The strains used in this study are shown in Table 1. E. coli DH5α was used to construct plasmids and was grown in LB (lysogeny broth) medium supplemented with antibiotics when necessary. E. coli BL21 (DE3) was used for C3G production and transporter expression. The plasmid pETM6-At3GT-m-PhANS was transformed into E. coli BL21 (DE3) to construct the baseline C3G-producing strain (Cress et al., 2017). The plasmid pACYCDuet-1 was used to express transporter genes. C3G production was performed in M9 medium (pH 5.0) containing 4 g/L of glucose (Lim et al., 2015).

The plasmids and primers used in this study are shown in Table 1 and Supplementary Table S1, respectively. The transporter genes were amplified using the genomic DNA of E. coli BL21 (DE3) as the template, and subsequently cloned into the plasmid pACYCDuet-1 by Nde I and Xho I. For the co-overexpression of two transporter genes, the single overexpression plasmids were used and the second gene was cloned into the second ORF between BamH I and Not I.

The transporter knockout strains were constructed using the CRISPR/Cas9 tool developed in a previous study (Jiang et al., 2015). The knockout strains were formed by homologous recombination between the genome and the homologous arms, with ~200 bp gene sequences deleted in the ORF of the target transporter genes in the repair plasmids. The deletion was finally confirmed by PCR amplification of the extracted genomic DNA from the knockout strains and by gene sequencing of the amplified PCR products. The complementation strains were constructed by introducing the pACYCDuet-1 plasmids carrying the transporter genes into the knockout strains.

For all the cloning work, the enzymes were purchased from New England Biolabs, and kits for plasmid miniprep and gene cleanup were from TIANGEN Biotech. Primer synthesis and gene sequencing were performed by Sangon Biotech.

The plasmid pETM6-At3GT-m-PhANS and pACYCDuet-1 plasmids expressing MDR transporters were co-transformed into E. coli BL21 (DE3) using the traditional calcium chloride method.

Cells were grown overnight at 37°C in 1 mL LB medium supplemented with 80 μg/mL ampicillin and 25 μg/mL chloramphenicol in 48-deep-well plates (VWR). Overnight cultures (20 μL) were sub-cultured into 1 mL of fresh LB medium containing the necessary antibiotics in 48-deep-well plates. Cells were grown at 37°C with shaking at 220 rpm, induced with 0.5 mM IPTG when OD₆₀₀ (the optical density at 600 nm) reached 0.6, and further grown at 30°C with shaking for 3 h. Cells were then collected by centrifugation at 4,000 rpm and 4°C and resuspended in 1 mL of M9 medium (pH 5.0) with 500 mg/L (+)-catechin. The conversion process was then carried out at 30°C with shaking for 19 h. At the end of the fermentation, 200 μL of the culture supernatants were mixed with an equal volume of acidified methanol and centrifuged at 12,000 rpm for 10 min. The supernatants were used to quantify residual catechin and extracellular C3G.

Glycerol stocks were inoculated into 3 mL of LB medium supplemented with 80 μg/mL ampicillin and 25 μg/mL chloramphenicol and grown overnight at 37°C. The overnight culture (1 mL) was sub-cultured into 50 mL of fresh LB medium containing the necessary antibiotics in 250 mL shake flasks. Cells were grown at 37°C with shaking at 200 rpm, induced with 0.5 mM IPTG when OD₆₀₀ reached 0.6, and further grown for 3 h at 30°C and 200 rpm. Cells were then harvested by centrifugation at 4,000 rpm and 4°C and resuspended in 10 mL of M9 medium (pH 5.0) with 500 mg/L (+)-catechin in 100 mL shake flasks. Cells were incubated at 30°C and 200 rpm for 19 h, and then 200 μL of the culture supernatant were mixed thoroughly with an equal volume of acidified methanol and centrifuged at 12,000 rpm for 10 min. The supernatant was used for the quantification of extracellular C3G and residual catechin.

The fermentation products and residual catechin were analyzed by a Shimadzu Essentia LC-16 HPLC system with a diode array detector. A reversed phase C18 column (5 μm, 4.6 × 250 mm) was used to separate and quantify the compounds. The column temperature was maintained at 25°C. Water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) were prepared as mobile phases at a flow rate of 1 mL/min. The elution program was as follows: 10–40% B at 0–10 min and 40–60% B at 10–15 min. C3G and catechin were detected and quantified by monitoring the absorbance at 520 nm and 280 nm, respectively. The C3G and (+)-catechin standards used in this study were purchased from Sigma-Aldrich.

Overnight cultures (1 mL) of diverse recombinant strains were sub-cultured into 50 mL of fresh LB medium containing the necessary antibiotics in 250 mL shake flasks, and were grown at 37°C and 200 rpm. Cell growth was monitored periodically by measuring OD₆₀₀ in the absence of IPTG. To study cell growth under the induction condition, cells were grown to OD₆₀₀ of ~0.6 and supplemented with 0.5 mM IPTG. The OD₆₀₀ was then tracked periodically.

MDR transporters facilitate the outward flow of toxic compounds from the cytoplasm and enhance cell survival under harsh environmental conditions such as exposure to antibiotics and heavy metals (Du et al., 2018). In our previous study, we found that overexpression of an MFS transporter MdtH could increase the extracellular C3G production by around 110% due to better efflux (Wu et al., 2024). To identify other MDR transporters beneficial for extracellular C3G biosynthesis in E. coli, we constructed a transporter library containing the known or putative MDR transporter genes (Nishino and Yamaguchi, 2001) in E. coli as well as the genes encoding the TolC porin and TolC-involving tripartite efflux systems that are important for drug resistance (Table 2). Some of the MDR transporters that were already analyzed in our previous study were not included here. We overexpressed each of these genes individually under the C3G-producing background and analyzed the extent of extracellular specific C3G production in 48-deep-well plates. Genes that are present in the same operon were overexpressed together. Since the precursor catechin and the product C3G both present antimicrobial activities (Nakayama et al., 2013; Lim et al., 2015) and since the product C3G is unstable under neutral or basic conditions, we adopted a two-step biosynthetic procedure, in which cells were first grown in LB medium and induced for pathway enzyme expression for 3 h, and then shifted to M9 medium (pH 5) containing 500 mg/L of catechin for C3G synthesis.

As shown in Table 2, overexpression of fsr, ydeE, ynfM, ydhC, ydiM, emrKY, mdtNOP, yebQ, sugE, emrE, mdlAB, yddA, yhiHJ, acrD, or cusCFBA increased the extracellular level of C3G per OD₆₀₀ by 0.37- to 1.72-fold, which was consistent with our hypothesis that the titer of C3G could be improved via efflux enhancement. Therefore, these transporter genes were selected for the subsequent analysis. It is interesting to note that overexpression of the transporter components that form tripartite efflux systems with TolC significantly impeded C3G biosynthesis, and the co-overexpression of tolC further enhanced such inhibitory effect, although a higher expression level of tolC alone had marginal effect on extracellular C3G concentration.

To verify the role of the selected MDR transporters in C3G biosynthesis, we conducted the bioconversion using the two-stage procedure in shake flasks. It is generally believed that the cultivation conditions in shake flasks can be controlled more stably than those in 48-well plates. Among the 15 selected transporter genes, overexpression of ynfM improved the extracellular synthesis per OD₆₀₀ by 4.80-fold compared to the control strain that harbored the empty plasmid (Table 3), and upregulation of fsr, yebQ, emrKY or mdlAB increased specific C3G production by 0.54- to 4.12-fold. In comparison, the other transporter genes that were tested effective in 48-well-plates did not show positive correlations at a higher expression level with extracellular C3G production in shake flasks. Such a discrepancy may be attributed to the different extents of oxygen supply and shear stress.

To investigate whether the phenotype of extracellular C3G production was indeed associated with the expression levels of the selected transporter genes, i.e., fsr, ynfM, yebQ, mdlAB, and emrKY, we constructed single-gene deletion and complementation strains, and analyzed C3G biosynthesis and catechin utilization by various recombinant strains. As shown in Figure 2A, overexpression of these genes greatly promoted extracellular C3G concentration compared with the control strain carrying the empty plasmid; deletion of yebQ, mdlAB, or emrKY decreased C3G production by 10–30% compared with the control strain, whereas deletion of fsr or ynfM had no impact on extracellular C3G concentration. Complementation of the corresponding genes in the knockout strains restored C3G production to levels similar to those in the overexpression strains. Moreover, despite large variations in C3G production by various overexpression strains, they consumed similar amounts of catechin except the mdlAB-related strains that utilized noticeably less catechin (Figure 2B). Taken into consideration both substrate usage and product formation, we found similar extracellular C3G yields obtained by the overexpression of ynfM, yebQ and mdlAB, which were 2.7- to 2.9-fold higher than that of the control strain. Interestingly, deletion of fsr or ynfM resulted in 0.61- or 0.82-fold higher yields, respectively (Figure 2C).

As C3G is naturally pink under acidic conditions, it is easy to judge the production using the colorimetric assay and by visual observation. Notably, the culture overexpressing ynfM or yebQ showed darker colors than the control strain and the other overexpression strains (Figure 2D). Additionally, the yebQ-overexpressing culture turned pink as early as 1 h after catechin supplementation (Supplementary Figure S1). In comparison, the control culture did not develop any pink color even after 4 h of biosynthesis.

Generally, overexpression of transporters involves extensive folding and translocation of an array of proteins and their translocation to the limited membrane space, thereby causing excessive burden to the central cell metabolism (Jensen et al., 2017; Zhu et al., 2020). To understand how cell growth was affected by the expression levels of the five transporters identified in the present study, we monitored the growth curves of the overexpression, deletion, complementation, and the control strains. To rule out the possible effect of the vector pACYCDuet-1 that was adopted for episomal expression of the transporters, both the deletion and the control strains harbored the empty plasmid. As shown in Figure 3, without IPTG to induce the gene expression, the growth of all the recombinant strains was comparable to that of the control strain, suggesting that single deletion of the transporter genes had no apparent impact on cell growth. In contrast, IPTG induction of the transporter genes and the pathway genes reduced cell growth in the late-log phase and biomass accumulation in the stationary phase for the overexpression and the complementation strains when compared with the deletion and the control strains, although all the strains grew at similar rates at the mid-log phase (Figure 4). In addition, the control strain expressing ANS and 3GT showed greatly reduced biomass compared to the condition without IPTG addition.

Given that the overexpression of ynfM, yebQ, mdlAB, and emrKY increased C3G production considerably, we explored whether the co-overexpression of these genes could further enhance the bio-production. The fsr gene was not included due to the fact that there was not much difference in the yield of extracellular C3G when this gene was overexpressed, deleted, or complemented. Compared with yebQ overexpression alone, the co-overexpression of yebQ together with ynfM, mdlAB, or emrKY reduced extracellular C3G production (Figure 5A). Similarly, overexpression of ynfM, which alone led to the highest extracellular C3G level, showed negative effects when co-expressed with mdlAB or emrKY. In contrast, the co-overexpression of mdlAB and emrKY resulted in a level of extracellular C3G production analogous to that when mdlAB alone was overexpressed. Co-expression of the transporter genes led to less utilization of catechin but did not cause much variation in the yield of extracellular C3G (Figures 5B,C).

Since single deletion of yebQ, mdlAB and emrKY reduced extracellular C3G production titer and the yields, and overexpression and complementation of each individual gene significantly improved both parameters, we wondered how critical these genes are for C3G production. To that end, we constructed double-knockout strains, and found that deletion of mdlAB/emrKY or mdlAB/yebQ combination reduced the extracellular C3G concentrations and the yields to levels lower than those in the single-knockout mutants, with the ΔmdlABΔemrKY double deletion leading to the largest extent of decline (Figure 6), while the substrate consumption of these two strains was similar to that of the control strain or the corresponding single deletion strains, suggesting that MdlAB and EmrKY are key to the secretion of C3G in E. coli. In contrast, ΔyebQΔemrKY mutation resulted in less extracellular C3G production than the Δyeb strain but more than the ΔemrKY strain. With slightly less catechin utilization, this double deletion strain generated C3G with an extracellular yield comparable to that in the ΔyebQ strain (Figure 6).