# Biorefinery: The Production of Isobutanol from Biomass Feedstocks
## 3 Biomass Isobutanol Production
### Isobutanol Production from Protein  
Biorefinery is focused on the industrial production of high-value compounds from biomass. Industrial waste like distillers' grains and okara waste cannot be integrated into the most existing strategies due to their primary component, proteins. For the process of protein hydrolysis to amino acids, some amino acids produce 2-keto acids, which are precursors of isobutanol. [PERSON]'s group [65; 86] has pioneered the use of proteins as feedstock for higher alcohols production. In _E. coli_, they used chemical mutagenesis to screen the _YH19_ strain that can utilize 13 amino acids as a sole carbon source. The restriction of ammonia assimilation, introduction of three exogenous transamines and deaminase cycles, and knocking out population-sensing genes (_luxS_ and _lsrA_) generated the strain YH83 [65]. Strain YH83 expresses the engineered isobutanol pathway that used algal protein hydrolysates to produce higher alcohols. Taking the more severe cofactor imbalance triggered by the use of protein biomass into consideration, [PERSON] group [66] performed cofactor-specific reconstructions of 2,3-dihydroxy isovalerate oxidoreductase (IlvC) and NADPH-dependent isobutanal dehydrogenase (YqhD) in the _YH83_ strain to obtain an isobutanol titer of 0.2 g/L from algae protein hydrolysate. [PERSON] group [67] then attempted to utilize both sugars and proteins from biomass by constructing an _E. coli_ microbial consortium, that is coculturing a proportional mix of strain _AY3_, which can produce heteraolcohols from proteins, and strain _BL2_, which can produce isobutanol from glucose and xylose. Protein utilization increased from 16.3% to 31.3% in a separate culture at a ratio of 1.5:1. The fermentation of distillers' grains hydrolysate under these conditions also resulted in the highest isobutanol titer of 6.5 g/L, which was higher than the 5.5 g/L achieved with strain _BL2_ fermentation alone. The fermentation of other biomass like algal protein hydrolysates was also investigated and the highest isobutanol titer of 2.38 g/L was achieved when strains _AY3_ and _BL2_ were fermented in a 4:1 ratio, demonstrating the industrial potential of this microbial consortium.  
Since _E. coli_ does not naturally produce the proteases required to process protein biomass, further attempts were made in _B. subtilis_, which secretes proteases that enable growth on polypeptides. Due to the different genetic backgrounds of _E. coli_ and _B. subtilis_, a series of experiments were performed to determine the importance of codY (a global regulator) deletion for inhibiting ammonia assimilation and improving branched-chain amino acid synthesis. Combined with a strategy of preventing the degradation of branched-chain amino acid by knocking out the dihydrolipoyl acyltransferase (_bklb_) in the branched-chain 2-keto acid dehydrogenase complex, a final biofuel titer of 0.72 g/L was obtained from protein biomass [86]. Based on this, [PERSON]'s group [68] knocked out _RelA_, expressing the regulatory protein responsible, that recognizes nutritional stress to activate the ilv-leu operon [87] and further promotes branched-chain amino acids synthesis. Comparing the results of spent coffee grounds and okara wastes fermentation, the triple deletion strain expressing KIVD and ADH produced almost no isobutanol with spent coffee grounds hydrolysate as a substrate, while okara wastes hydrolysate fermented about 0.02 g/L of isobutanol in eight days. The microbial consortium strategy was also applied to this engineered strain and isobutanol was successfully obtained from the hydrolysates of watermelon rind and soybean residue by coculturing with _E. coli AY3_[88]. The highest isobutanol titer was 0.88 g/L at an _E. coli_ to _Bacillus subtilis_ ratio of 4:1. From these studies that used proteins to produce isobutanol, it was found that differences in the source of biomass could lead to large changes in product ratios, which require further research.