# Biorefinery: The Production of Isobutanol from Biomass Feedstocks
## 2 Research on Isobutanol Production  
The isobutanol synthesis pathway (Figure 1) starts when pyruvate is converted to 2-ketoisovalerate (KIV) by acetolactate synthase (AHAS), acetohydroacyacid reductoisomerase (AHARI), and dihydroxyacid dehydatase (DHAD), respectively. Next, KIV is converted to isobutyraldehyde by the 2-ketoacid decarboxylase (KIVD), and finally alcohol dehydrogenase (ADH) produces isobutanol (we call the pathway consisting of these five enzymes the engineered isobutanol pathway below). Microorganisms that are widely used to biosynthesize isobutanol include _Escherichia coli_, _Corynebacterium glutamicum_, _Bacillus subtilis_, and _Saccharomyces cerevisiae_. Most microorganisms do not produce, or only produce trace amounts of isobutanol on their own. Now several strategies can increase isobutanol production, such as the overexpression of key enzymes of the isobutanol synthesis pathway, the inhibition of byproduct production, cofactor engineering, and microbial robustness enhancement. We selected some representative studies to briefly introduce current microbial isobutanol production strategies. In 2008, [PERSON]'s group [16] validated the potential of microbes to produce higher alcohols using _E. coli_. By introducing KIVD from _Lactococcus lactis_ and ADH from _S. cerevisiae_, the engineered strain JCL260 successfully produced isobutanol using glucose as a substrate. Then increasing the pyruvate and 2-ketoacid concentrations, JCL260 produced 22 g/L of isobutanol, which, at 86% of the theoretical maximum, demonstrated this strategy's potential. This is the first time that researchers have used metabolic engineering strategies to produce isobutanol with microorganisms. The cytotoxicity of isobutanol causes high concentrations of isobutanol to inhibit cell growth, thus limiting the maximum titer. [PERSON]'s group [21] further combined an in situ product removal strategy (gas stripping) with isobutanol production to improve the final titer of isobutanol. The final isobutanol production reached 50 g/L after 72 h of fed-batch fermentation in a bioreactor. They also used the chemical mutagen N'-nitro-N-nitrosoguanidine (NTG) to induced sequential mutagenesis in _E. coli_ and screened for a high-yielding isobutanol strain. Repairing an inhibitory mutation resulted in a final isobutanol titer of 21.2 g/L isobutanol in 99 h [22]. Another factor that limits the isobutanol titer is a cofactor imbalance. [PERSON]'s group [23] removed the dependence of the isobutanol synthesis pathway on NADPH in _E. coli_ and achieved 100% of the theoretical maximum isobutanol production during growth in anaerobic conditions. \begin{table}
\begin{tabular}{c c c c c} \hline  & **Ethanol** & **1-butanol** & **Isobutanol** & **Gasoline** \\ \hline Lower Heating Value (MJ/kg) & 27.0 & 33.1 & 33.3 & 43.5 \\ Flash point (\({}^{\circ}\)C) & 13 & 37 & 28 & \(-\)43 \\ Solubility (20 \({}^{\circ}\)C in water, wt \%) & Miscible & 7.7 & 8.7 & negligible \\ Boiling temperature (\({}^{\circ}\)C) & 78.4 & 117.7 & 108 & 25–215 \\ Vapor toxicity & Toxic & Moderate & Moderate & Moderate \\ \hline \end{tabular}
\end{table} 
Table 1: Major characteristics comparison of several common biofuels with gasoline. _C. glutamicum_ is widely used to produce various amino acids, so some researchers have explored isobutanol production by _C. glutamicum_. In 2010, [PERSON]'s group [24] tried to produce isobutanol with _C. glutamicum_ for the first time. They overexpressed the engineered isobutanol pathway, and on this basis deleted the _PYC_ gene (encoding pyruvate carboxylase) and _LDH_ gene (encoding lactate dehydrogenase). This engineered strain they obtained produced 4.9 g/L of isobutanol when fermented for 96 h. [PERSON]'s group [25] found that the main byproducts of _C. glutamicum_ during isobutanol production are lactic and succinate. By knocking out the byproduct synthesis genes, restoring the redox balance in combination with heterologous expression of transhydrogenase (_pntAB_ from _E.