1,3-propanediol at a higher concentration (1104 mM) and productivity than the natural producer C. butyricum VPI 3266. Furthermore, this strain was also successfully used for very long term continuous production of 1,3-propanediol at high volumetric productivity (3 g L-1 h-1) and titer (788 mM).     

丙酮丁醇梭菌的遗传操作系统

丙酮丁醇梭菌是极具潜力的替代燃料——生物丁醇的合成菌,受到各国研究者的普遍关注。丙酮丁醇梭菌菌株改造是生物丁醇产业化进程中的一项重要工作,其中遗传操作是核心内容之一。以下对丙酮丁醇梭菌的遗传操作系统的发展历史、种类和原理进行了综述,分析了目前几种遗传操作系统的局限性,并对其发展进行了展望。     

Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity

A typical characteristic of the butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, Clostridium acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain HCBEKW (pta⁻, buk⁻, ctfB⁻ and adhE1⁻) at pH 6.0 resulted in the production of 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/L of glucose. By further knocking out the hydA gene (encoding hydrogenase) in the HCBEKW strain, the butyric acid titer was not further improved in batch fermentation. However, the BA/AA ratio (28.5 g/g) obtained with the HYCBEKW strain (pta⁻, buk⁻, ctfB⁻, adhE1⁻ and hydA⁻) was 1.6 times higher than that (18.2 g/g) obtained with the HCBEKW strain at pH 5.0, while no improvement was observed at pH 6.0. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum.     

Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production

The biosynthesis of the solvents 1-butanol and acetone is restricted to species of the genus Clostridium, a diverse group of Gram-positive, endospore forming anaerobes comprising toxin-producing strains as well as terrestrial non-pathogenic species of biotechnological impact. Among solventogenic clostridia, Clostridium acetobutylicum represents the model organism and general but yet important genetic tools were established only recently to investigate and understand the complex life cycle-accompanied physiology and its regulatory mechanisms. Since clostridial butanol production regained much interest in the past few years, different metabolic engineering approaches were conducted--although promising and in part successful strategies were employed, the major breakthrough to generate an optimum phenotype with superior butanol titer, yield and productivity still remains to be expected.