Metabolic engineering and process development in butanol production with clostridium tyrobutyricum

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Date
2016
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University of Alabama Libraries
Abstract

As a sustainable and environmentally friendly biofuel, biobutanol is a potential substitute for gasoline without any engine modification. The multiple Omics studies were applied to evaluate the change of the expression of host protein and intracellular metabolism in Clostridium tyrobutyricum in response to butanol production. The key enzymes related to carbon balance (i.e. acid and solvent end products and carbohydrates in central pathway), redox balance, energy balance, and cell growth has been studied. It was found that rebalancing both carbon and redox was critical to improve butanol production. These findings were used to achieve high production of biobutanol via integrated metabolic cell-process engineering (MCPE). In a comparative genomics study, the wild type C. tyrobutyricum, the metabolically engineered mutant with down-regulated acetate kinase and evolutionarily engineered strain showing fast cell growth were used to evaluated in butyrate fermentation at pH 6.0 and 37 oC. It was found that the cell growth rate was increased by 61-100% and butyrate productivity was improved by 44-102% by the evolutionarily engineered strain. To understand the mechanism of butyric acid production and cell growth regulation in engineered C. tyrobutyricum mutant, a comparative genomics study was performed. It was concluded that the genome mutations in transcription, translation, amino acid and phosphate transportation and cofactor binding might play important role in regulating cell growth and butyric acid production. Comparative proteomics, which covered 78.1% of open reading frames and 95% of core enzymes, was performed using wild type, mutant producing 37.30 g/L of butyrate and mutant producing 16.68 g/L of butanol. Carbon regulation enzymes in the central metabolic pathway that correlated with butanol production were identified, including thiolase (thl), acetyl-CoA acetyltransferase (ato), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt). The apparent imbalance of energy and redox was also observed due to the downregulation of acids production and the addition of butanol synthesis pathway. The understanding of the mechanism of carbon redistribution enabled the rational design of metabolic cell and process engineering strategies were revealed to achieve high butanol production in C. tyrobutyricum. With the fundamental understanding, the C. tyrobutyricum was metabolically engineered by rebalancing carbon and redox simultaneously. The overexpression of aldehyde/alcohol dehydrogenase (adhE2) and formate dehydrogenase (fdh) improved butanol titer by 2.15 fold in serum bottle and 2.72 fold in bioreactor. In addition, the proteomics study and metabolite analysis showed that more than 90% of the amino acid in the medium was consumed before the cell entered the stationary phase and some enzymes involved in amino acid metabolism had low expression in butanol producing mutant. Extra yeast extract or casamino acids was fed to the free-cell fermentation the mid-log phase, improving the butanol titer to more than 18 g/L compared to 14 g/L without extra nitrogen supplement. The rational metabolic cell-process engineering facilitated with systems biology understanding was demonstrated a powerful approach in butanol production. Finally, the C. tyrobutyricum was further rationally engineered by integrating multiple regualtors, including 1) heterologous NAD+-fdh that provides extra reducing power, 2) the thiolase (thl) that redirects metabolic flux from C2 to C4, and 3) AdhE2. Two novel mutatns, ACKKO-adhE2-fdh and ACKKO-thl-adhE2-fdh, were constructed and produced 18.37 g/L and 19.41 g/L, respectively. This study demonstrated that systems biology-based metabolic cell-process engineering of C. tyrobutyricum enabled a high production of butanol.

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Electronic Thesis or Dissertation
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Chemical engineering
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