丙酮丁醇梭菌的基因编辑工具及代谢工程改造

丙酮丁醇梭菌作为极具潜力的新型生物燃料丁醇的生产菌,受到各国研究学者的广泛关注。通过丙酮丁醇梭菌(ABE)发酵生产丁醇,由于生产成本高,限制了其工业化应用。随着基因组学和分子生物学的快速发展,适用于丙酮丁醇的基因编辑工具不断发展并应用于提高菌株的发酵性能。本文对丙酮丁醇梭菌基因编辑工具和代谢工程改造取得的进展进行综述。     

Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation

As a promising alternative biofuel, biobutanol can be produced through acetone/butanol/ethanol (ABE) fermentation. Currently, ABE fermentation is still a small-scale industry due to its low production and high input cost. Moreover, butanol toxicity to the Clostridium fermentation host limits the accumulation of butanol in the fermentation broth. The wild-type Clostridium acetobutylicum D64 can only produce about 13 g butanol/L and tolerates less than 2% (v/v) butanol. To improve the tolerance of C. acetobutylicum D64 for enhancing the production of butanol, nitrogen ion beam implantation was employed and finally five mutants with enhanced butanol tolerance were obtained. Among these, the most butanol tolerant mutant C. acetobutylicum NT642 can tolerate above 3% (v/v) butanol while the wide-type strain can only withstand 2% (v/v). In batch fermentation, the production of butanol and ABE yield of C. acetobutylicum NT642 was 15.4 g/L and 22.3 g/L, respectively, which were both higher than those of its parental strain and the other mutants using corn or cassava as substrate. Enhancing butanol tolerance is a great precondition for obtaining a hyper-yield producer. Nitrogen ion beam implantation could be a promising biotechnology to improve butanol tolerance and production of the host strain C. acetobutylicum.     

Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts

Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone–butanol–ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.     

In situ biobutanol recovery from clostridial fermentations: a critical review

Butanol is a precursor of many industrial chemicals, and a fuel that is more energetic, safer and easier to handle than ethanol. Fermentative biobutanol can be produced using renewable carbon sources such as agro-industrial residues and lignocellulosic biomass. Solventogenic clostridia are known as the most preeminent biobutanol producers. However, until now, solvent production through the fermentative routes is still not economically competitive compared to the petrochemical approaches, because the butanol is toxic to their own producer bacteria, and thus, the production capability is limited by the butanol tolerance of producing cells. In order to relieve butanol toxicity to the cells and improve the butanol production, many recovery strategies (either in situ or downstream of the fermentation) have been attempted by many researchers and varied success has been achieved. In this article, we summarize in situ recovery techniques that have been applied to butanol production through Clostridium fermentation, including liquid–liquid extraction, perstraction, reactive extraction, adsorption, pervaporation, vacuum fermentation, flash fermentation and gas stripping. We offer a prospective and an opinion about the past, present and the future of these techniques, such as the application of advanced membrane technology and use of recent extractants, including polymer solutions and ionic liquids, as well as the application of these techniques to assist the in situ synthesis of butanol derivatives.     

Prospective and development of butanol as an advanced biofuel

Butanol has been acknowledged as an advanced biofuel, but its production through acetone–butanol–ethanol (ABE) fermentation by clostridia is still not economically competitive, due to low butanol yield and titer. In this article, update progress in butanol production is reviewed. Low price and sustainable feedstocks such as lignocellulosic residues and dedicated energy crops are needed for butanol production at large scale to save feedstock cost, but processes are more complicated, compared to those established for ABE fermentation from sugar- and starch-based feedstocks. While rational designs targeting individual genes, enzymes or pathways are effective for improving butanol yield, global and systems strategies are more reasonable for engineering strains with stress tolerance controlled by multigenes. Compared to solvent-producing clostridia, engineering heterologous species such as Escherichia coli and Saccharomyces cerevisiae with butanol pathway might be a solution for eliminating the formation of major byproducts acetone and ethanol so that butanol yield can be improved significantly. Although batch fermentation has been practiced for butanol production in industry, continuous operation is more productive for large scale production of butanol as a biofuel, but a single chemostat bioreactor cannot achieve this goal for the biphasic ABE fermentation, and tanks-in-series systems should be optimized for alternative feedstocks and new strains. Moreover, energy saving is limited for the distillation system, even total solvents in the fermentation broth are increased significantly, since solvents are distilled to ~ 40% by the beer stripper, and more than 95% water is removed with the stillage without phase change, even with conventional distillation systems, needless to say that advanced chemical engineering technologies can distil solvents up to ~ 90% with the beer stripper, and the multistage pressure columns can well balance energy consumption for solvent fraction. Indeed, an increase in butanol titer with ABE fermentation can significantly save energy consumption for medium sterilization and stillage treatment, since concentrated medium can be used, and consequently total mass flow with production systems can be reduced. As for various in situ butanol removal technologies, their energy efficiency, capital investment and contamination risk to the fermentation process need to be evaluated carefully.     

Integrated butanol recovery for an advanced biofuel: current state and prospects

Butanol has recently gained increasing interest due to escalating prices in petroleum fuels and concerns on the energy crisis. However, the butanol production cost with conventional acetone–butanol–ethanol fermentation by Clostridium spp. was higher than that of petrochemical processes due to the low butanol titer, yield, and productivity in bioprocesses. In particular, a low butanol titer usually leads to an extremely high recovery cost. Conventional biobutanol recovery by distillation is an energy-intensive process, which has largely restricted the economic production of biobutanol. This article thus reviews the latest studies on butanol recovery techniques including gas stripping, liquid–liquid extraction, adsorption, and membrane-based techniques, which can be used for in situ recovery of inhibitory products to enhance butanol production. The productivity of the fermentation system is improved efficiently using the in situ recovery technology; however, the recovered butanol titer remains low due to the limitations from each one of these recovery technologies, especially when the feed butanol concentration is lower than 1 % (w/v). Therefore, several innovative multi-stage hybrid processes have been proposed and are discussed in this review. These hybrid processes including two-stage gas stripping and multi-stage pervaporation have high butanol selectivity, considerably higher energy and production efficiency, and should outperform the conventional processes using single separation step or method. The development of these new integrated processes will give a momentum for the sustainable production of industrial biobutanol.     

Mathematical models of ABE fermentation: review and analysis

Among different liquid biofuels that have emerged in the recent past, biobutanol produced via fermentation processes is of special interest due to very similar properties to that of gasoline. For an effective design, scale-up, and optimization of the acetone–butanol–ethanol (ABE) fermentation process, it is necessary to have insight into the micro- and macro-mechanisms of the process. The mathematical models for ABE fermentation are efficient tools for this purpose, which have evolved from simple stoichiometric fermentation equations in the 1980s to the recent sophisticated and elaborate kinetic models based on metabolic pathways. In this article, we have reviewed the literature published in the area of mathematical modeling of the ABE fermentation. We have tried to present an analysis of these models in terms of their potency in describing the overall physiology of the process, design features, mode of operation along with comparison and validation with experimental results. In addition, we have also highlighted important facets of these models such as metabolic pathways, basic kinetics of different metabolites, biomass growth, inhibition modeling and other additional features such as cell retention and immobilized cultures. Our review also covers the mathematical modeling of the downstream processing of ABE fermentation, i.e. recovery and purification of solvents through flash distillation, liquid–liquid extraction, and pervaporation. We believe that this review will be a useful source of information and analysis on mathematical models for ABE fermentation for both the appropriate scientific and engineering communities.     

Problems with the microbial production of butanol

With the incessant fluctuations in oil prices and increasing stress from environmental pollution, renewed attention is being paid to the microbial production of biofuels from renewable sources. As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hygroscopicity. A variety of cheap substrates have been successfully applied in the production of biobutanol, highlighting the commercial potential of biobutanol development. In this review, in order to better understand the process of acetone–butanol–ethanol production, traditional clostridia fermentation is discussed. Sporulation is probably induced by solvent formation, and the molecular mechanism leading to the initiation of sporulation and solventogenesis is also investigated. Different strategies are employed in the metabolic engineering of clostridia that aim to enhancing solvent production, improve selectivity for butanol production, and increase the tolerance of clostridia to solvents. However, it will be hard to make breakthroughs in the metabolic engineering of clostridia for butanol production without gaining a deeper understanding of the genetic background of clostridia and developing more efficient genetic tools for clostridia. Therefore, increasing attention has been paid to the metabolic engineering of E. coli for butanol production. The importation and expression of a non-clostridial butanol-producing pathway in E. coli is probably the most promising strategy for butanol biosynthesis. Due to the lower butanol titers in the fermentation broth, simultaneous fermentation and product removal techniques have been developed to reduce the cost of butanol recovery. Gas stripping is the best technique for butanol recovery found so far.     

Genomic Analysis of Carbon Monoxide Utilization and Butanol Production by Clostridium carboxidivorans Strain P7T

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7^T possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO₂ fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7^T chromosome. The functionality of these pathways was also confirmed by growth of P7^T on CO and production of CO₂ as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7^T was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7^T genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans''s natural capacity to produce potential biofuels from syngas.     

Ex situ product recovery for enhanced butanol production by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In situ butanol recovery fermentation has been intensively studied as an effective alternative to conventional butanol production, which is limited due to the cellular toxicity of butanol. However, the low biocompatibility of adsorbents often leads to failure of in situ recovery fermentations. In this study, Clostridium beijerinckii NCIMB 8052 was cultured in flasks without shaking and in situ recovery fermentation was performed by using an adsorbent L493. The amounts of acetone, butanol, and ethanol (ABE) increased by 34.4 % in the presence of the adsorbent. In contrast, cell growth and production of organic acids and ABE were retarded in the 7-L batch fermentations with in situ butanol recovery. Cell damage occurred in the fermentor upon agitation in the presence of the adsorbent, unlike in static flask cultures with in situ recovery. Ex situ recovery fermentation using circulation of fermentation broth after mid-exponential phase of cell growth was developed to avoid adsorbent-cell incompatibility. No apparent cell damage was observed and 25.7 g/L of ABE was produced from 86.2 g/L glucose in the fed-batch mode using 7 L fermentors. Thus, ex situ recovery fermentation with C. beijerinckii is effective for enhancing butanol fermentation.     

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2–2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD^D485G variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad^D485G) ABE products resulted in a blend with nearly 50 wt%≤C₉ products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9 g L⁻¹ while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80 wt% of catalysis products having a carbon chain length≥C₁₁ which amounts to 9.8 g L⁻¹ of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.     

Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

Background

Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.

Results

KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g?·?L^-1?·?h^-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.

Conclusions

Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.     

Comparative analysis of high butanol tolerance and production in clostridia

2016, was the 100 years anniversary from launching of the first industrial acetone-butanol-ethanol (ABE) microbial production process. Despite this long period and also revival of scientific interest in this fermentative process over the last 20 years, solventogenic clostridia, mainly Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum and Clostridium pasteurianum, still have most of their secrets. One such poorly understood mechanism is butanol tolerance, which seems to be one of the most significant bottlenecks obstructing industrial exploitation of the process because the maximum achievable butanol concentration is only about 21 g/L. This review describes all the known cellular responses elicited by butanol, such as modifications of cell membrane and cell wall, formation of stress proteins, extrusion of butanol by efflux pumps, response of regulatory pathways, and also maps both random and targeted mutations resulting in high butanol production phenotypes. As progress in the field is inseparably associated with emerging methods, enabling a deeper understanding of butanol tolerance and production, progress in these methods, including genome mining, RNA sequencing and constructing of genome scale models are also reviewed. In conclusion, a comparative analysis of both phenomena is presented and a theoretical relationship is described between butanol tolerance/high production and common features including efflux pump formation/activity, stress protein production, membrane modifications and biofilm growth.     

Acetone–butanol–ethanol fermentation of corn stover by Clostridium species: present status and future perspectives

Sustainable vehicle fuel is indispensable in future due to worldwide depletion of fossil fuel reserve, oil price fluctuation and environmental degradation. Microbial production of butanol from renewable biomass could be one of the possible options. Renewable biomass such as corn stover has no food deficiency issues and is also cheaper in most of the agricultural based countries. Thus it can effectively solve the existing issue of substrate cost. In the last 30 years, a few of Clostridium strains have been successfully implemented for biobutanol fermentation. However, the commercial production is hindered due to their poor tolerance to butanol and inhibitors. Metabolic engineering of Clostridia strains is essential to solve above problems and ultimately enhance the solvent production. An effective and efficient pretreatment of raw material as well as optimization of fermentation condition could be another option. Furthermore, biological approaches may be useful to optimize both the host and pathways to maximize butanol production. In this context, this paper reviews the existing Clostridium strains and their ability to produce butanol particularly from corn stover. This study also highlights possible fermentation pathways and biological approaches that may be useful to optimize fermentation pathways. Moreover, challenges and future perspectives are also discussed.     

Acetone production in solventogenic <Emphasis Type="Italic">Clostridium</Emphasis> species: new insights from non-enzymatic decarboxylation of acetoacetate

Development of a butanologenic strain with high selectivity for butanol production is often proposed as a possible route for improving the economics of biobutanol production by solventogenic Clostridium species. The acetoacetate decarboxylase (aadc) gene encoding acetoacetate decarboxylase (AADC), which catalyzes the decarboxylation of acetoacetate into acetone and CO₂, was successfully disrupted by homologous recombination in solventogenic Clostridium beijerinckii NCIMB 8052 to generate an aadc ⁻ mutant. Our fermentation studies revealed that this mutant produces a maximum acetone concentration of 3 g/L (in P2 medium), a value comparable to that produced by wild-type C. beijerinckii 8052. Therefore, we postulated that AADC-catalyzed decarboxylation of acetoacetate is not the sole means for acetone generation. Our subsequent finding that non-enzymatic decarboxylation of acetoacetate in vitro, under conditions similar to in vivo acetone–butanol–ethanol (ABE) fermentation, produces 1.3 to 5.2 g/L acetone between pH 6.5 and 4 helps rationalize why various knock-out and knock-down strategies designed to disrupt aadc in solventogenic Clostridium species did not eliminate acetone production during ABE fermentation. Based on these results, we discuss alternatives to enhance selectivity for butanol production.     

New insights into the butyric acid metabolism of Clostridium acetobutylicum

Biosynthesis of acetone and n-butanol is naturally restricted to the group of solventogenic clostridia with Clostridium acetobutylicum being the model organism for acetone-butanol-ethanol (ABE) fermentation. According to limited genetic tools, only a few rational metabolic engineering approaches were conducted in the past to improve the production of butanol, an advanced biofuel. In this study, a phosphotransbutyrylase-(Ptb) negative mutant, C. acetobutylicum ptb::int(87), was generated using the ClosTron methodology for targeted gene knock-out and resulted in a distinct butyrate-negative phenotype. The major end products of fermentation experiments without pH control were acetate (3.2?g/l), lactate (4.0?g/l), and butanol (3.4?g/l). The product pattern of the ptb mutant was altered to high ethanol (12.1?g/l) and butanol (8.0?g/l) titers in pH?≥?5.0-regulated fermentations. Glucose fed-batch cultivation elevated the ethanol concentration to 32.4?g/l, yielding a more than fourfold increased alcohol to acetone ratio as compared to the wildtype. Although butyrate was never detected in cultures of C. acetobutylicum ptb::int(87), the mutant was still capable to take up butyrate when externally added during the late exponential growth phase. These findings suggest that alternative pathways of butyrate re-assimilation exist in C. acetobutylicum, supposably mediated by acetoacetyl-CoA:acyl-CoA transferase and acetoacetate decarboxylase, as well as reverse reactions of butyrate kinase and Ptb with respect to previous studies.     

Recent progress on industrial fermentative production of acetone–butanol–ethanol by Clostridium acetobutylicum in China

China is one of the few countries, which maintained the fermentative acetone–butanol–ethanol (ABE) production for several decades. Until the end of the last century, the ABE fermentation from grain was operated in a few industrial scale plants. Due to the strong competition from the petrochemical industries, the fermentative ABE production lost its position in the 1990s, when all the solvent fermentation plants in China were closed. Under the current circumstances of concern about energy limitations and environmental pollution, new opportunities have emerged for the traditional ABE fermentation industry since it could again be potentially competitive with chemical synthesis. From 2006, several ABE fermentation plants in China have resumed production. The total solvent (acetone, butanol, and ethanol) production capacity from ten plants reached 210,000 tons, and the total solvent production is expected to be extended to 1,000,000 tons (based on the available data as of Sept. 2008). This article reviews current work in strain development, the continuous fermentation process, solvent recovery, and economic evaluation of ABE process in China. Challenges for an economically competitive ABE process in the future are also discussed.     

非离子表面活性剂对生物丁醇发酵的影响

传统的丙酮-丁醇发酵的产物浓度过低(丁醇终浓度约为1.3 wt%),导致后期分离成本过高,从而影响了该过程的经济性,限制了其工业化进程。本文研究了高添加量的小分子非离子表面活性剂对生物丁醇发酵的影响。以吐温80为例,实验表明,当表面活性剂添加量超过其临界胶束浓度后,丁醇发酵的终浓度会随着表面活性剂添加量的增加而增加。当添加量达到5 wt%时,丁醇终浓度可以达到1.6 wt%,远高于该菌种的抑制浓度(0.8 wt%)。为阐明表面活性剂的作用机理,实验考察了吐温80对丁醇的增溶效应以及对发酵菌体表面亲疏水性的影响。结果表明,吐温80对丁醇的增溶效果很小,而对菌体表面的亲疏水性有较明显的影响。     

Efficient butanol production under aerobic conditions by coculture of Clostridium acetobutylicum and Nesterenkonia sp. strain F

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone–butanol–ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful “coculture” for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.