# Biorefinery: The Production of Isobutanol from Biomass Feedstocks
## 2 Research on Isobutanol Production
coli_), and overexpression _adhA_, the final isobutanol titer reached 13 g/L at 48 h. Recently, [PERSON]'s group [26] has used different promoter combinations to confirm the importance of higher activity of AHAS and KDC for isobutanol synthesis in _C. glutamicum_. In combination with a cofactor strategy (altering the cofactor specificity of AHARI and ADH) and enhanced glycolytic flux strategy (overexpression of endogenous glycolytic genes and the phosphoenolpyruvate:carbohydrate phosphotransferase system  
Figure 1: Schematic diagram of microorganism isobutanol synthesis pathways. Part (**A**) shows the general pathways of isobutanol biosynthesis; Part (**B**) shows a partial cofactor balancing strategy for successful optimization of isobutanol synthesis; Part (**C**) shows the common by-product synthesis pathways of isobutanol synthesis; and Part (**D**) shows the sugar metabolism pathway using non-glucose fermentation to produce isobutanol. The dashed line indicates the source of the precursor substance for the byproduct; AHAS: Acetolactate synthase; AHARI: Acetohydroyacid reductoisomerase; DHAD: Dihydroxyacid dehydratase; KIVD: Keto acid decarboxylase; ADH: Alcohol dehydrogenase; G6 PD: Glucose-6-phosphate dehydrogenase; 6 PGD: 6-phosphogluconate dehydrogenase; XR: Xylose reductase; XDH: Xylitol dehydrogenase; XI: Xylose isomerase; XKS: Xylulokinase; AI: L-arabinose isomerase; RK: L-Ribulokinase; RSPE: L-Ribulose-5-P-4-epimerase. (PTS), plus introduction of the Entner-Doudoroff pathway from _Zymomonas mobilis_), 20.8 g/L isobutanol was produced at 24 h to reach 84% of the theoretical value. [PERSON]'s group [27] proved that _B. subtilis_ could be used as a cell factory for isobutanol synthesis through an isobutanol tolerance test. In 2011, they introduced the engineered isobutanol pathway into _B. subtilis_, resulting in strain BSUL03. The concentration of the isobutanol reached 2.63 g/L at 54 h. Next, they performed an elementary mode analysis (EMA) on the engineered strain BSUL03 to identify targets in the metabolic network that could be optimized, lactate dehydrogenase and pyruvate dehydrogenase complexes. By knocking out the _ldh_ and _pdhC_ genes, they engineered the strain BSUL05 and obtained an isobutanol titer of 5.5 g/L after 60 h of fermentation [28]. Afterwards, they performed a metabolic flux analysis and comparison of the two engineered strains. To increase the concentration of NADPH and achieve a redox balance, the engineered strain BSUL08 was obtained by knocking out _pgi_ (encoding glucose 6-phosphate isomerase), overexpressing _zwf_ (encoding glucose 6-phosphate dehydrogenase), and further overexpression the transhydrogenase gene (_udhA_ from _E. coli_). The production performance was tested with fed-batch fermentation and the isobutanol titer reached 6.12 g/L at 60 h, which was 63% of the theoretical maximum [29]. _S. cerevisiae_ can produce a small amount of higher alcohols through the biosynthetic pathway of various amino acids. It also has a natural tolerance to alcohols that is higher than other microorganisms [30]. In recent years, _S. cerevisiae_ has been widely used to produce higher alcohols. In 2011, [PERSON]'s group [31] overexpressed of the genes _ILV2_ (encoding acetlactate synthase), _ILV3_ (encoding dihydroxy acid dehydatase), and _ILV5_ (encoding diacetolate reductase) to obtain an isobutanol yield of 3.86 mg/g glucose. This is the first record of _S. cerevisiae_ being used to produce isobutanol. Based on that, [PERSON]'s group [32] truncated the N-terminal mitochondrial targeting sequence of the _ILV_s to achieve expression of _ILV2_, _ILV3_, and _ILV5_ in the cytoplasm, and optimized the codons of these three genes. They studied the activities of KIVD and ADH and determined that _ARO10_ and _ADH2_ were the most active enzymes in the synthesis of isobutanol. The final concentration of isobutanol produced by the strain they obtained was 0.63 g/L.