The alcohol consumption and tolerance of the ascomycete Neurospora crassa was investigated in this study. This fungus is able to utilize both native alcohol and non-native alcohols as carbon sources, yet little is known about the enzymes involved in these processes. The deletion of alcohol dehydrogenase 1 gene (adh-1) from the genome can efficiently prohibit both ethanol and isobutanol metabolism, while the deletion of the alcohol dehydrogenase 3 gene (adh-3) does not have an observable effect on the prevention of alcohol consumption. Both wild type N. crassa and the N. crassa Δadh-1 strain can tolerate up to 48 g/L ethanol and 8.5 g/L isobutanol when grown on glucose or Avicel.
Keywords: Ethanol consumption; Isobutanol consumption; Alcohol tolerance; Neurospora crassa
The use of biofuels produced from renewable lignocellulosic biomass has been proposed as a potential solution to worldwide challenges related to a rapidly diminishing fossil fuel supply and global climate change. Although lignocellulosic biomass is available in large abundance and at low cost, the emergence of cellulosic biorefineries is hindered by the lack of low cost processing technologies [1,2]. The canonical conversion process for biofuels production from lignocellulosic biomass consists of five steps: pretreatment, cellulase production, hydrolysis, fermentation, and product recovery. Consolidated bioprocessing (CBP), the conversion of lignocellulosic biomass into products (such as ethanol) in one step without added enzymes, is a transformative technology, offering potentially breakthrough solutions to these current high cost methods. The CBP enabling microorganism can be constructed via either a native or recombinant strategy [3,4]. The native strategy of CBP microbial construction begins with a naturally efficient cellulase producer (category I CBP microorganism) and entails engineering the ability to produce designated biofuels or chemicals effectively within the organism. The recombinant strategy starts with a powerful biofuels producer (category II CBP microorganism), and consists of conveying the ability to utilize cellulose via cellulase production.
Cellulolytic bacteria and fungi are the main candidates for category I CBP microorganisms . Compared to cellulolytic bacteria, cellulolytic fungi can naturally produce cellulases in the quantity and with the quality suitable for complete saccharification of pretreated lignocellulose, as well as have better tolerance toward inhibitors from lignocellulosic biomass pretreatment. They have great potential to be used as CBP enabling microorganisms either alone or in cooperation with other microorganisms, and they have been used previously to produce alcoholic biofuels [6,7]. N. crassa is amongst the most attractive candidates for CBP application either as the sole producer or a member in a microbial consortium for producing native or non-native alcoholic biofuels directly from cellulosic biomass [5,8].
N. crassa is a model filamentous fungus, with its genetics, biochemistry, and biology having been extensively studied for more than 70 years . It is an efficient plant cell wall degrader, because of its wide array of cellulases and hemi-cellulases . N. crassa produces ethanol as a fermentation product and has two major predicted alcohol dehydrogenase genes (adh-1 and adh-3) present in the genome . The ADH1 and ADH3 peptide sequences showed 65% identity and 79% similarity . Both have >50% identity to S. cerevisiae ADH-1 and ADH-2, which are responsible for ethanol degradation and synthesis, respectively (Xie et al., 2004). Xie et al. suggested that N. crassa ADH-1 would be responsible for ethanol consumption, and ADH-3 would be responsible for ethanol production due to their homology to the S. cerevisiae alcohol dehydrogenases and their regulatory response to glucose. Subsequently, N. crassa ADH-1 and ADH-3 were hetero-expressed in E. coli and the recombinant ADH-1 gave a specific activity of 283.8 ± 8.9 mU/mg toward ethanol as a substrate, while ADH-3 only gave a specific activity of 4.1 ± 0.1 mU/mg. The 70-fold higher specific activity toward using ethanol provides in vitro evidence that ADH-1 in N. crassa may be responsible for ethanol consumptio. However, in vivo evidence for the function of these enzymes has not been previously explored .
Alcohol consumption and tolerance levels are important issues that need to be addressed if N. crassa is used as a CBP host for alcohol production. Herein, we studied the consumption of a native alcohol (ethanol) and a non-native alcohol (isobutanol) using different ADH knock out strains of N. crassa . Thus, we provide the first in vivo evidence for the roles of ADH-1 and ADH-3 in N. crassa . The alcohol tolerance of the N. crassa strains are also investigated.
Wild type N. crassa FGSC 2489, N. crassa Δadh-1 (ΔNCU01754, FGSC 12935), N. crassa Δadh-3 (ΔNCU02476, FGSC 12920) were obtained from the Fungal Genetics Stock Center. Strain N. crassa Δadh-1Δadh-3 was constructed through genetic crossing following a standard mating protocol [13,14]. The single knockout strains and resulting double knockout strains were verified by PCR genotyping. The three engineered N. crassa strains have similar growth rate to the wild type strain in the Vogel’s media contained sucrose.
N. crassa strains’ growth conditions for alcohol consumption
N. crassa strains were grown in 250 mL Erlenmeyer flasks on 1x Vogel’s solid medium supplemented with 15 g /L sucrose at 30 °C with constant light for 3 days . They were then moved to the bench in room temperature for an additional 7 to 10 days. Conidia were subsequently harvested and re-suspended in sterile water (20 mL). Fermentation experiments were conducted in 250 mL Erlenmeyer flasks with a 50 mL working volume, containing 1x Vogel’s salts medium and 0.5 g /L glucose to initiate growth. Ethanol or isobutanol was added to a final concentration of 20 g/L or 5 g/L respectively. 50 mL water in 250 mL Erlenmeyer flasks contained same amount of ethanol or isobutanol without any conidia inoculated were used as control. Flasks were incubated at 28 °C in a rotary shaker at 200 rpm in constant light. Samples were taken out after 5 days for measurement of the alcohol concentrations via High Performance Liquid Chromatography (HPLC).
Alcohol tolerance of N. crassa with glucose and Avicel as carbon sources
1x Vogel’s media containing 40 g/L glucose or 20 g/L Avicel were inoculated with conidial suspensions of N. crassa FGSC 2489 and N. crassa Δadh-1 to a final OD420 of 0.1. They were then were cultured at 28 ºC for 12 h. Varying amounts of ethanol or isobutanol were added at 12 h. Samples were taken out at 0, 12, 18, 24, 36, 48, and 60 h time points to analyze the concentrations of glucose and alcohol. A duplicate experiment was conducted identically to the above experiment, in which cultures were harvested for mycelial biomass quantification.
Quantification of mycelial biomass
The mycelial biomass of the fungus grown in glucose medium was harvested by filtration through filter paper, washed with 50 mL distilled water, then dried at 105°C overnight and quantified by weighing the dried residues. The dry weight of the mycelial biomass of the fungus grown in the Avicel medium was measured by extracting ergosterol from the mycelia and measuring the amount by HPLC [16,17].
Concentrations of glucose, ethanol, and isobutanol were analyzed using a Shimadzu LC-20AD HPLC equipped with a refraction index detector and a Transgenomic ICSep ION-300 column (Transgenomic, San Jose, CA, USA) at 80°C. 5 mM sulfuric acid at a flow rate of 0.6 mL/min was used as the mobile phase.
Deletion of ADH-1 can efficiently prevent ethanol and isobutanol utilization
N. crassa FGSC 2489, N. crassa Δadh-1, N. crassa Δadh-3, and N. crassa Δadh-1Δadh-3 strains were used to characterize the consumption of ethanol and isobutanol as the carbon source in flasks. As shown in Figure 1, the wild type N. crassa FGSC 2489 strain consumed about 12.7 g/L ethanol or 3.2 g/L isobutanol in 5 days. The strain N. crassa Δadh-3 consumed a similar amount of alcohols compared to the wild type stain. However, the ethanol and isobutanol concentrations in the flasks containing N. crassa Δadh-1 or N. crassa Δadh-1Δadh-3 only decreased approximately 4.0 g/L and 1.8 g/L, respectively.
These consumptions show no statistical difference compared to control flasks without any inoculation where ethanol and isobutanol are lost solely through evaporation. The results provide in vivo evidence that ADH-1 is responsible for the alcohol consumption in N. crassa FGSC 2489 and N. crassa Δadh-3, and the deletion of adh-1 can partially eliminate this metabolic pathway. Contrarily, ADH-3 appears to have little contribution to the alcohol consumption. Since the adh-3 deletion in N. crassa did not affect alcohol consumption, the Δadh-1 strain was used for further study.
Alcohol tolerance of wild type and Δadh-1 deficient N. crassa strains on glucose as a carbon source
The ethanol and isobutanol tolerance of N. crassa FGSC 2489 and strain Δadh-1 were analyzed (Figure 2). As shown in Figures 2a and 2c, the biomass accumulation and rate of glucose utilization decreased with increasing concentration of alcohol in the culture for both strains. When 32 g/L ethanol was added at 12 h, biomass produced at 60 h was about 55% of that of without any ethanol addition, and the glucose utilization rate was about 62% of the control. When the added ethanol reached 56 g/L, the growth and glucose utilization for both strains ceased as there was no obvious increase of biomass or decrease of glucose concentration since the time of alcohol addition (Figure 2c). Our results indicate that N. crassa FGSC 2489 and N. crassa Δadh-1 can tolerate up to 48 g/L ethanol in the medium, respectively. The growth phenotypes of N. crassa Δadh-1 with different concentrations of alcohols are similar to those of N. crassa FGSC 2489.
Results of isobutanol tolerance of N. crassa FGSC 2489 and N. crassa Δadh-1 are shown in Figures 2b and 2d, respectively, which demonstrated similar isobutanol tolerance between the strains. Comparing a culture containing 6.5 g/L isobutanol to the cultures containing no added isobutanol the biomass and glucose utilization rate of N. crassa FGSC 2489 and N. crassa Δadh-1 decreased 56% and 63%, respectively. When 8.5 g/L isobutanol was added, approximately 10 g/L glucose was consumed after the addition of isobutanol at 12 h, and 100 mg of additional mycelial biomass was produced. However, there was neither detectable glucose utilization nor biomass production when 9.0 g/L isobutanol was added. These results indicate that the isobutanol g/L tolerance of both N. crassa FGSC 2489 and N. crassa Δadh-1 is about 8.5 g/L when grown on glucose.
Alcohol tolerance of wild type and N. crassa Δadh-1 strains on Avicel as a carbon source
As we would like to use N. crassa as a candidate of CBP host, we are also interested in investigating the alcohol tolerance of N. crassa Δadh-1 when Avicel, a commonly used model cellulose product representing cellulosic biomass, is used as the carbon source. As shown in Figure 3, growth was readily detected when 50 g/L ethanol was added, or 8.5 g/L isobutanol was added. However, the fungal growth diminished when the ethanol or isobutanol were added at higher concentrations. Therefore, the ethanol and isobutanol tolerance of N. crassa Δadh-1 when grown on Avicel were 50 g/L and 8.5 g/L, respectively, comparable to the data acquired when strains are grown on glucose media (Figures 2 & 3).
The two major alcohol dehydrogenases (ADH-1 and ADH-3) of N. crassa FGSC 2489 were previously hetero-expressed in E. coli. In this study it was shown that ADH-1 had much higher in vitro specific activity toward ethanol conversion than ADH-3, indicating ADH-1 in N. crassa may be responsible for ethanol consumption. Our results provide in vivo experimental verification that ADH-1 in N. crassa is the primary alcohol dehydrogenase responsible for alcohol metabolism (>99%). The presence of ADH-1 enables N. crassa to use a native alcohol (ethanol), and a non-native alcohol (isobutanol) as the carbon source. The deletion of adh-1 in the genome thus leads to the elimination of >99% of the total alcohol consumption, while the deletion of adh-3 has no effect on alcohol utilization. The deletion of adh-1 does not lead to an obvious growth phenotype in N. crassa (data not shown).
High concentrations of alcohols inhibit microbial growth by inactivating the cytosolic enzymes and damaging cell membranes . This intolerance to high concentrations of alcohol in microorganisms limits the final titer and the productivity in fermentation, both of which have significant impacts on the economics of a bio-refinery system. Most current designs for production methods of ethanol from lignocellulosic substrates featuring the enzymatic hydrolysis step aim for ethanol concentrations of at least 50 g/L due to the constraints associated with slurry handling . Both N. crassa FGSC 2489 and N. crassa Δadh-1 can tolerate ethanol 48 g/L. ADH-1 is the enzyme responsible for alcohol consumption, but it did not seem to affect the ethanol tolerance of N. crassa FGSC 2489. Although the ethanol tolerance level of N. crassa is far inferior to that of Saccharomyces cerevisiae and Zymomonas mobilis (Category II CBP microorganisms, tolerate 150-200 g/L ethanol), it is higher than those of naturally occurring cellulolytic bacteria (e.g. Clostridium thermocellum tolerates 10 ethanol g/L), and in the range of what could be achieved from the adapted or engineered cellulolytic bacteria . The ethanol tolerance level is suitable for industrial ethanol production from cellulosic biomass.
Isobutanol is a non-native alcohol, and it was able to be produced by only a handful of recombinant hosts in recent studies . The responses to isobutanol and ethanol vary depending on the microorganism. Z. mobilis, for example grows at about 60% of its maximal growth rate when the strain was grown on 12 g/L isobutanol [20,21]. In contrast, S. cerevisiae , which has high tolerance to ethanol, was not be able to grow when 8 isobutanol g/L was added. Thermoanaerobacterium saccharolyticum could not grow when ethanol concentration was as high as 20 g/L, while it maintained about half the growth rate when isobutanol concentration was as high as 12 g/L. Both N. crassa Δadh-1 and N. crassa FGSC 2489 can tolerate up to 8.5 g/L isobutanol, which is in a similar range as naturally occurring E. coli which has a tolerance level of 8 g/L and S. cerevisiae , as mentioned above [22-24].
N. crassa is able to metabolize both a native alcohol, ethanol, and a non-native alcohol, isobutanol, as a carbon source under aerobic conditions. Deletion of the adh-1 gene in N. crassa can efficiently prevent alcohol consumption under aerobic conditions. N. crassa Δadh-1 can tolerate up to 48 g /L ethanol and 8.5 g /L isobutanol when grown on glucose or Avicel as the carbon source.
This project was supported by Agriculture and Food Research Initiative Competitive Grant No. 2011-67009-20060 from the USDA National Institute of Food and Agriculture. The authors thank Eric Walters and Amanda Hildebrand for reading through the paper, and Edyta Szewczyk for her technical support.