Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance

The primary alcohol/aldehyde dehydrogenase (coded by the aad gene) is responsible for butanol formation in Clostridium acetobutylicum. We complemented the non-sporulating, non-solvent-producing C. acetobutylicum M5 strain (which has lost the pSOL1 megaplasmid containing aad and the acetone-formation genes) with aad expressed from the phosphotransbutyrylase promoter and restored butanol production to wild type levels. Because no acetone was produced, no acids (acetate or butyrate) were re-assimilated leading to high butyrate but especially acetate levels. To counter acetate production, we examined thiolase overexpression in order reduce the acetyl-CoA pool and enhance the butyryl-CoA pool. We combined thiolase overexpression with aad overexpression aiming to also enhance butanol formation. While limiting the formation of acetate and ethanol, the butanol titers were not improved. We also generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5 using a modified protocol to increase the antibiotic-resistance gene expression. These strains exhibited greater than 60% reduction in acetate or butyrate formation, respectively. We complemented the AKKO M5 strain with aad overexpression, but could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. These data suggest that loss of the pSOL1 megaplasmid renders cells resistant to changes in the two acid-formation pathways, and especially so for butyrate formation. We argue that the difficulty in generating high butanol producers without acetone and acid production is hindered by the inability to control the electron flow, which appears to be affected by unknown pSOL1 genes.     

限制葡萄糖、葡萄糖/乙酸双底物条件下自由控制丙丁梭菌ABE发酵丙酮浓度和丙酮/丁醇比

提出一种可以提高和自由控制丙丁梭菌ABE发酵丙酮浓度与丙酮/丁醇比的方法。（1）通过控制糖化酶用量、反应时间和温度调节玉米培养基初始葡萄糖浓度,使发酵进入到产溶剂期后,残留葡萄糖浓度降至接近于0 g/L的水平;（2）在葡萄糖受限的条件下,诱导丙丁梭菌合成分泌糖化酶,分解寡糖,将葡萄糖维持于低浓度,进而限制梭菌胞内糖酵解途径的碳代谢和NADH生成速度。与此同时,外添乙酸形成葡萄糖/乙酸双底物环境。在能量代谢基本不受破坏、丁醇未达到抑制浓度的条件下,适度抑制丁醇生产,有效地利用外添乙酸强化丙酮合成;（3）在外添乙酸的基础上,添加适量酿酒酵母,形成丙丁梭菌/酿酒酵母混合发酵体系,提高梭菌对高丁醇浓度的耐受能力。整个发酵体系可以将丙酮浓度和丙酮/丁醇比自由控制在5~12 g/L和0.5~1.0的水平,最大丙酮浓度和丙酮/丁醇比达到11.74 g/L和1.02,并可维持丁醇浓度于10~14 g/L的正常水平,充分满足工业ABE发酵对于丙酮和丁醇产品的不同需求。     

丙酮丁醇梭菌发酵菊芋汁生产丁醇

对丙酮丁醇梭菌Clostridium acetobutylicum L7发酵菊芋汁酸水解液生产丁醇进行了初步研究。实验结果表明,以该水解液为底物生产丁醇,不需要添加氮源和生长因子。当水解液初始糖浓度为48.36 g/L时,其发酵性能与以果糖为碳源的对照组基本相同,发酵终点丁醇浓度为8.67 g/L,丁醇、丙酮和乙醇的比例为0.58∶0.36∶0.06,但与以葡萄糖为碳源的对照组相比,发酵时间明显延长,表明该菌株葡萄糖转运能力强于果糖。当水解液初始糖浓度提高到62.87 g/L时,发酵终点残糖浓度从3.09 g/L增加到3.26 g/L,但丁醇浓度却提高到11.21 g/L,丁醇、丙酮和乙醇的比例相应为0.64∶0.29∶0.05,表明适量糖过剩有助于C.acetobutylicum L7胞内代谢从丙酮合成向丁醇合成途径调节;继续提高水解液初始糖浓度,发酵终点残糖浓度迅速升高,丁醇生产的技术经济指标受到明显影响。     

Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113

Aims: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG‐CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG‐CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l^?1 ethanol and 296 mmol l^?1 acetate and started producing 1·8 mol l^?1 acetone in single‐stage continuous syngas fermentation. Conclusions: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone–butanol–ethanol fermentation of sugars. Significance and Impact of the Study: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl‐CoA to acetone via a non‐naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.     

Physiological Events in Clostridium acetobutylicum during the Shift from Acidogenesis to Solventogenesis in Continuous Culture and Presentation of a Model for Shift Induction

The pH of continuous cultures of Clostridium acetobutylicum growing at pH 5.6 was allowed to decrease to 4.3 after acid production and thereby to shift the cultures from acetate and butyrate to acetone and butanol formation. Several parameters were determined during the shift. An increase in the intracellular acid concentration to 440 mM was recorded. An excess of undissociated butyric acid but not of acetic acid just before the shift to solventogenesis was followed by a decline in acid production and subsequently by the uptake of acids. The intracellular ATP concentration reached a minimum before the onset of solventogenesis; this presumably reflects the ATP-consuming proton extrusion connected with the increase in the ΔpH from 0.7 to 1.4 units. The pool of NADH plus NADPH exhibited a drastic increase until solventogenesis was induced. The changes in the ATP and ADP and NADH plus NADPH pools during these pH shift experiments were the beginning of a stable metabolic oscillation which could also be recorded as an oscillation of the culture redox potential under steady-state solventogenic conditions. Similar changes were observed when the shift was induced by the addition of butyrate and acetate (50 mM each) to the continuous culture. However, when methyl viologen was added, important differences were found: ATP levels did not reach a minimum, acetoacetate decarboxylase activity could not be measured, and butanol but not acetone was produced. A model for the shift is proposed; it assumes the generation of two signals, one by the changed ATP and ADP levels and the other by the increased NAD(P)H level.     

添加有机酸对Clostridium acetobutylicum合成丙酮和丁醇的影响

为提高丙酮-丁醇梭菌厌氧发酵生产丙酮和丁醇的能力,在发酵过程中添加有机酸（乙酸和丁酸）,考察其对菌体生长、溶剂合成影响。实验表明：当添加1.5 g/L乙酸时能够促进菌体的生长,促进丙酮的合成,在600 nm处的最大OD值比参照值高出18.4%,丙酮的最终质量分数提高了21.05%,但不能促进丁醇的合成;当添加1.0g/L丁酸时能够促进菌体生长,促进丁醇的合成,在600 nm处的最大OD比参照值高22.29%,丁醇的最终质量分数比对照组提高了24.32%,但不能促进丙酮的合成。     

Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

ABSTRACT: BACKGROUND: Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum, Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of constitutive thl promoter. RESULTS: The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach led to the complete conversion of acetone into isopropanol, achieving a total alcohol titer of 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. CONCLUSIONS: The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 thus can be considered as a good host for further engineering of solvent/alcohol production.     

Increasing butanol/acetone ratio and solvent productivity in ABE fermentation by consecutively feeding butyrate to weaken metabolic strength of butyrate loop

In this study, we attempted to increase butanol/acetone ratio and total solvent productivity in ABE fermentations with corn- and cassava-based media, by consecutively feeding a small amount of butyrate/acetate during solventogenic phase to weaken the metabolic strengths in butyrate/acetate closed-loops. Consecutively feeding a small amount of butyrate (a total of 3.0 g/L-broth) is most effective in improving performance of corn-based ABE fermentations, as it simultaneously increased average butanol/acetone ratio by 23 % (1.92–2.36) and total solvent productivity by 16 % (0.355–0.410 g/L/h) as compared with those of control. However, the butyrate feeding strategy could not improve butanol/acetone ratio and total solvent productivity in cassava-based ABE fermentations, where the metabolic strength of butyrate closed-loop had already been very low.     

Enhanced butanol production in a microbial electrolysis cell by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> IB4

Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD⁺ levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD⁺, whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production.     

乙、丁酸添加条件下丁醇发酵图论模型的构建

有机酸代谢途径在丁醇发酵过程中具有重要的作用,对细胞内碳流的分配和产物的合成影响显著。在7 L厌氧发酵罐中,进行了间歇添加乙酸或丁酸的发酵实验。结果表明,乙、丁酸的添加显著提高了总溶剂的生产效率,分别提高了47.1%和39.2%;此外,丁醇/丙酮比在添加丁酸的批次中提高了21.7%,在添加乙酸的批次中降低了16.2%;厌氧瓶中的发酵实验也证实了以上结果。有机酸代谢计算的结果表明,乙、丁酸的添加基本上阻断了相应有机酸闭环的吸收途径。基于相关报道和代谢计算结果,构建了针对乙、丁酸添加批次的图论模型,并利用该模型对不同发酵条件下的溶剂浓度和丁醇/丙酮比进行了计算。结果表明,该模型很好地预测了实验结果,合理地构建了乙、丁酸添加批次的信号传递线图。     

木薯和玉米原料丁醇发酵中丁醇丙酮质量比的图论理论计算及其验证

在丁醇发酵产溶剂阶段,乙酸和丁酸的生成途径、消耗途径同时存在,各自形成一个闭环路径。本研究利用图论对丁醇发酵中丁醇丙酮质量比进行了理论计算,并对以木薯和玉米为原料的丁醇发酵进行了模拟计算,结果表明:丁酸闭环路径(L2环)的代谢强度是影响丁醇丙酮质量比的主要因素,并且L2环的代谢强度越弱,丁醇丙酮质量比越高;与玉米原料丁醇发酵相比,木薯原料发酵的m(丁醇)/m(丙酮)提高了16.7%。实验结果证实了以上计算结果:在传统发酵、油醇萃取发酵和生物柴油萃取发酵中,以木薯(适时添加酵母浸粉)为原料的发酵批次与以玉米为原料的发酵批次相比,由于其丁酸闭环路径代谢强度较弱,相应发酵方式下丁醇丙酮质量比分别提高了12.9%、61.4%和6.7%,而且两种原料相应发酵方式的丁醇总产量和生产效率基本持平。另外,高丁醇丙酮质量比的木薯发酵所得改良型生物柴油中丁醇浓度与玉米发酵的相比提高了16%,性能得到进一步提高。     

Development and characterization of butanol — Resistant strain ofClostridium acetobutylicum in molasses medium

The production of acetone—butanol—ethanol solvents from cane molasses by locally isolated culture ofClostridium acetobutylicum was limited by butanol toxicity (1.6 mol/L). The butanol tolerance of the isolated culture was increased up to 4.8 mol/L by a serial enrichment method. The butanol-resistant strain had greater efficiency for the conversion of saccharides to mixed solvents and produced 52% more butanol at the expense of acetone and ethanol than the original strain. Moreover, the fermentation profile of parent and butanol-resistant strains in anerobic fermentation of cane molasses demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, sugar utilization, final butanol concentration and other parameters.     

Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH > or = 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH > or = 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation.     

Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio

A possible way to improve the economic efficacy of acetone–butanol–ethanol fermentation is to increase the butanol ratio by eliminating the production of other by-products, such as acetone. The acetoacetate decarboxylase gene (adc) in the hyperbutanol-producing industrial strain Clostridium acetobutylicum EA 2018 was disrupted using TargeTron technology. The butanol ratio increased from 70% to 80.05%, with acetone production reduced to approximately 0.21 g/L in the adc-disrupted mutant (2018adc). pH control was a critical factor in the improvement of cell growth and solvent production in strain 2018adc. The regulation of electron flow by the addition of methyl viologen altered the carbon flux from acetic acid production to butanol production in strain 2018adc, which resulted in an increased butanol ratio of 82% and a corresponding improvement in the overall yield of butanol from 57% to 70.8%. This study presents a general method of blocking acetone production by Clostridium and demonstrates the industrial potential of strain 2018adc.     

Economical challenges to microbial producers of butanol: feedstock, butanol ratio and titer

Butanol is an important solvent and transport fuel additive, and can be produced by microbial fermentation. Attempts to generate a superior microbial producer of butanol have been made through different metabolic engineering strategies. However, to date, butanol bio-production is still not economically competitive compared to petrochemical-derived production because of its major drawbacks, such as, high cost of the feedstocks, low butanol concentration in the fermentation broth and the co-production of low-value by-products acetone and ethanol. Here we analyze the main bottlenecks in microbial butanol production and summarize relevant advances from recently reported studies. Further needs and directions for developing real industrially applicable strains in butanol production are also discussed.     

Genomic analysis of carbon monoxide utilization and butanol production by Clostridium carboxidivorans strain P7

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 CO(2) 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 CO(2) 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.     

Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Enhancement of Butanol Formation by Clostridium acetobutylicum in the Presence of Decanol-Oleyl Alcohol Mixed Extractants

Extractive fermentation has been proposed to enhance the productivity of fermentations that are end product inhibited. Unfortunately, good extractants for butanol, such as decanol, are toxic to Clostridium acetobutylicum. The use of mixed extractants, namely, mixtures of toxic and nontoxic coextractants, was proposed to circumvent this toxicity. Decanol appeared to inhibit butanol formation by C. acetobutylicum when present in a mixed extractant that also contained oleyl alcohol. However, maintenance of the pH at 4.5 alleviated the inhibition of butanol production and the consumption of butyrate during solventogenesis. A mixed extractant that contained 20% decanol in oleyl alcohol enhanced butanol formation by 72% under pH-controlled conditions. The production of acetone and acetoin was also increased, even though these two products were not extractable. The enhancement of butanol formation was not limited by the toxicity of decanol. Supplementation of glucose and butyrate in the extractive fermentation yielded a 47% increase in butanol. The enhancement of butanol formation appeared to be dependent on the presence of dissolved decanol in the broth but was not observed unless an organic phase was present to extract butanol. A mechanism for the effects of decanol on product formation is proposed.