# Biorefinery: The Production of Isobutanol from Biomass Feedstocks
## 4 Conclusions  
In recent years, researchers have made considerable effort to increase the yield of microbial biofuel production and continued to explore chemical synthesis from different biomass materials [13]. In addition, some achievements have been made in the production of bioethanol [14; 89]. In this paper we summarized current metabolic engineering strategies applied to biomass isobutanol production and recent advances in the production of isobutanol from different biomass feedstocks.  
It can be seen that in the last decade, there was a significant improvement in the production of cellulosic isobutanol while the synthesis of isobutanol from protein biomass was demonstrated. Biomass isobutanol is no longer at the theoretical stage. However, the efforts made in the broader context of biorefining were not enough. Since isobutanol is a secondary metabolite, controlling the competing pathway to maximize the conversion of pyruvate to isobutanol is difficult, and removing the largest competitor, the ethanol pathway, can lead to severe growth defects. Coupled with the fact that biomass utilization efficiencies are low, the combination of the two problems leads to very low yields. Efficient inhibition of ethanol synthesis has been accomplished using light-controlled genetic systems [34], but the in vivo metabolic backgrounds of different microorganisms are very different, and further investment in research is needed to accomplish an efficient inhibition of the competing pathways. Another issue is how to enhance the robustness of engineered microorganisms, as both isobutanol and organics from biomass hydrolyses can cause considerable cellular damage. This issue includes how to improve the redox imbalance caused by isobutanol production, as it has been shown an improved redox significantly increases strain isobutanol tolerance [90] or furfural tolerance [91; 92; 93] and thus isobutanol production. Adaptive laboratory evolutionary applications for tolerance enhancement have obtained some results, with a large number of target genes being identified. However, the tolerance mechanisms are still opaque for both isobutanol and lignocellulosic-derived microbial inhibitors. Research on the application of tolerance enhancement strategies to biomass isobutanol production is also quite scarce.  
In particular, it should be noted that of all the current studies on the production of isobutanol from biomass, the highest titers reach 23 g/L by pervaporation-coupled fermentation, while the highest titers of isobutanol production are currently over 50 g/L due to in situ product removal. The timely isolation of isobutanol from the medium reduces the inhibitory effect of isobutanol on cells, reduces cytotoxicity, and significantly enhances the final titer. Fermentation processes also have a considerable influence on the production of isobutanol, but the existing research on microbial isobutanol synthesis and product separation [92; 93] is weakly linked. This aspect also needs to be strengthened in future research on microbial isobutanol production. We believe that the synthesis of biomass isobutanol will make significant progress with the further research on these issues.  
Conceptualization, Y.S., W.Z. and W.S.; writing--original draft preparation, Y.S. and W.Z.; writing--review and editing, Y.S.; supervision, A.Z. All authors have read and agreed to the published version of the manuscript. This work was supported by the Chunhui program of the Ministry of Education (Grant No.Z2017012). The authors declare no conflict of interest.