Microorganisms exocytose certain metabolites for maintaining homeostasis as well as for regulating overflow metabolism 1. When metabolic pathways are highly imbalanced, intracellular metabolites can diffuse, sometimes assisted by membrane transport proteins, across membranes into an extracellular medium. Secretion of toxic metabolic products offers an effective way for host cells to relieve the feedback inhibition of metabolic pathways by replenishing intracellular reservoirs 4-6. This indicates that regardless of toxicity, value-added metabolites can be consistently produced and transported out of genetically modified microbial cells 7. The desired compounds can then be easily recovered from an extracellular liquid medium, without any harvesting or cell disruption. Thus, metabolite secretion engineering holds great potential for many applications in synthetic biology, including continuous flow production of valuable metabolites in an efficient and cost-effective manner 2,8.
Despite industrial and pharmaceutical importance, microbial engineering for selective metabolite secretion is exceptionally challenging. Recently, the use of ATP-binding cassette transporters, known to export lipids or drugs, was explored to mediate biofuel efflux in Escherichia coli 9-12. However, since noncognate transmembrane proteins are polyspecific, exocytosis of isoprenoids was indiscriminate, and the degree of efflux was insignificant (~25 mg/L) 13. In this regard, interorganellar protein trafficking in eukaryotes is unique. Thousands of cytoplasmic proteins are encrypted with sorting signals, and due to these address labels, each protein can accurately be delivered to intracellular compartments or extracellular milieu. This is the most significant systemic difference between secretion of proteins and metabolites.
In this study, we investigated whether metabolite secretion could be selectively guided by discrete sorting signals, similar to those of the elaborate protein trafficking system. It is well known that all metabolites interact with proteins. They serve as either substrates or products in enzymatic reactions and are required by allosteric proteins as specific ligands. Molecular recognition can appropriately be exploited for metabolite trafficking; specific proteins that bind to desired metabolites can be chosen and readily tagged with signal peptides for further sorting. Thus, the fusion proteins, capable of carrying the target metabolites, are designated certain destinations, within or outside cells. By integrating a metabolite-binding protein with its sorting tag, we successfully engineered a metabolite trafficking system. As a proof of concept, we systematically designed an exclusive pathway for secretion of medicinal terpenes, large and hydrophobic high-value chemicals, and demonstrated that terpene production was substantially boosted in Saccharomyces cerevisiae (e.g., ~226 mg/L for 5-day batch fermentation and ~700 mg/L for 15-day semi-continuous culture). To the best of our knowledge, this is the first programmed cognate pathway for selective metabolite secretion in microorganisms, thus enabling the intracellularly accumulated target compounds to pass through otherwise impermeable membranes.
Our fusion protein design involved coupling of a terpene-binding protein with an export signal peptide. Since supernatant protein factor (SPF) is involved in the regulation of cholesterol biosynthesis in the human liver 14, we first identified this cytosolic lipid-binding protein as a terpene carrier. To facilitate the hydrophobic interaction of SPF with lipophilic metabolites, we only employed the lipid-binding domain of SPF (tSPF, amino acids 1-278) by eliminating the C-terminal Golgi dynamics domain (Supplementary Fig. 1) 15. In parallel, we examined the ability of sucrose transport protein (invertase, Suc2), derived from yeast, for export signaling via co-translational translocation 16. When the signal peptide of Suc2 is cleaved from a nascent protein in the endoplasmic reticulum (ER) lumen by signal peptidases, the remaining secretory protein is immediately translocated into the ER membrane for subsequent secretion. Taking advantage of this mechanism, we fused the Suc2 signal peptide to the tSPF N-terminal to obtain Suc2-tSPF. This tailored fusion protein could perform a series of programmed actions for selective terpene secretion (Fig. 1 and Supplementary Fig. 2). Briefly, once Suc2-tSPF was translated in the ER (step a), signal peptidases subsequently cleaved the Suc2 signal peptide (step b). The mature tSPF was specifically loaded with a terpene (step c) and then transported to the Golgi (step d). To complete terpene trafficking, the terpene-loaded tSPF was exported into the extracellular medium (step e).
We successfully expressed Suc2-tSPF in an engineered S. cerevisiae strain for squalene overproduction and achieved extracellular secretion of hitherto membrane-impermeable terpenes (Fig. 2). Briefly, the Suc2-tSPF gene was systematically incorporated into our original squalene producer (SQ03-INO2; subsequently referred to as SQ), which is known to the most productive yeast strain, capable of producing up to 69 mg/g dry cell weight (DCW) squalene in 144 h 17. Unlike conventional methods, a two-phase cultivation system comprising culture medium and 10% (v/v) immiscible dodecane was adopted (Fig. 2A) 13,18. Hydrophobic metabolites are soluble in the organic phase, but not in the aqueous medium; thus, the secreted squalene can be fully recovered by simply collecting the dodecane (see Methods). High-performance liquid chromatography (HPLC) revealed that successful overexpression of Suc2-tSPF resulted in remarkably high levels of squalene in the dodecane phase (Fig. 2B (navy) and Supplementary Fig. 3). In contrast, no squalene secretion was observed when the control strain SQ was used (blue).
Surprisingly, the most efficient production of squalene was demonstrated by the newly engineered yeast strain (Suc2-tSPF-expressing SQ, Suc2-tSPF/SQ) to date. Time-dependent quantification of squalene secretion (Fig. 2C and Supplementary Table 1) revealed a significant increase in extracellular squalene secretion by Suc2-tSPF/SQ compared with the original SQ. After 144 h of cultivation, the titer of extracellular squalene produced by Suc2-tSPF/SQ was 226 mg/L, which was ~26-fold more than that by SQ (8 mg/L). We noted that long-term cultivation (6 d) was accompanied by the natural death of old cells, which were inevitably included in the dodecane phase. Thus, SQ yielded markedly low levels of extracellular squalene via inadequate squalene secretion. Along with unprecedented squalene secretion, Suc2-tSPF/SQ also exhibited increased intracellular squalene accumulation (Supplementary Table 1); it produced 97 mg/g DCW of intracellular squalene after 144 h of fermentation, and this value showed a ~40% increase over that produced by the original SQ (69 mg/g DCW). Without the signal peptide, tSPF alone could not secrete squalene (Fig. 2B – C and Supplementary Table 1). The tSPF-expressing SQ strain (tSPF/SQ) showed negligible squalene secretion (13 mg/L), similar to that by the control SQ strain (8 mg/L), even after fermentation for 144 h.
Selective metabolite secretion depends on the level of each intracellular product, and only surplus squalene can be exclusively secreted. Unlike squalene, 2,3-oxidosqualene, an important metabolic intermediate in the squalene biosynthesis pathway, was not secreted. Considering that SPF has no significant preference for either squalene or 2,3-oxidosqualene, we speculated that their intracellular accumulation would determine whether they could be loaded onto the export carrier, Suc2-tSPF. Since 2,3-oxidosqualene was rapidly consumed in our engineered yeast cells, its intracellular concentration was not sufficient to trigger Suc2-tSPF capture for subsequent secretion (Supplementary Table 2). Conversely, the extracellular transport of the desired product, squalene, increased in a time-dependent manner. On comparing the results of Suc2-tSPF/SQ cultivation for 72 and 144 h, we found that extracellular squalene was markedly increased by ~36%, confirming that squalene titers gradually increased over time (Fig. 2C and Supplementary Table 1).
Even when other export signal peptides were coupled with tSPF, the selective squalene secretory pathway remained active. Two different proteins, viz., acid phosphatase (Pho5) and yeast α-mating factor (MFα) (Supplementary Table 3), were chosen on the basis of translocation modes 19-21. Pho5 uses the same co-translational translocation pathway as Suc2, but its signal peptide is cleaved by different ER peptidases. In contrast, MFα is transported to the ER membrane via post-translational translocation, which involves two-step cleavage of the signal peptide (Supplementary Fig. 2). Following the removal of the export signal peptide of MFα by signal peptidases in the ER lumen, Kex2 and Ste13 proteases further cleave the remnant of the signal peptide in the Golgi. Owing to the same translocation mode, squalene secretion by Pho5-tSPF was comparable to Suc2-tSPF (Fig. 2B); however, total squalene secretion after 144 h of cultivation (190 mg/L) was lower (Fig. 2C and Supplementary Table 1). Interestingly, MFα-tSPF, which is trafficked via a route distinct from that of Suc2-tSPF, showed significantly lower squalene secretion (Fig. 2B – C and Supplementary Table 1). Although the fundamental mechanism underlying this is unclear, we speculated that the inherent complexity of signal peptide cleavage might have affected the loading efficiency of squalene onto tSPF before secretion 22.
To clarify further whether the presence of squalene in the dodecane phase was caused by programmed secretion or cell lysis-dependent release, we performed a western blot assay (Fig. 2D, see Methods) using actin as a loading control. In principle, with negligible cell lysis, cytosolic actin should not be detected in the extracellular medium, but it must be possible to detect tSPF by signal peptide-guided secretion. Based on the actin bands of the culture supernatant, we verified that the presence of tSPF in the extracellular space was not caused by cell lysis. Without signal peptides, tSPF was enriched only inside the cells (Fig. 2D, upper). However, when the cells overexpressed tSPF that was systematically integrated with signal peptides, tSPF was successfully secreted out of the cells (Fig. 2D, lower). Among the three different signal peptides, Suc2 exported tSPF most efficiently, which was consistent with the finding that Suc2-tSPF/SQ secreted squalene most productively.
We further validated the role of signal peptide-guided tSPF in squalene secretion by fusing green fluorescent protein (GFP) to the C-terminal of tSPF and observed its localization using confocal fluorescence microscopy (Fig. 2E and Supplementary Fig. 4). Corroborating the western blot results, the intracellular GFP signal was only intense for the signal peptide-lacking tSPF, whereas no fluorescence inside the cells were observed for the signal peptide-tagged tSPF. From this observation, we concluded that GFP-fused Suc2-tSPF and Pho5-tSPF (Suc2-tSPF-GFP and Phot-tSPF-GFP, respectively) enabled the secretion of tSPF-GFP after cleavage of the signal peptide. We noted that MFα-tSPF-GFP could not be completely transported outside the cells owing to its poor secretion capability. However, without the export signaling tag, the tSPF-GFP was destined for intracellular retention.
Indeed, the selective capture of tSPF was responsible for squalene secretion, which was validated by the use of control proteins incapable of squalene binding. For this purpose, we prepared Suc2-tagged GFP (Suc2-GFP), since GFP can neither perform extracellular secretion nor capture terpene (Supplementary Table 1) 23. Overexpression of Suc2-GFP in the SQ strain clearly showed its extracellular export (Supplementary Fig. 5). However, there was no evidence of squalene secretion, thereby indicating that the engineered squalene secretion pathway was the exclusive result of synchronizing two different molecular functions, namely protein hitchhiking and metabolite trafficking.
Additionally, we implemented a semi-continuous fermentation using Suc2-tSPF/SQ, the best squalene-secreting strain, to prove that our metabolite trafficking system could be used to produce and secrete the target metabolite in a continuous and efficient manner (Fig. 2F, Supplementary Fig. 6, and Supplementary Table 4; see Methods). Briefly, we sampled yeast cells and collected dodecane, replenishing with fresh culture medium once every 3 days. During five repeated cycles, intracellular squalene remained at ~60 mg/g DCW, indicating no further increase in production. However, extracellular squalene accumulated consistently, and by the fifth cycle, its titer exceeded 700 mg/L, the highest titer of squalene production reported to date. Moreover, the graph for total squalene production over time was linear, thereby indicating the potential for continuous flow production.
Given the binding promiscuity of SPF towards other hydrophobic terpenes 15, we further expanded our innovative concept of metabolite secretion by applying it for production of other terpenes. Thus, β-carotene, a tetra-terpene, was chosen to demonstrate the engineered secretion owing to its simplistic colorimetric detection 13. For production of β-carotene in S. cerevisiae (Fig. 3A), we used the p415-BC plasmid containing four different β-carotene biosynthetic genes: tHMG1 (truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1) from S. cerevisiae, and crtE (geranyl diphosphate synthase), crtYB (phytoene synthase), and crtI (carotene desaturase) from Xanthophyllomyces dendrorhous. As ascertained by squalene secretion, we overexpressed Suc2-tSPF using yeast cells containing p415-BC for β-carotene production. Interestingly, we observed a ~23-fold increase (1.4 mg/L) in β-carotene secretion compared to that by the control, which was incapable of secreting tSPF (0.06 mg/L) (Fig. 3B – D, Supplementary Fig. 3, 7, and Supplementary Table 5).
Finally, we identified the metabolites that could benefit from our SPF-driven programmed secretion, using molecular docking with AutoDock Vina in PyRx 24,25. We scrutinized 43 different terpenes that microbial cells have produced as production hosts 26,27, and the predicted binding energy was calculated for each compound (Fig. 3E and Supplementary Table 6, see Methods). The molecular interaction energy between SPF and each terpene varied from -10.7 to - 6.1 kcal/mol. Squalene and 2,3-oxidosqualene interaction energies were -10.6 and -10.5 kcal/mol, respectively, confirming their comparable binding capabilities to the SPF. In contrast, glutamate and pyruvate, key intermediates of metabolic pathways in yeast, exhibited much lower affinities (-4.4 and -3.6 kcal/mol, respectively), suggesting that the SPF carrier protein is terpene-specific. Furthermore, we thoroughly analyzed the pattern of SPF-terpene interactions after the docking simulation; the positions of all terpenes overlapped within the SPF binding pocket (Supplementary Fig. 8), although end groups of several tetra-terpenes (e.g., an isophorone group of zeaxanthin) protruded from the SPF protein surface. These simulations validated that SPF could serve as a suitable vehicle for various terpenes. Thus, the signal peptide-guided SPF would enable precise delivery of target terpenes to a desired location, including the extracellular space, thereby actualizing the synthetic pathway of metabolite trafficking.
In summary, we first demonstrated that a common multiplexed protein secretion pathway could mediate sustainable and efficient extracellular transport of target metabolites. The integration of protein hitchhiking with metabolite trafficking was highly synergistic. Membrane-impermeable terpenes could be rescued from the extracellular medium without cell disruption and subsequent extraction, thereby demonstrating a potential cost-effective, high-yielding continuous flow process for production of valuable chemicals on an industrial scale. Unlike previous noncognate transmembrane engineering 13, our cognate secretion pathway programming achieved exceptionally efficient terpene secretion (~226 mg/L for squalene and ~1.6 mg/L for β-carotene), the highest values reported in microorganisms for the first time in this study.
To maximize terpene production and secretion by optimal cell growth, our metabolite trafficking pathway can be fine-tuned by employing various synthetic biology tools and strategies 3,28,29. For this proof-of-concept study, we overexpressed Suc2-tSPF using the strongest yeast TDH3 promoter on a high-copy number plasmid, likely causing the protein burden effect 23,30. However, this could be mitigated by balancing or tuning the expression levels of relevant proteins. Furthermore, synthetic regulatory systems with diverse operational modes 31,32, such as auto-regulatory feedback loops, toggle switches, and engineered riboswitches, may permit the decoupling or integration of cellular growth and terpene secretion.
Importantly, our metabolite trafficking strategy is versatile. Despite the promiscuity of SPF, its concentration-dependent binding characteristic enables the desired metabolic product to be loaded only onto the carrier protein, thereby indicating that metabolic pathway engineering can allow us to readily choose the terpene to be secreted exclusively. Furthermore, by systematically combining other carrier proteins and signal peptides, various high-value biomolecules of economic, environmental, or therapeutic importance could be precisely delivered to desired intracellular locations, as well as to extracellular spaces, thereby pioneering the unprecedented avenues of synthetic biology in controlling cellular life.