Sustainable production of natural products using synthetic biology: Ginsenosides.

Synthetic biology approaches offer potential for large-scale and sustainable production of natural products with bioactive potency, including ginsenosides, providing a means to produce novel compounds with enhanced therapeutic properties. Ginseng, known for its non-toxic and potent qualities in traditional medicine, has been used for various medical needs. Ginseng has shown promise for its antioxidant and neuroprotective properties, and it has been used as a potential agent to boost immunity against various infections when used together with other drugs and vaccines. Given the increasing demand for ginsenosides and the challenges associated with traditional extraction methods, synthetic biology holds promise in the development of therapeutics. In this review, we discuss recent developments in microorganism producer engineering and ginsenoside production in microorganisms using synthetic biology approaches.

Metabolic recycling of storage lipids promotes squalene biosynthesis in yeast.

BACKGROUND: Metabolic rewiring in microbes is an economical and sustainable strategy for synthesizing valuable natural terpenes. Terpenes are the largest class of nature-derived specialized metabolites, and many have valuable pharmaceutical or biological activity. Squalene, a medicinal terpene, is used as a vaccine adjuvant to improve the efficacy of vaccines, including pandemic coronavirus disease 2019 (COVID-19) vaccines, and plays diverse biological roles as an antioxidant and anticancer agent. However, metabolic rewiring interferes with inherent metabolic pathways, often in a way that impairs the cellular growth and fitness of the microbial host. In particular, as the key starting molecule for producing various compounds including squalene, acetyl-CoA is involved in numerous biological processes with tight regulation to maintain metabolic homeostasis, which limits redirection of metabolic fluxes toward desired products. RESULTS: In this study, focusing on the recycling of surplus metabolic energy stored in lipid droplets, we show that the metabolic recycling of the surplus energy to acetyl-CoA can increase squalene production in yeast, concomitant with minimizing the metabolic interferences in inherent pathways. Moreover, by integrating multiple copies of the rate-limiting enzyme and implementing N-degron-dependent protein degradation to downregulate the competing pathway, we systematically rewired the metabolic flux toward squalene, enabling remarkable squalene production (1024.88 mg/L in a shake flask). Ultimately, further optimization of the fed-batch fermentation process enabled remarkable squalene production of 6.53 g/L. CONCLUSIONS: Our demonstration of squalene production via engineered yeast suggests that plant- or animal-based supplies of medicinal squalene can potentially be complemented or replaced by industrial fermentation. This approach will also provide a universal strategy for the more stable and sustainable production of high-value terpenes.

Chain flexibility of medicinal lipids determines their selective partitioning into lipid droplets.

In guiding lipid droplets (LDs) to serve as storage vessels that insulate high-value lipophilic compounds in cells, we demonstrate that chain flexibility of lipids determines their selective migration in intracellular LDs. Focusing on commercially important medicinal lipids with biogenetic similarity but structural dissimilarity, we computationally and experimentally validate that LD remodeling should be differentiated between overproduction of structurally flexible squalene and that of rigid zeaxanthin and ß-carotene. In molecular dynamics simulations, worm-like flexible squalene is readily deformed to move through intertwined chains of triacylglycerols in the LD core, whereas rod-like rigid zeaxanthin is trapped on the LD surface due to a high free energy barrier in diffusion. By designing yeast cells with either much larger LDs or with a greater number of LDs, we observe that intracellular storage of squalene significantly increases with LD volume expansion, but that of zeaxanthin and ß-carotene is enhanced through LD surface broadening; as visually evidenced, the outcomes represent internal penetration of squalene and surface localization of zeaxanthin and ß-carotene. Our study shows the computational and experimental validation of selective lipid migration into a phase-separated organelle and reveals LD dynamics and functionalization.

Metabolite trafficking enables membrane-impermeable-terpene secretion by yeast.

Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells. We investigate whether engineered microbes can exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner. In this study, we demonstrate a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking is highly achievable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically couple a terpene-binding protein with an export signal peptide and subsequently demonstrate efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for ß-carotene). Other carrier proteins can also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the most efficient cognate pathway for metabolite secretion by microorganisms.

Pairing of orthogonal chaperones with a cytochrome P450 enhances terpene synthesis in Saccharomyces cerevisiae.

The supply of terpenes is often limited by their low extraction yield from natural resources, such as plants. Thus, microbial biosynthesis has emerged as an attractive platform for the production of terpenes. Many strategies have been applied to engineer microbes to improve terpene production capabilities; however, functional expression of heterologous proteins such as cytochrome P450 enzymes (P450s) in microbes is a major obstacle. This study reports the successful pairing of cognate chaperones and P450s for functional heterologous expression in Saccharomyces cerevisiae. This chaperone pairing was exploited to facilitate the functional assembly of the protopanaxadiol (PPD) biosynthesis pathway, which consists of a P450 oxygenase and a P450 reductase redox partner originating from Panax ginseng and Arabidopsis thaliana, respectively. We identified several chaperones required for protein folding in P. ginseng and A. thaliana and evaluated the impact of the coexpression of the corresponding chaperones on the synthesis and activity of PPD biosynthesis enzymes. Expression of a chaperone from P. ginseng (PgCPR5), a cognate of PPD biosynthesis enzymes, significantly increased PPD production by more than 2.5-fold compared with that in the corresponding control strain. Thus, pairing of chaperones with heterologous enzymes provides an effective strategy for the construction of challenging biosynthesis pathways in yeast.

Engineering Cell Wall Integrity Enables Enhanced Squalene Production in Yeast.

Microbial production of many lipophilic compounds is often limited by product toxicity to host cells. Engineering cell walls can help mitigate the damage caused by lipophilic compounds by increasing tolerance to those compounds. To determine if the cell wall engineering would be effective in enhancing lipophilic compound production, we used a previously constructed squalene-overproducing yeast strain (SQ) that produces over 600 mg/L of squalene, a model membrane-damaging lipophilic compound. This SQ strain had significantly decreased membrane rigidity, leading to increased cell lysis during fermentation. The SQ strain was engineered to restore membrane rigidity by activating the cell wall integrity (CWI) pathway, thereby further enhancing its squalene production efficiency. Maintenance of CWI was associated with improved squalene production, as shown by cell wall remodeling through regulation of Ecm33, a key regulator of the CWI pathway. Deletion of ECM33 in the SQ strain helped restore membrane rigidity and improve stress tolerance. Moreover, ECM33 deletion suppressed cell lysis and increased squalene production by approximately 12% compared to that by the parent SQ strain. Thus, this study shows that engineering of the yeast cell wall is a promising strategy for enhancing the physiological functions of industrial strains for production of lipophilic compounds.

Circulating Tumor Cell Number Is Associated with Primary Tumor Volume in Patients with Lung Adenocarcinoma.

BACKGROUND: Circulating tumor cells (CTCs) are frequently detected in patients with advanced-stage malignant tumors and could act as a predictor of poor prognosis. However, there is a paucity of data on the relationship between CTC number and primary tumor volume in patients with lung cancer. Therefore, our study aimed to evaluate the relationship between CTC number and primary tumor volume in patients with lung adenocarcinoma. METHODS: We collected blood samples from 21 patients with treatment-naive lung adenocarcinoma and 73 healthy individuals. To count CTCs, we used a CTC enrichment method based on fluid-assisted separation technology. We compared CTC numbers between lung adenocarcinoma patients and healthy individuals using propensity score matching, and performed linear regression analysis to analyze the relationship between CTC number and primary tumor volume in lung adenocarcinoma patients. RESULTS: CTC positivity was significantly more common in lung adenocarcinoma patients than in healthy individuals (p<0.001). The median primary tumor volume in CTC-negative and CTC-positive patients was 10.0 cm³ and 64.8 cm³, respectively. Multiple linear regression analysis showed that the number of CTCs correlated with primary tumor volume in lung adenocarcinoma patients (ß=0.903, p=0.002). Further subgroup analysis showed a correlation between CTC number and primary tumor volume in patients with distant (p=0.024) and extra-thoracic (p=0.033) metastasis (not in patients with distant metastasis). CONCLUSION: Our study showed that CTC numbers may be associated with primary tumor volume in lung adenocarcinomas patients, especially in those with distant metastasis.

Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway.

The endoplasmic reticulum (ER) is a dynamic organelle that synthesizes and folds proteins. An imbalance between the ER protein synthesis load and its folding capacity triggers the unfolded protein response, thereby restoring normal ER functions via size adjustment. Inspired by such inherent genetic programming events, we engineered Saccharomyces cerevisiae to expand the ER by overexpressing a key ER size regulatory factor, INO2. ER space expansion enhanced ER protein synthesis and folding capacity, and relieved metabolic constraints imposed by the limited enzyme abundance. Harnessing the yeast ER for metabolic engineering, we ultimately increased the production of squalene and cytochrome P450-mediated protopanaxadiol by 71-fold and 8-fold, compared to their respective control strains without overexpression of INO2. Furthermore, genome-wide transcriptome analysis of the ER-expanded strain revealed that the significant improvement in terpene production was associated with global rewiring of the metabolic network. Therefore, the yeast ER can be engineered as a specialized compartment for enhancing terpene production, representing new possibilities for the high-level production of other value-added chemicals.

Development of Dissociation-Enhanced Lanthanide Fluoroimmunoassay for Measuring Leptin.

Development of a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) for measuring leptin, a satiety hormone of appetite control, was conducted in sandwich assay format exploiting a microplate immobilized with an anti-leptin antibody and another antibody raised against leptin and tagged with an europium chelate. In the leptin DELFIA of this study, amounts of antibody coated to the microplate and of the bioconjugate for the second immune reaction were optimized as 0.5 µg and 200 ng per well, respectively. When plotted in double-logarithmic scale, a linear relationship of y (log10 response signal) = 0.6023× (log10 leptin concentration) + 3.4084 (r(2) = 0.9646) was obtained at the leptin concentrations of 0.01─50 ng/mL with the limit of detection of 0.01 ng/mL. Individual leptin concentrations in various samples were well convergent to the calibration curve of the current assay. When applied to the measurement of leptin in a rat serum, the present assay was found quite effective and was competitive to a commercial sandwich-type ELISA.

Enzymatic hydrolysis of anchovy fine powder at high and ambient pressure, and characterization of the hydrolyzates.

BACKGROUND: At specific conditions of high pressure, the stability and activity of some enzymes are reportedly known to increase. The aim of this study was to apply pressure-tolerant proteases to hydrolyzing anchovy fine powder (AFP) and to determine product characteristics of the resultant hydrolyzates. RESULTS: Anchovy fine powder enzyme hydrolyzates (AFPEHs) were produced at 300 MPa and ambient pressure using combinations of Flavourzyme 500MG, Alcalase 2.4L, Marugoto E and Protamex. When the same protease combination was used for hydrolysis, the contents of total soluble solids, total water-soluble nitrogen and trichloroacetic acid-soluble nitrogen in the AFPEHs produced at 300 MPa were conspicuously higher than those in the AFPEHs produced at ambient pressure. This result and electrophoretic characteristics indicated that the high-pressure process of this study accelerates protein hydrolysis compared with the ambient-pressure counterpart. Most peptides in the hydrolyzates obtained at 300 MPa had molecular masses less than 5 kDa. Functionality, sensory characteristics and the content of total free amino acids of selected hydrolyzates were also determined. CONCLUSION: The high-pressure hydrolytic process utilizing pressure-tolerant proteases was found to be an efficient method for producing protein hydrolyzates with good product characteristics.

Development of Antigen-Immobilized Metallothionein Sensor that Exploits Gold Nanoparticle-Based Enhancement of Signal.

An antigen-immobilized indirect-competitive immunosensor that detects metallothionein (MT), a potent biomarker of contamination with heavy metals, was developed exploiting enhancement of signal based on an additional binding of gold nanoparticles to an anti-MT antibody through the biotin-avidin interaction. The sensor was constructed by the immobilization of MT at 1 mg/mL on a 9-MHz quartz crystal microbalance and the concentration of the antibody for competitive reaction was optimized as 10 µg/mL based on the degree of sensor response. At this moment, the control response of the sensor obtained with enhancement of signal was 343.8 Hz and was larger than that without enhancement of signal 2.47 fold. The sensor responses decreased gradually with increasing analyte concentrations, and a linear relationship between analyte concentration and sensor response was acquired in the range of 0.005-1 ng/mL MT in double-logarithmic scales with a correlation coefficient (r) of 0.9858. The limit of detection of the present sensor was presumed to be present below 5 pg/mL MT.