Artificial cell factory design for shikimate production in Escherichia coli

Abstract

Shikimate is a key intermediate in high-demand for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu®). Microbial-based shikimate production strategies have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. Although shikimate biosynthesis has been reported in several engineered bacterial species, the shikimate production yield is still unsatisfactory. This study designed an Escherichia coli cell factory and optimized the fed-batch culture process to achieve a high titer of shikimate production. Using the previously constructed dehydroshikimate (DHS)-overproducing E. coli strain, two genes (aroK and aroL) responsible for converting shikimate to the next step were disrupted to facilitate shikimate accumulation. The genes with negative effects on shikimate biosynthesis, including tyrR, ptsG, and pykA, were disrupted. In contrast, several shikimate biosynthetic pathway genes, including aroB, aroD, aroF, aroG, and aroE, were overexpressed to maximize the glucose uptake and intermediate flux. The shiA involved in shikimate transport was disrupted, and the tktA involved in the accumulation of both PEP and E4P was overexpressed. The rationally designed shikimate-overproducing E. coli strain grown in an optimized medium produced approximately 101 g/L of shikimate in 7-L fed-batch fermentation, which is the highest level of shikimate production reported thus far. Overall, rational cell factory design and culture process optimization for microbial-based shikimate production will play a key role in complementing traditional plant-derived shikimate production processes.

Engineered Escherichia coli cell factory for anthranilate over-production

“…First, a well-known shikimate pathway transcriptional regulator gene, trpR , was deleted from the Inha254 chromosome, and the aroE was integrated into the Inha254 chromosome under the control of strong oppA promoter ( Supplementary Figures 2 , 3 ; Gu et al, 2016 ). Second, to increase the erythrose-4-phosphate (E4P) pool, one of the essential precursors of the shikimate pathway, the tktA involved in the pentose phosphate pathway was over-expressed under the control of the strong promoter J23119 ( Supplementary Figure 4 ; Shen et al, 2012 ; Lee et al, 2021 ). Third, the aroL and arolK genes were coupled translationally and expressed under the control of the strong promoter J23119 ( Supplementary Figure 5 ).…”

“…All E. coli strains were grown in Luria-Bertani (LB) medium at 30 or 37°C with the appropriate antibiotics. Small-scale cultivation and fed-batch fermentation for anthranilate production were conducted, as described previously ( Lee et al, 2021 ). For small-scale cultivation in a 24-well culture plate, a single colony was inoculated in 1.3 ml LB medium at 30°C for 15 h. The culture broth was inoculated with 1% (v/v) in the same medium at 30°C for 15 h. The secondary culture broth was inoculated in 1.3 ml of E. coli production medium (EPM) at 30°C for 4 days.…”

“…The feeding medium was supplied using a peristaltic pump when glucose was depleted. The pH was adjusted with 10 N NaOH or 3 M HCL at 7.0, and the DO level was maintained above 40% by controlling the rpm, aeration, and feeding rates ( Lee et al, 2021 ).…”

“…Previous studies published the world’s highest shikimate high-production E. coli strain. The current study attempted to develop an anthranilate high-production strain by optimizing the shikimate-to-anthranilate pathway based on a secured shikimate high-production strain ( Lee et al, 2021 ). The present study performed additional engineering to produce anthranilate starting from the shikimate-overproducing strain.…”

Bifunctional optogenetic switch for improving shikimic acid production in E. coli

“…For the biosynthesis of shikimic acid, several static strategies were used to improve the titer of shikimic. For instance, two-stage fermentation [ 49 ], gene knockout [ 50 ], and key pathway gene optimization [ 51 ] were used to increase shikimic acid titer, producing 7.98, 13.1, and 101 g/L, respectively. Recently, dynamic regulation was used to rescue the use of expensive aromatic amino acids.…”