Construction of sabinene synthetic pathway in S. cerevisiae
Initially, sabinene synthetic pathway was established on centromeric plasmid-based system (Additional file 1: Fig. S1a). The sabinene synthase (SabS1) from S. pomifera, which was reported to demonstrate better catalytic specificity than those from other sources [21], was chosen and introduced along with ERG20WW into our former constructed strain YJGZ1 [17] (Fig. 2a). Based on this host harboring overexpressed tHMGR (truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase) and IDI1 (isopentenyl diphosphate isomerase) (Fig. 2a), a gram grade titer of geraniol has been achieved [17], which would guarantee a promising GPP supply for sabinene production. In the meanwhile, yeasts derived from strain YJGZ1 (△GAL80 background) adopt inducible GAL promoters to control heterologous genes (Additional file 1: Fig. S1a). In that case, the fermentation process would be divided into cell growth stage and sabinene accumulation stage, to relieve the cytotoxicity brought by sabinene. Also to reduce product toxicity, 20% (v/v) isopropyl myristate (IPM) was supplemented into the culture before fermentation to enrich sabinene. As shown in Additional file 1: Fig. S2, the products of the initial sabinene producing strain Sc041001 and its parental strain YJGZ1 (Control) were analyzed by GC-TOF-MS after 96-h cultivation. For strain Sc041001, it was detected a chromatographic peak whose retention time (3.99 min) as well as the mass fragment (91, 93, 136 m/z) were consistent with those of the sabinene standard, respectively, while no such peak was detected in the control (Additional file 1: Fig. S2). This data indicated that the sabinene biosynthesis pathway was successfully functioned in S. cerevisiae.
The N-terminal transit peptide of monoterpene synthetase is proteolyzed after targeting to plastid [14, 15]. When expression of the full-length ones in S. cerevisiae, it might result in reduction of enzyme activities due to the lack of plastid targeting and cutting mechanism of transit peptides. In order to further increase sabinene titer, sabinene synthase was attempted expressed in their N-truncated mature forms, and the proper truncation position was investigated according to the putative disordered region of SabS1 (M1-R43, Additional file 1: Fig. S3) predicted by the PSIPRED Workbench (http://www.cbs.dtu.dk/services/Chlorop/). As shown in Fig. 2b, SabS1 was truncated at three different positions within its N-terminus (i.e. L34, R43 and L52), generating proteins t34SabS1, t43SabS1 and t52SabS1, respectively. These three N-truncated SabS1 were introduced into strain YJGZ1 to measure their corresponding sabinene output. Besides, each N-truncated SabS1 was further fused with RFP and expressed in strain YJGZ1 to determine their expression level. As a result, compared with the full-length SabS1, these three ways of N-truncation did not weaken the expression level of SabS1 (Fig. 2c). However, none sabinene could be detected in the strains harboring t43SabS1 or t52SabS1 (Fig. 2c). That might due to the damage to the RR(X)8W motif (R43-W53 within SabS1, Additional file 1: Fig. S3) which is highly conserved among most of monoterpene synthetases and is critical to their catalytic activities [14]. Whereas, among those tested SabS1s, t34SabS1 achieved the highest sabinene production as well as the highest expression level, which were increased by 99.2% (reach to 5.28 mg/L) and 96.1% than the sabinene titer and the RFU (relative fluorescence units) of the strains harboring the full-length SabS1, respectively (Fig. 2c). This data suggested that truncation at L34 not only improve the expression level of soluble proteins, but also be benefit to its catalytic activity. Further dynamically down-regulated the transcription of wild-type gene ERG20 via replace the endogenous promoter of ERG20 by glucose-dependent weak promoter HXT1, increased the sabinene titer by 2.67-flod (to 19.4 mg/L, Fig. 2d). Correspondingly, the wild-type ERG20 in the host strain YJGZ1 was also down-regulated to generated strain YCTH1 for further optimization.
Investigation of the subcellular location of sabinene synthase and GPP
Yeast has many subcellular organelles, each of which has a unique physiological environment and cofactors to support different metabolic pathways [22]. Among these organelles, peroxisomes and mitochondria represent the typical ones enclosed by lipid monolayer and bilayer membranes, respectively. Both of these two organelles are rich in cofactors (like ATP and NADPH) for terpene synthesis. Notably, according to Liu et al. [23], the peroxisomal lumen was consider as an efficient site for reactions involved hydrophobic chemicals like fatty acids. Through harnessing peroxisomes as subcellular compartments for triterpenes synthesis, they dramatically increase squalene titer to an extremely high level [23]. Besides, the detoxification function of peroxisomes might be helpful to produce the cytotoxicity brought by monoterpenes. Thus, peroxisomes might suit monoterpene synthesis. Meanwhile, the high pH environment in mitochondria might be benefit to formation and maintain the critical carbocation intermediate within the reaction catalyzed by monoterpene and sesquiterpene synthase [24]. Farhi et al. [12] once reported that individually overexpression of valencene synthase or amorphadiene synthase in yeast mitochondria gained much more valencene and amorphadiene production than overexpression in the cytosol, suggesting that mitochondria provide a preferable environment for activities of sesquiterpene synthase. Correspondingly, the catalytic environment mitochondria might also be benefit to monoterpene synthase. Therefore, peroxisomes and mitochondria were select as the candidate organelles for sabinene production.
As known, free GFP expressed in yeasts usually uniformly dispersed in cytosol [25]. When introducing GFP into the strain harboring RFP fused t34SabS1, the overlapping green and red fluorescence signals observed by confocal laser scanning microscope indicated that t34SabS1 was located at cytosol (Fig. 3a). This cytosolic protein is hard to attach any precursor enclosed by organelle membranes. If GPP could pass through lipid membranes to form enough precursor pools in peroxisomes and mitochondria, simply expressing t34SabS1 in these two organelles would obtain satisfactory sabinene output. As reported, the signal peptides SKL (GGGSSKL) [26] and MLS (LSLRQSIRFFKPATRTLCSSRYLLQ) [27] have been successfully used to locate enzymes to peroxisomes and mitochondria, respectively. Here, these two protein-targeting signals were adopted and individually attached to the C-terminal and N-terminal of t34SabS1, respectively, and then expressed in strain YCTH1 in a fully chromosome integrated form (Additional file 1:Fig. S1c), while gene ERG20ww was carried by centromeric plasmid. In order to verify whether SKL or MLS fused t34SabS1 was targeting into the desired location, RFP was directly fused to the C-terminal of t34SabS1 (Additional file 1: Fig. S1d). And the GFP fused PEX3 [28] and COX4 [29] were co-expressed with t34SabS1–RFP as the specific marks of peroxisomes and mitochondria, respectively. As demonstrated by fluorescence microscopy, SKL and MLS directed t34SabS1s appeared shapes of dots and rings, respectively, which were consistent to the reported typical patterns of peroxisomes [26] and mitochondria [27], and overlapped with the corresponding specific markers (Fig. 3a). Thus, t34SabS1–SKL and MLS–t34SabS1 were successfully located in peroxisomes and mitochondria, respectively. In terms of sabinene production, unanticipated, expression of peroxisomal (P) or mitochondrial (M) t34SabS1 was not demonstrated significantly superior to utilization of cytosolic (C) t34SabS1 (Fig. 3b). Targeting t34SabS1 to peroxisomes or mitochondria achieved comparable titer to the one produced by cytosolic t34SabS1 (Fig. 3b). This data confirmed the existence of GPP pools in peroxisomes and mitochondria. And these parts of GPP should be utilized for sabinene production in addition to the cytosolic supply.
Combined subcellular compartmentalization of sabinene synthase
Subcellular targeting the key enzyme towards precursor storage location has been proved to be promising to achieve high level production. Lv et al. [27] once promote the synthesis of isoprene in S. cerevisiae through dual metabolic engineering of cytoplasmic and mitochondrial acetyl-CoA. Yang et al. [25] obtained highly effectively compartmentalized biosynthesis of triacylglycerol derived products in Yarrowia lipolytica via simultaneously targeted lipase dependent pathways directed towards three lipid related organelles. Herein, in order to realize adequate utilization of the GPP pools dispersed within the cell, t34SabS1s were tempted to be simultaneously targeted to different combination of locations. Firstly, three strains were constructed, i.e. strain Sc041063 for targeting t34SabS1 to cytosol as well as peroxisomes (CP), strain Sc041064 for targeting to cytosol as well as mitochondria (CM) and strain Sc041066 for targeting to peroxisomes as well as mitochondria (PM). As a result, all the stains harnessing double subcellular locations (CP, CM and PM) made significantly higher sabinene output than those achieved by cytosolic t34SabS1 with the same copy numbers (Fig. 3c). Among these constructed strains, strain Sc041064 (CM) gained the highest sabinene titer of 64.6 mg/L, which was 85.1% and 79.9% higher than those of strain Sc041063 (CP) and strain Sc041066 (PM), respectively (Fig. 3c). And this sabinene titer for CM was 1.53-fold and 1.73-fold higher than those of strains Sc041065 and Sc041067 with double copies of t34SabS1s in cytosol and mitochondria, respectively (Fig. 3c). However, supplement of another copy of peroxisomal t34SabS1 into strain Sc041064 (obtaining strain Sc041069 for CPM) could not further improve sabinene output, and even resulted in the larger errors on sabinene production (Additional file 1:Fig. S4), suggesting strain Sc041069 was extreme instability. That probably was due to the cell burden brought by high level expression of t34SabS1 in three subcellular location. And mitochondria might be sensitive in such case because them involved in energy supplies and central metabolism which are all vital to yeast survival [30]. Thus, strain Sc041064 harboring cytosolic and mitochondrial t34SabS1 was adopted for further optimization.
Regulation of mitochondria dynamics
Low capacity for substrates/enzymes is a limitation for many organelles [31]. Increasing local concentrations of substrates/enzymes in organelles may results in faster reaction rates and higher productivity. Through combining overexpression of PEX34 and deletion of PEX31 and PEX32, Zhou et al. [31] increased the peroxisome population and improve alkane production by 2-fold. This shows that genetically control cellular physiology to alter organellar number, volume and shape is critical to enhance the compartmentalized pathway. In our case, double the copy number of mitochondrial t34SabS1 did not alter the sabinene titer (Fig. 3b and 3c), indicating the concentration of GPP in mitochondria tended to be saturation relative to the concentration of mitochondrial t34SabS1. Thus, we should tempt to enlarge the number, size or dispersion of mitochondria to improve sabinene synthesis.
Biogenesis, growth and division of organelles are highly controlled by different mechanisms. In yeast, mitochondrial morphology depends on its dynamic behavior (called mitochondrial dynamics) including continuously move along cytoskeletal tracks as well as frequently balancing between fusion (interconnected networks) and fission (distinct small spherical particles) activities [32, 33]. Mitochondrial dynamics are important for their metabolism and many important functions. In order to attempt to improve sabinene production via altering mitochondrial morphology, ten proteins associated with mitochondrial dynamics (Table 1) were selected and individually overexpressed in their centromeric plasmid-based form in the t34SabS1 CM-targeted strain. These overexpressed proteins covered FIS1 [34] for mitochondrial division; MGM1 [35] for mitochondrial fusion; MMM1 [36] and SNF1 [37] for mitochondrial tubulation; ARC18 [38], LSB3 [39] and JSN1 [40] for mitochondrial motility; as well as other functional protein like GEM1 [41], MBA1 [42] and AIM25 [43]. Among these proteins, only overexpression of FIS1, LSB3, MBA1 and AIM25 significantly enhanced sabinene production (Additional file 1: Fig. S5).
Table 1
Proteins involved in mitochondrial dynamics
Process | Protein | Systematic Name | Proposed function | Reference |
Division | FIS1 | YIL065C | Mitochondrial fission protein, assembles with DNM1 and MDV1 into a ternary complex that mediates mitochondrial outer membrane division. | [34] |
Fusion | MGM1 | YOR211C | Mitochondrial GTPase, presents in complex with UGO1 and FZO1, required for mitochondrial inner membrane fusion. | [35] |
Tubulation | MMM1 | YLL006W | A member of ER-mitochondria encounter structure, assembles with MDM10 and MDM12 into a ternary complex in mitochondrial outer membrane for maintenance of mitochondrial DNA nucleoids and mitochondrial tubular shape. | [54] |
SNF1 | YDR477W | AMP-activated protein kinase in response to glucose depletion, involved in mitotic spindle alignment along the mother-bud axis. | [37] |
Motility | ARC18 | YLR370C | Subunit of the actin-related proteins ARP2/3 complex, required for actin polymerization-driven mitochondrial motility. | [38] |
JSN1 | YJR091C | RNA-binding proteins on the mitochondrial surface, co-localizes with ARP2/3 complex and supports them targeting to mitochondria. | [40] |
LSB3 | YFR024C | Binding to LAS17 which involved in actin patch assembly and actin polymerization. | [39] |
Other | GEM1 | YAL048C | Outer mitochondrial membrane GTPase, a member of ER-mitochondria encounter structure, requires for maintenance of mitochondrial morphology. | [41] |
MBA1 | YBR185C | Mitochondrial ribosome-binding protein localizes to mitochondrial inner membrane, involved in organization of the mitochondrial inner membrane and required for assembly of mitochondrial respiratory chain complexes. | [42] |
AIM25 | YJR100C | Mitochondrial protein involved in the regulation of chronological lifespan, and responses to both heat shock and oxidative stress, required for maintaining the integrity of the mitochondrial network. | [43] |
As demonstrated in Additional file 1: Fig. S6, overexpression of FIS1, LSB3, MBA1 and AIM25 did not affect cell growth, indicating these alternations on mitochondrial morphology did not damage the energy metabolism and central carbon metabolism in mitochondria. In order to determine the expression level of t34SabS1 located in cytosol and mitochondria, RFP was individually fused with the tested proteins expressed in these two locations. In the meanwhile, the mitochondria within the strains harboring RFP fused mitochondria t34SabS1 was specifically dyed by Rhodamine 123 [44]. As shown in Fig. 4b, when individual overexpression of FIS1, LSB3, MBA1 and AIM25, MLS–t34SabS1 still located only in mitochondria. Before tuning mitochondrial morphology, there was no significant difference between the expression level of cytosol and mitochondria-targeted t34SabS1 (Fig. 4a). And individual overexpression of these four proteins did not significantly altered the ratio between cytosol and mitochondria-targeted t34SabS1 (Fig. 4a), suggesting tuning mitochondrial morphology did not affect protein subcellular localization and expression level.
FIS1 is a mitochondrial fission protein mediating mitochondrial outer membrane division [34]. Compared with the mitochondria in the control strain, the mitochondria under FIS1 overexpression exhibited normal size but larger number, and diffused towards the center of cytoplasm (Fig. 4b). Differently, LSB3 interacts with the protein associated with actin assembly [39]. Overexpression of LSB3 did not affect mitochondrial subcellular dispersion, but enlarge the volume (Fig. 4b). Meanwhile, overexpression of MBA1 and AIM25 demonstrated similar pattern, i.e. dispersed mitochondria with larger numbers (Fig. 4b). These data suggested that increase on mitochondrial number and size might make the compartmentalized sabinene synthesis pathway to better cooperate. Among these four targets significantly associated with sabinene output, overexpression of AIM25 achieved the highest sabinene production, which was 1.16-fold increase (to 90.4 mg/L) of the sabinene titer of the control stain (Fig. 4a). Further insertion of all the engineered genes including ERG20ww, t34SabS1, MLS–t34SabS1 and AIM25 into the chromosome of the host strain YCTH1, boosted sabinene production to 154.9 mg/L (generating strain Sc0512020) (Fig. 4c). This titer was 58.5-fold of the one of our original stain.