Engineering Clostridium saccharoperbutylacetonicum for Enhanced Isopropanol-Butanolethanol (IBE) Production from Lignocellulosic Biomass through Acetic Acid Pretreatment
Type of DegreePhD Dissertation
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The objective of this study was to achieve efficient biofuel production from lignocellulosic biomass through the development of a novel biomass pretreatment method in combination with the metabolic engineering of microbial strains for efficient conversion of biomass feedstock. First, an innovative biomass pretreatment method was developed using acetic acid (AA) as the treatment reagent considering its various advantages compared to the conventional dilute acid pretreatment method and the benefit of AA for biobutanol production; then, the hyper-butanol producing strain Clostridium saccharoperbutylacetonicum N1-4 was engineered for enhanced acid re-assimilation and acetone-butanol-ethanol (ABE) production from the acetic-acid-pretreated biomass; further, C. saccharoperbutylacetonicum was engineered for enhanced production of isopropanol-butanol-ethanol (IBE; which can be used directly as a fuel source rather than ABE) from the acetic-acid-pretreated biomass. For the biofuel production from lignocellulosic biomass, most biomass pretreatment processes need to use some chemical reagent as the catalyst to overcome the biomass recalcitrance barrier. Such reagents are usually severe inhibitors for the subsequence fermentation process. Therefore, in many cases, the liquid prehydrolysates fraction (LPF) after the pretreatment is discarded, which is a tremendous wasting of materials and leads to additional pollution. Biobutanol produced from ABE fermentation process has been of great interests recently due to its high value as a biofuel or biochemical. During the ABE fermentation, AA is produced and then re-assimilated as a carbon source. Thus, AA is a substrate rather than an inhibitor for biobutanol production. In this study, we employed AA as the chemical catalyst for the pretreatment of switchgrass which then be used for ABE production through simultaneous saccharification and fermentation (SSF) with hyper-butanol producing C. saccharoperbutylacetonicum N1-4. Through systematic investigation of pretreatment conditions and fermentation, we concluded that the pretreatment with 3 g/L AA at 170 oC for 20 min is the optimal conditions for switchgrass pretreatment leading to efficient biobutanol production. Both LPF and solid cellulosic fraction (SCF) of the pretreatment biomass are highly fermentable. In the fermentation with the LPF/SCF mixture, 8.6 g/L butanol (corresponding to a yield of 0.16 g/g) was obtained. Overall, here we demonstrated an innovative biomass pretreatment strategy for efficient carbon source utilization and biobutanol production. ABE fermentation generally has two phases: in the acidogenesis phase, fatty acids (acetic acid and butyric acid) are accumulated, while in the solventogenic phase, fatty acids are re-assimilated and converted into solvents. Therefore, the improvement of acid re-assimilation capability in the Clostridium host can possibly enhance the solvent production. In addition, acetic acid is often a significant component in the biomass prehydrolysates after pretreatment (especially when acid-based biomass pretreatment approach is employed). Thus, the enhancement of acid re-assimilation in Clostridium has practical significance for biofuel production from lignocellulosic biomass. Here, we overexpressed key genes of the ABE fermentation pathways in C. saccharoperbutylacetonicum to enhance the acid re-assimilation and solvent production. First, the native sol operon (ald-ctfA-ctfB-bcd) was overexpressed under the strong constitutive thiolase promoter (Pthl), generating PW2 strain. Fermentation results demonstrated that the acid re-assimilation was improved in the host strain and ABE production has been increased to 31.4 g/L (vs. 26.4 g/L in JZ100 strain as the control). Although the ethanol production has been increased by six times, the butanol production has not been significantly increased in the engineered strain. In order to further drive the carbon flux from C2 metabolites to C4 metabolites and ultimate butanol production, the key genes including hbd, thl, crt and bcd (expression cassette, or EC) in the butanol production pathway was further overexpressed under Pthl besides the sol operon overexpression as in PW2, generating PW3 strain. Compared to the control, the butanol and acetone production in PW3 was increased by 8% and 18% respectively. The final total solvent production increased by 12.4% than the control, but was 10% lower than PW2 (mainly because of the dramatic increase of ethanol production in PW2). In PW3, both sol operon and EC were overexpressed with Pthl, which could lead to competition for the same RNA polymerase for the expression of multiple genes. To avoid this issue and further improve ABE production, a new strain PW4 was constructed to express sol operon with Pthl but EC with ferredoxin gene promoter (Pfdx). The fermentation results demonstrated that, however, the production of all the solvents in PW4 was actually slightly lower than those in PW3. Moreover, we evaluated the effect of acetic acid concentrations on the solvent production in the engineered strains, and the maximum level of solvent production was achieved when 4.6 g/L acetate was supplemented. Therefore, SSF was carried out with PW2 and PW3 using switchgrass biomass pretreated with 3 g/L acetic acid (which ends up with approximately 4.6 g/L in the fermentation medium). 15.4 g/L total ABE (with a yield of 0.31 g/g) was produced in both PW2 and PW3, which was significantly higher than that in JZ100. This study demonstrated that the overexpression of key genes for acid re-assimilation and solvent production can significantly enhance ABE production in solventogenic clostridia. Acetone is highly corrosive to engine parts, and thus cannot be used as a fuel source. For this reason, the acetone produced during ABE fermentation is often considered as an undesirable byproduct. Biologically, acetone can be converted into isopropanol by the secondary alcohol dehydrogenase. Isopropanol, and thus the isopropanol-butanol-ethanol (IBE) mixture, can be used a valuable biofuel. In this study, we attempt to metabolically engineer the hyper-ABE producing C. saccharoperbutylacetonicum N1-4 strain for IBE production. First, we overexpressed the secondary alcohol dehydrogenase (sadh) gene from C. beijerinckii B593 in C. saccharoperbutylacetonicum on a plasmid, generating PW5 strain. A hydG gene (encoding a putative electron transfer protein) is right downstream of sadh within the same operon in the C. beijerinckii B593 genome. Therefore, additionally, we overexpressed sadh-hydG gene cluster together in C. saccharoperbutylacetonicum to evaluate the effect of hydG for isopropanol production, generating PW6 strain. Fermentation results indicated that in both PW5 and PW6, high levels of isopropanol were produced with no acetone production was detected. Comparatively, PW6 produced slightly higher isopropanol (10.2 g/L vs. 9.4 g/L in PW5) and total IBE. However, overall the performance of PW6 for solvent production is very similar to that of PW5. To eliminate the issue with plasmid-based overexpression such as instability and the requirement of antibiotics for cell cultivation and fermentation, we further integrated sadh or sadh-hydG into the chromosome of C. saccharoperbutylacetonicum, and generated strains PW8 and PW9. In PW8, there was 4.8 g/L acetone and 4.0 g/L isopropanol produced, while in PW9, up to 9.5 g/L isopropanol was produced with only 0.4 g/L acetone was detected. This indicated that the co-overexpression of hydG with sadh through chromosomal integration had significant positive effects on the conversion of acetone to isopropanol. In order to further enhance the solvent production, we additionally overexpressed in PW9 the sol operon (ald-ctfA-ctfB-adc), the expression cassette EC (thl-hbd-crt-bcd), or sol in combination with EC, generating strains PW10, PW11, and PW12, respectively. The fermentation characterization indicated that PW10 had significantly elevated ethanol production, as well as 25% higher isopropanol with slightly decreased butanol production, leading to a significant increase in total solvent titer (34.2 g/L vs. 27.6 g/L in PW9) and yield (0.48 g/g vs. 0.40 g/g in PW9). In PW11, the butanol production increased to 17.9 g/L while ethanol production decreased to 0.4 g/L; however, the isopropanol and final total solvent production was very similar to that in PW9. In PW12, with the co-overexpression of sol operon and EC, the production of isopropanol, butanol, and ethanol increased to 11.7 g/L, 17.3 g/L, and 1.1 g/L respectively comparing to PW9, resulting in a slight increase in total solvent yield. Finally, SSF was carried out with PW9 and PW10 using the acetic-acid-pretreated switchgrass as the feedstock, and the final solvent titer reached 13.7 g/L and 16.2 g/L, corresponding to the solvent yield of 0.27 g/L and 0.32 g/g in PW9 and PW10, respectively. The engineered strains in this study (PW9, PW10, PW11) produced the highest total IBE that has ever been reported in the batch fermentation with solventogenic clostridia. Our results indicated that the acetic-acid-pretreated biomass can be efficiently converted into biofuel using the metabolically engineered Clostridium hosts. Overall, this study demonstrated an innovative approach for biofuel production by combining a tailored biomass pretreatment method and metabolic engineering of microbial workhorse for enhanced conversion of lignocellulosic carbon source for biofuel production.