Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST)
Research objective
Microbial production of isobutanol (IB), which is a raw material for polyethylene terephthalate, has attracted atention in response to the recent trend toward bio-based chemicals. Among them, almost all studies that have used Escherichia coli as a host have employed anaerobic conditions during the stationary phase as an evaluation target to ensure reduction power and for avoiding competition during growth. Under these conditions, while the IB production yield can be increased, both the low Glucose AceE amount of biomass due to the anaerobic conditions and V 145 → 6 Pyruvate the low substrate uptake rate due to the stationary phase AlsS substantially  limit  rate  of  IB  production. can 9 → 10 → Alternatively, although a substantial amount of biomass Acetyl-CoA Acetolactate and a high substrate uptake rate can be guaranteed >> 2,3-dihydroxylsovalerate under aerobic conditions during the log phase, the IB v Growth 2-ketoisovalerate production yield will decline because of the increase in CO' >> CO 2 generated by complete substrate oxidation (Fig. 1). Isobutylaldehyde → Exogenous IB In the present study, I focused on pyruvate, the biosynthesis pathway IB branching point of carbon flow, and aimed to improve Fig. 1. A strategy focusing on the the flux to the IB side by limiting the flux to the growth catalytic efficiency (kcat/Km) of side. Specifically, I adopted a strategy in which the enzymes involved in pyruvate distribution efficiency of pyruvate was changed by distribution. The numbers in weakening AceE and strengthening AlsS using the dicate kcat/Km values. catalytic efficiency ( kcat / K m ) value as an index.
Methods
Escherichia coli MG1655 was transduced with T7 RNA polymerase gene using a λDE3 Lysogenization kit. A site-specific mutagenesis by pORTMAGE 11 was employed to construct the aceE mutant ²). The plasmid for IB production (pIB) was constructed by linking five genes of the IB biosynthetic pathway 3 ) which were codon-optimized for E. coli into pIB under a T7 promoter. Site-specific mutagenesis by PCR was used to construct the alsS mutant 4 ). E. coli harboring pIB was cultured aerobically in a modified M9 medium using glucose as the sole carbon source. The supernatant of the culture was analyzed by HPLC to quantify the concentrations of glucose, IB, and organic acids. Intracellular pyruvate levels were measured using an EnzyChrom Pyruvate Assay kit.
Results
1) IB production using the aceE mutant strain
In a previous study, the kcat/Km value of the AceE variant (AceE k 403A ) was approxymately 4% of AceE 2 ). It is considered that the decrease in AceE activity leads to a reduced amount of flux to the TCA cycle, which is the main energy production pathway, thereby resulting in a lower growth rate. As expected, the specific growth rate of the aceE k403A strain decreased to approximately 17% of that of the wild-type strain. This result indicates that the weakening of AceE suppressed pyruvate distribution to the growth side. However, the growth rate as well as the IB production and glucose consumption decreased in the aceE* 403A strain harboring pIB (Fig. 2). The reason for this is currently unknown; however, it is considered that certain enzymes supplemented the flux from pyruvate to acetyl-CoA instead of AceE because the levels of biomass and acetate production were increased relative to glucose consumption. Therefore, to improve the distribution efficiency of pyruvate to the IB side, further identification and suppression of the outflow pathway to acetate will be necessary to prevent pyruvate distribution to the growth side. 2) IB production using the alsS mutant strain AlsS, which catalyzes the initial reaction to the IB production pathway, uses pyruvate as a substrate in a similar manner to AceE catalyzing the initial reaction of the TCA cycle. The intracellular pyruvate concentrations of the alsS strain constructed in the present study under aerobic and anaerobic conditions were 1.2 mM and 3.1 mM, respectively. In contrast, it was reported that the K m and kcat values of AlsS were 13.6 mM and 121 s- 1 , while those of the AlsS variant (AlsS Q487S ) were 1.1 mM and 10 s −1 , respectively 4) . From these results, it is hypothesized that the high kcat/Km value of AlsSQ4875 can be utilized under aerobic conditions because the intracellular pyruvate concentration was close to the K m value of AlsS♀487S (1.1 mM) and was considerably lower than the of AlsS. In fact, the alsS♀487S strain showed higher IB productivity than the alsS strain despite the low biomass ( Table 1 ; biomass: 0.9-fold, IB production: 1.2-fold, specific IB production rate: 1.5-fold). This result indicates that the increase in kcat/K m contributes to the enhancement of the pyruvate distribution to the IB side. However, the amount of IB produced in the log phase was approximately 13% of that produced in the entire culture phase. This result is consistent with the fact that the kcat /Km values of AlsS and AlsSQ487S are extremely low compared to those of AceE, implying that IB production under aerobic conditions during the log phase must be integrated with AceE weakening.
Table 1. Effects of alsS mutation on IB productivity.
Conclusion
As expected, the growth rate of the aceE × 403A strain decreased to 17%. The specific IB production rate of the alsSQ487S strain was 1.5-fold higher than that of the alsS strain, and 13% of the final titer of IB was obtained during the log phase. Further investigation is warranted to understand the mechanisms regulating the decrease in glucose consumption and IB production in the aceEK403A strain harboring pIB. Additionally, it is also necessary to evaluate the IB productivity of strains harboring the aceE × 403.4 and als.524875 mutation.