In silico analysis of Eat1 N-terminal sequences
Optimal function of Eat1 in E. coli likely depends on introducing the mature, truncated form of the enzyme. This form is not known for any of the Eat1 homologs that are able to produce ethyl acetate and can only be determined in the native yeast hosts through laborious experiments. Instead, we searched in silico for predicted cleavage sites within the N-termini of 15 Eat homologs from various yeast species using MitoFates (Fukasawa et al., 2015). All but the S. cerevisiae Eat1 N-termini contained an amphipathic region that is typically observed in N-terminal sequences of mitochondrial proteins (Figure 1a). Several sequences also had predicted MPP/Icp55 cleavage sites. Curiously, the predicted MPP/Icp55 cleavage sites would not fully remove the destabilising amphipathic region of their respective Eat1 N-termini. Since the amphipathic regions destabilise protein folding (Wiedemann and Pfanner, 2017), they are presumably removed during enzyme processing in the mitochondria. This may indicate that additional cleavage events, such as Oct1 cleavage in Cja Eat1 (Figure 1a) occur in Eat1.
We focused on N-termini of the Wickerhamomyces anomalus (Wan) Eat1 and Kluyveromyces marxianus (Kma) Eat1. Both enzymes are derived from yeasts that are able to produce high amounts of ethyl acetate. Efficient ethyl acetate synthesis by unmodified (but codon-harmonized) Wan Eat1 has already been demonstrated in E. coli (Kruis et al., 2017). However, the composition of the Wan and Kma Eat1 N-termini is remarkably different. The longer Kma Eat1 contained a clear pre-sequence and recognition sites for two mitochondrial peptidases, MPP and Icp55 at amino acid (AA) positions 19 and 20, respectively (Figure 1a). The shorter N-terminus of Wan still showed the characteristic amphipathic region, but no clear mitochondrial peptidase motifs were detected (Figure 1a). We therefore initially focused on optimising the N-terminus of the Kma Eat1. We designed 14 truncated versions of Kma Eat1 (trEat1) based on predicted cleavage sites, as well as arbitrary positions within the N-terminus. The truncated variants are denoted by the first AA appearing after the cleavage position (Figure 1b), although in reality, all proteins begin with M.
Expression of truncated Kma Eat1 variants in E. coli.
Ethyl acetate production from glucose by the truncated Kma Eat1 (Kma trEat1) variants was assessed in E. coli. The cells were grown under anaerobic conditions to stimulate production of ethanol, which is required by Eat1 to produce ethyl acetate. The carbon flux was channelled towards ethyl acetate production by disrupting the lactate dehydrogenase (ldhA) and acetate kinase (ackA) genes. This eliminated lactate production and lowered acetate formation, respectively (results not shown). The resulting E. coli BW25113 ΔackAΔldhA (DE3) strain was used to express the eat1 gene variants under the control of the lacI/T7 promoter.
We induced gene expression with 0.01 mM IPTG and 0.1 mM IPTG (Figure 2). At the lowest concentration, a profoundly positive effect on the final ethyl acetate titre was observed with several truncated variants compared to the untruncated (up) Eat1 (Figure 2ab). At 0.1 mM IPTG, the differences in ethyl acetate titres produced by Kma upEat1 and the Kma trEat1 variants were less apparent (Figure 2cd). Since 0.1 mM IPTG is a high inducer concentration, it is likely that ethyl acetate production was not limited by the AAT activity of Eat1, but instead by other metabolic bottlenecks. However, the low ethyl acetate production at 0.01 mM IPTG suggests that ethyl acetate production was limited by the activity of Kma Eat1. Any changes in the ethyl acetate production can therefore be linked directly to the in vivo activity of the enzymes.
The BW25113 ΔackAΔldhA (DE3) strains producing Kma trEat1 F-26, N-27, Q-28 and K-30 all formed ethyl acetate within 24h of cultivation, whereas no ethyl acetate was detected in the strains producing the unprocessed Kma Eat1 and most other Kma trEat1 variants (Figure 2a). During the second time point (144 hours) all Eat1 variants produced detectable amounts of ethyl acetate, except Kma trEat1 T-15, P-36 and I-37. Nevertheless, Kma trEat1 F-26, N-27, Q-28 and K-30 produced substantially more ethyl acetate compared to the unprocessed control and other Eat1 variants. The best performer was E. coli BW25113 ΔackAΔldhA (DE3) producing Kma trEat1 K-30, which formed 11.8-fold more ethyl acetate than the unprocessed variant (Figure 2b). E. coli BW25113 ΔackAΔldhA (DE3) producing Kma trEat1 P-9, Y-19, S-20, P-34 and P-35 formed approximately the same amounts of ethyl acetate as the unprocessed Kma1 (Figure 2).
Most trEat1 variants either led to increased ethyl acetate production or did not affect it significantly (Figure 2ab). The exceptions were the strains producing Kma trEat1 T-15, P-36 and I-37, which formed only traces of ethyl acetate. The Kma trEat1 P-36, and I-37 removed the first conserved region that is present in all Eat1 homologs from various yeasts (Kruis et al., 2017), which indicates that this conserved region is critical for ethyl acetate formation by Eat1. It is unclear why ethyl acetate formation was severely reduced in the strain producing Kma trEat1 T-15 (Figure 2).
Improved in vivo performance of Kma trEat1 F-26 and K-30 is likely the consequence of improved protein folding. An alternative explanation may be that truncating the 5’ coding sequence of Kma eat1 affected the translation initiation rates of the ribosome binding sites (RBS) used for protein translation. To exclude this possibility, we calculated the translation initiation rates for each Kma trEat1 gene using the RBS Calculator (Salis, Mirsky and Voigt, 2010). We compared the translation initiation rates with the ethyl acetate titres achieved by E. coli BW25113 ΔackAΔldhA (DE3) producing the Kma trEat1 variants with 0.01 mM IPTG (Figure 2a) and found little correlation between them (r = -0.14, Supplementary Figure 1). This supports the hypothesis that truncating the N-terminus of Kma Eat1 affected its function on the protein level.
Improved in vitro stability of Kma trEat1 variants
The improved ethyl acetate production was presumably caused by changes to Eat1 on the protein level, either by a higher specific activity or by an enhanced stability. To test this, we purified the unprocessed Kma Eat1, Kma trEat1 F-26, and Kma trEat1 K-30, and measured their initial 1-naphthyl acetate (1-NA) hydrolysis rates at 30 °C, 35 °C and 40 °C. (Figure 3a). Hydrolysis of 1-NA releases free 1-naphthol, which can be measured spectrophotometrically. The specific esterase activities of the three proteins moderately increased with temperature, with a 10°C increase leading to a 3-fold higher specific activity (Figure 3a). The truncated variants of Kma Eat1, however, did not exhibit a higher specific activity compared to unprocessed Eat1. We then tested whether truncating Kma Eat1 affected the stability of the proteins by determining their half-lives at 45 °C, 50 °C and 55 °C. For both Kma trEat1 F-26 and K-30, the half-lives were significantly higher at all tested temperatures compared to the unprocessed Kma Eat1 (Figure 3b, Supplementary Figure 2). Kma trEat1 F-26 and K-30 were thus more thermostable. Apparently, the N-terminal region has a weakening effect on the thermostability of Eat1.
Expression of truncated Wan Eat1 variants in E. coli
We examined whether the function of Wan Eat1 could also be improved by truncating its N-terminus. Predicting the structure of the N-terminal localisation sequence of Wan Eat1 using MitoFates did not result in clearly defined protease cleavage positions. Therefore, we used the conserved region at AA positions 36 and 37 within the Kma N-terminus as a guide to create two Wan trEat1 variants. Kma trEat1 Q-28 and K-30 were used to generate their Wan trEat1-V11 and N-13 counterparts, respectively (Figure 4a). The variants were produced in E. coli BW25113 ΔackAΔldhA (DE3) under the control of the lacI/T7 promoter. Interestingly, ethyl acetate titres exceeded measured values of Kma Eat1 already after 24 h when induced with 0.01 mM IPTG. All three strains producing the Wan Eat1 variants formed approximately 4 mM ethyl acetate, and no significant difference could be observed between unprocessed and truncated Eat1s (Figure 4b). After 120 h of fermentation, ethyl acetate concentrations varied between 9 and 11 mM, which was higher than all values obtained with the Kma Eat1 variants at 0.1 mM IPTG (Figure 2cd, 3b). This suggests that 0.01 mM IPTG was sufficient to fully induce Wan eat1 expression to a point where the activity of Eat1 did not limit ethyl acetate synthesis.
To more accurately study the effect of the truncations, we lowered the IPTG concentration to 0.001 mM IPTG (Figure 4c). The ethyl acetate formation trends in E. coli BW25113 ΔackAΔldhA (DE3) producing Wan trEat1 N-13 at 0.001 mM IPTG were similar to those observed in strains producing the Kma K-30 at 0.01 mM IPTG (Figures 2ab and 4c). At both sampling points, the strain producing Wan trEat1 N-13 reached a 2-fold higher ethyl acetate concentration than the unprocessed Wan Eat1 (Figure 4b). No difference was found between the Wan trEat1 V-11 and the unprocessed Wan Eat1 after 24 h, while over a longer time period the truncated variant even produced less ethyl acetate than the other two tested strains (Figure 4b, c).