Preface
Considering that the long biosynthetic pathways of terpenoids (e.g., geraniol, citronellol, linalool) may impose metabolic burden on a single chassis organism, we plan to employ a co-culture system of yeast and Escherichia coli (E. coli) for the collaborative production of customized essential oil components, thereby distributing the metabolic load (Fig. 1).
Fig.1 Strategy of two microorganisms for collaborative production.
We aim to establish a one-stop production system for customized mixed essential oils, eliminating the need for post-production mixing of individual essential oil components. The solution lies in the division of labor among different chassis organisms: Saccharomyces cerevisiae (S. cerevisiae), the most widely used eukaryotic model organism, is typically employed for producing complex molecules such as artemisinin[1]; while E. coli, though evolutionarily simpler than S. cerevisiae, exhibits higher efficiency in manufacturing simple products (e.g., those requiring no post-synthetic processing)[2].
Accordingly, our strategy involves the co-cultivation of S. cerevisiae and E. coli (Fig. 2), with pathway splitting at the geraniol node: yeast is engineered to produce the precursor geraniol, which accumulates intracellularly before being secreted into the culture medium. Geraniol then diffuses into E. coli cells, where it serves as a substrate for the synthesis of citronellol or linalool. Through this co-cultivation system, yeast and E. coli collaborate to accomplish the production of distinct essential oil components.
Fig.2 Distribution the biosynthesis pathways of geraniol, citronellol and linalool between S. cerevisiae and E. coli.
Geranyl Pyrophosphate Synthase, GPPS; Geraniol Synthase, GES; Geraniol Reductase, GER; Linalool isomerase, Lis.
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1. Construction of Microbial Genetic Circuits
1.1 Design of Yeast Genetic Circuits
Yeast cells possess a robust native MVA pathway, which can supply abundant IPP and DMAPP for GPP synthesis (Fig. 3). Therefore, S. cerevisiae was selected as the chassis organism for GPP and geraniol production.
GPPS is a key enzyme for GPP synthesis and also a gatekeeping enzyme linking central metabolism (MVA/MEP pathways) to the biosynthesis of the large monoterpenoid family[3]. However, due to the instability of GPP, it is difficult to accumulate intracellularly or excrete extracellularly. Thus, we introduced GPPS and GES into S. cerevisiae, enabling GPP to be converted into geraniol for intracellular accumulation and subsequent transport to the extracellular environment.
Fig.3 S. Cerevisiae has native MVA pathway to synthesize GPP using IPP and DMAPP, then GPP is converted to geraniol which will be accumulated and tranported into the medium.
Geranyl Pyrophosphate Synthase, GPPS, GPPS; Geraniol Synthase, GE.
To obtain large amounts of GPP for subsequent geraniol production, we intend to overexpress GPPS and GES in S. cerevisiae. For genetic circuit construction, the pYES2 plasmid was selected as the expression vector for GPPS and GES, based on the following reasons:
① High copy number: The pYES2 plasmid contains a 2μ replicon, with approximately 20-40 copies per cell.
②Facilitated screening: It carries the URA3 selectable marker, which is fully compatible with the auxotrophic S. cerevisiae strain INVSc1. This allows plate screening on synthetic complete medium lacking uracil (SD-Ura).
③Strong inducible promoter: Its key advantage is controllable expression. In glucose medium without galactose, the expression of exogenous genes is completely repressed; upon galactose addition, expression levels become extremely high. Using an inducible promoter enables "one-click activation" of geraniol synthesis after cells reach an appropriate density, avoiding growth inhibition in the early growth stage and ultimately achieving higher cell density and product yield.
We constructed the two genes (GPPS and GES) under the control of a single promoter, and also generated pYES2-EGFP as the experimental control group (Fig. 4 and 5).
Fig.4 GPPS and GES genes were constructed into the pYES2 plasmid, followed by transformation into S. Cerevisiae.
Fig.5 S. EGFP gene was constructed into the pYES2 plasmid, followed by transformation into S. Cerevisiae, serving as a control.
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1.2 Design of E. coli Genetic Circuits
Expression vectors harboring the gene encoding geraniol reductase (which reduces geraniol to citronellol) from Rosa damascena (Damask rose) and the gene encoding linalool isomerase (which converts geraniol to linalool) from bacteria were constructed and then transformed into E. coli. Once geraniol accumulates in S. cerevisiae and diffuses into the culture medium, it can be taken up by E. coli and utilized for the synthesis of citronellol and linalool.
Fig.6 Designed pathway within E. coli. Linalool isomerase, Lis; Geraniol Reductase, GER.
1.2.1 Construction of recombinant plasmid pACYCDuet-1-GER-Lis
The "Duet" series of plasmids are powerful tools for multi-gene co-expression. To reduce competition between plasmids, we cloned geraniol reductase and linalool isomerase into the two multiple cloning sites (MCS) of the high-copy plasmid pACYCDuet-1, respectively. This enables the co-expression of these two genes under the control of the same strong promoter (T7/lac), allowing the "one-click activation" of the synthesis of multiple essential oil components in E. coli.
Fig.7 GER and Lis genes were constructed into the plasmid of pACYCDuet-1, followed by transformation into E. coli. Geraniol Reductase, GER, Linalool isomerase, Lis.
Prior to gene synthesis, we optimized the gene sequences based on the codon preference of E. coli. The constructed recombinant plasmid pACYCDuet-1-GER-Lis was then transformed into competent BL21(DE3) cells, and positive clones were screened on agar plates containing chloramphenicol. The genome of the BL21 strain harbors the T7 RNA polymerase gene (controlled by the lacUV5 promoter) and also contains the gene encoding the LacI repressor protein. Upon induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), the strain can efficiently express recombinant proteins, which are used for the synthesis of citronellol and linalool.
1.2.2 Design of the Orthogonal Expression System
On the pACYCDuet-1-GER-Lis plasmid, although the "one-click activation" synthesis can be achieved since both GER and Lis genes are co-controlled by the T7 promoter and IPTG induction, it fails to meet the requirement for regulating the personalized production of different essential oil components. We optimized the previous design using an orthogonal expression system to enable separate control over the synthesis of different essential oil components.
A vector that allows two promoters to be induced by two different inducers on the same plasmid is generally referred to as a "dual-expression vector" or "orthogonal expression system". The core of this design lies in the use of two orthogonal promoter-inducer systems. The term "orthogonal" means there is no cross-reactivity between the two systems: inducer A only activates promoter A without affecting promoter B, and vice versa. However, there is currently no "ready-to-use" commercial single plasmid that directly integrates two such promoters, so we have to construct an orthogonal combinatorial plasmid ourselves.
The T7/lac system consists of the T7 promoter and the repressor protein LacI, which is induced by IPTG to achieve high expression levels and is one of the most commonly used expression systems. The araBAD (pBAD) system comprises the pBAD promoter and the repressor protein AraC, which is induced by L-arabinose. It offers highly precise regulation, with induction levels linearly adjustable according to arabinose concentration, and is completely orthogonal to the T7/lac system. Therefore, we plan to construct an orthogonal combinatorial plasmid containing both T7/lac and pBAD promoters for the expression of GER and Lis genes.
The procedure was performed in two steps: first, two recombinant plasmids, pET28a(+)-GER and pBAD-Lis, were constructed. pET28a(+) contains the T7/lac promoter and is induced for expression by IPTG; pBAD contains the araBAD promoter and is induced for expression by L-arabinose. Subsequently, the fusion of the two plasmids, namely the orthogonal combination plasmid-GER-Lis, was constructed using homologous recombination PCR. This plasmid contains KanR, GER, T7/lac promoter and LacI genes from pET28a(+)-GER, as well as AraC, pBAD promoter, Lis，ApR genes，and Ori site from pBAD-Lis, with a total length of 9926 base pairs.
Fig 8 Construction of Orthogonal combination plasmid-GER-Lis using recombinant PCR method.
Geraniol Reductase, GER, Linalool isomerase, Lis.
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2. Optimization Strategies for Metabolic Engineering
Although certain optimization strategies have been adopted in the design of this project, further optimizations from multiple aspects of metabolic engineering are still required to improve the yield. These specifically include the following: enhancing precursor supply, engineering key enzymes, regenerating cofactors, reducing cytotoxicity, increasing product secretion, optimizing culture conditions, etc.
2.1 Modifying Genome and Enhancing Precursor Supply
Natural GPP serves not only as a precursor for monoterpene synthesis but also as a precursor for farnesyl pyrophosphate (FPP). However, the endogenous metabolic flux of S. cerevisiae naturally tends to flow toward FPP and sterol synthesis. To achieve efficient GPP synthesis, a combinatorial strategy must be adopted.
The ERG20 gene encodes FPP synthase, a bifunctional enzyme that acts as the core enzyme for the synthesis of the C15 terpenoid precursor (FPP) in yeast cells. This enzyme catalyzes two consecutive condensation reactions:
① condensing IPP and DMAPP into GPP;
② further condensing GPP with another molecule of IPP into FPP. As the "core factory" for FPP synthesis, it strongly converts GPP into FPP.
The conversion of GPP to FPP can be reduced by knocking out or attenuating the endogenous ERG20 gene. However, complete knockout of ERG20 is lethal (since FPP is an essential substance). Therefore, we introduced a mutant FPP synthase (ERG20-F96W-N127W) into the genome of S. cerevisiae via homologous recombination. This mutant exhibits significantly reduced FPP synthase activity while retaining a certain level of enzymatic activity to maintain cell viability[5,6].
Fig.9 Introduction of the mutant ERG20-F96W-N127W into the genome of S. Cerevisiae can increase GPP biosynthesis to accumulate geraniol.
Against the background of introducing the aforementioned ERG20 mutant into S. cerevisiae, GPPS and GES were further overexpressed via the plasmid pYES2-GPPS-GES. The endogenous and exogenous genes act synergistically to maximize the conversion of IPP/DMAPP into GPP, thereby achieving high-level accumulation of geraniol.
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2.2 Reducing Product Cytotoxicity
A core challenge in microbial monoterpene synthesis is product toxicity. These terpenoid compounds possess cell membrane permeability; when accumulated to a certain concentration in the culture medium, they exert significant growth inhibition or even lethal effects on microbial hosts, thereby severely limiting the final yield and production efficiency. To overcome this bottleneck, in-situ product removal (ISPR) technology has emerged. Among various ISPR strategies, two-phase extractive fermentation is an efficient one: it involves introducing a water-immiscible organic solvent as the second phase into the fermentation system to extract the produced terpenoid products from the aqueous phase (culture medium) in real time. This maintains the product concentration in the aqueous phase at a low-toxicity level, ensures cell growth and metabolic activity, and ultimately achieves a substantial increase in total yield[7].
We intend to use isopropyl myristate (IPM), n-dodecane, and n-hexane as organic overlay layers respectively to determine which one exhibits the best performance.
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2.3 Optimization of Culture Conditions
When constructing a S. cerevisiae-E. coli co-culture system for the efficient production of citronellol and linalool, optimizing the culture conditions is crucial.
2.3.1 Culture Medium and Carbon Source
A unified culture medium is required for co-culture to meet the basic growth needs of both microorganisms. Generally, a minimal salt medium (e.g., M9 medium) is used with glucose added as the main carbon source[8]. The key is to ensure a sufficient carbon source concentration; if necessary, a fed-batch fermentation mode can be adopted to continuously supply the carbon source.
2.3.2 Inoculation Ratio and Timing
Inoculation ratio and timing are core factors affecting the balance between the two strains and the efficiency of metabolite transfer. It is necessary to optimize the initial inoculation ratio of the two strains through experiments[9] to achieve a dynamic balance between the supply and consumption of geraniol, and avoid overgrowth or inhibition of either strain.
2.3.3 Temporal Control
A staged inoculation strategy can be adopted: first, yeast is inoculated and allowed to grow to a certain density and start geraniol synthesis, after which E. coli is inoculated. This ensures that when E. coli begins to express recombinant enzymes, there is already a sufficient amount of geraniol precursor available, thereby avoiding resource competition and improving production efficiency.
2.3.4 Temperature Control
Temperature significantly affects the growth rate and enzyme activity of the two strains. A temperature of 33.5°C has been reported as an optimal choice for the E. coli-S. cerevisiae co-culture system, as it well balances the growth and metabolic needs of both microorganisms [11]. Experiments are required to determine the optimal temperature for the specific production system.
2.3.5 pH Regulation
Maintaining a suitable pH environment is crucial for cell activity and enzyme function. Studies have shown that maintaining the pH of the culture system at approximately 7.0 (neutral condition) is beneficial for the stability and production efficiency of the co-culture system[10]. A buffer system or real-time control by adding acid/alkali should be used.
2.3.6 Inducer Concentration and Induction Time
The concentration of the inducer IPTG is typically optimized within the range of 0.1-1.0 mM; the concentration of L-arabinose is usually tested between 0.1% and 0.2% (w/v). In addition, samples need to be taken at specific time points to determine the optimal induction time.
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References
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