# Advances in the Metabolic Engineering of _Escherichia coli_ for the Manufacture of Monoterpenes
## 6 Improvement of the Toxicity Tolerance for the Host Strain  
Almost all monoterpenes exert critical toxicity to the host cell. Thus, continuously elevated production of monoterpenes may exacerbate the exhaustion of the cell factory. Their toxicity could inhibit the growth of producing hosts, lower the biotransformation activity of enzymes, and impede the availability of the metabolic route. In the end, the total production of monoterpenes by microbial strains is eventually reduced [21; 77; 78]. Commonly used approaches to improve toxicity tolerance include modification of membrane proteins, expression of efflux pumps, and activation of the stress response system, such as expression and regulation of heat shock proteins [79; 80; 81]. [PERSON] et al. employed a cellular export system to alleviate biofuel toxicity and enhance the host's tolerance. They screened bacterial genomes and read out all the efflux pumps, a class of membrane transport proteins driven by the proton motive force [82; 83; 84]. Afterward, 43 of the pumps were selected and cloned into a plasmid library, which were subsequently transformed into _E. coli_ for heterologous expression. This export mechanism shows significant protective effects of host survival for five chosen biofuels, including geranyl acetate, geraniol, \(\alpha\)-pinene, limonene, and farnesyl hexanoate [31]. The elevated tolerance to the product toxicity and the significant improvement of product yields might be due to the efficient transportation of toxins out of the host and the maintenance of biomass accumulation by the efflux pumps. However, the combination of more than one type of efflux pumps could inhibit cell growth, implying that antagonistic mechanism is involved in the multi efflux pump effects [85]. Nevertheless, this approach has a profound effect on the toxicity tolerance of the prokaryotic hosts and has a great potential to be applied in monoterpene production. Alternatively, another intelligent strategy called in situ product recovery (ISPR) based on anion exchange resin was developed [86; 87]. This method uses a column containing a fluidized bed of resin combined with a bioreactor to specifically trap toxic products and remove them from the culture media. This manner could relieve the effect of toxic inhibition on bioconversion and finally improve the production of the target monoterpene. [PERSON] et al. tested four commercially available resins and then found the Amberlite IRA 410 Cl, a most suitable resin for POH recovery. Amberlite resin traps the POH, which can maintain a low final product concentration in the media and maintain a high rate of metabolic flux toward it. Their results showed 1.5-fold higher POH production than that in the resin-absent group. Moreover, combination of this method with overexpression of the cytochrome P450 system could increase the ultimate concentration of POH with 2.5-fold in total and 3.5-fold in specific production [32].  
Meanwhile, a method using an aqueous-organic two-phase system for host cell culture produces similar protective effects. Rational utilization of in situ separation and extraction for two-phase culture medium not only alleviates the product toxicity to cells but also prevents the target monoterene from volatilizing [88; 89]. By taking advantage of the in situ two-phase extraction, the bacteria harboring the heterologous MEV pathway, GPPS and MS could produce myrcene to a maximum of 58.19 \(\pm\) 12.13 mg/L [38]. However, these progresses for toxic tolerance improvement are mainly focused on the effective transfer of products. Other ways of optimizing the metabolic systems or engineering the host cells in a more robust condition should also be considered in future studies.