OBJECTIVE To study the improvement effect and mechanism of Tripterygium wilfordii multiglucoside （TWM） on nephrotic syndrome in rats. METHODS The nephrotic syndrome model was established by intravenous injection of adriamycin via the tail vein. The modeling rats were randomly divided into the model group （distilled water）， prednisone group （10 mg/kg）， and TWM high- and low-dose groups （10 and 5 mg/kg， respectively）. Additionally， blank group （distilled water） without model induction was established. Each group consisted of 9 rats. Rats in each group were administered the corresponding drugs or distilled water by gavage， once a day， for 6 consecutive weeks. The histopathological morphology of kidney tissues in rats was observed； the levels of 24-hour urinary protein （24 h-UTP） and serum biochemical indicators [albumin （ALB）， blood urea nitrogen （BUN）， serum creatinine （SCr）， cholesterol （CHOL）， and triglyceride （TG）] in rats were determined； the levels of oxidative stress indicators [superoxide dismutase （SOD）， malondialdehyde （MDA）] in kidney tissue of rats were determined； expressions of hypoxia-inducible factor-1α （HIF-1α）/microRNA-155-5p （miR-155-5p）/nuclear factor erythriod 2- related factor 2 （Nrf2） signaling pathway-related mRNA and protein in the renal tissues of rats were detected. RESULTS Compared with the blank group， the rats in the model group exhibited disordered renal tissue structure， with a small amount of glomerular necrosis and edema of the renal tubular epithelial cells. 24 h-UTP， serum levels of SCr， BUN， CHOL and TG， MDA content， mRNA and protein expressions of HIF-1α and Keap1 as well as the expression of miR-155-5p in renal tissues were increased significantly （ P ＜0.05）. Serum level of ALB， SOD level in renal tissue as well as mRNA and protein expressions of Nrf2 were decreased significantly （ P ＜0.05）. Compared with the model group， TWM high-dose and low-dose groups exhibited significant improvements in renal injury， with notable reversals in the levels of the above quantitative indicators （ P ＜0.05）. CONCLUSIONS TWM can alleviate oxidative stress-induced damage and thereby improve nephrotic syndrome in rats by regulating the HIF-1α/miR-155-5p/Nrf2 signaling pathway.

ObjectiveTaking the cuproptosis/oxidative stress pathway as the entry point， this study investigated the effect and mechanism of Liangyi Paste on hepatic lipid deposition in naturally aged mice fed with a high-fat diet. MethodsAfter adaptive feeding， 80 ten-week-old male C57BL/6 mice were used. Thirty of them were randomly divided into three groups （10 mice per group）： The 12-month-old control group （12MCON）， the 15-month-old control group （15MCON）， and the 15-month-old group with a high-fat diet （15MHFD）. The 12MCON and 15MCON groups were continuously fed a standard diet， while the 15MHFD group started receiving a high-fat diet at 12 months of age. Tissue samples were collected at the corresponding time points for each group. The remaining 50 mice were randomly divided into five groups （10 mice per group）： the 20-month-old control group （20MCON）， the model group， and the low-， medium-， and high-dose Liangyi Paste groups （2.91 ， 5.82 ， 11.64 g·kg^-1·d^-1， respectively）. The 20MCON group was continuously fed a standard diet， while the other groups started receiving a high-fat diet at 15 months of age. At 18 months of age， the Liangyi Paste groups were administered the corresponding doses of Liangyi Paste by gavage， while the 20MCON and model groups were given an equal volume of saline by gavage. After 8 weeks of continuous gavage （when the mice reached 20 months of age）， tissue samples were collected. Hepatic TG levels were measured using assay kits； liver histology and lipid deposition were observed via hematoxylin-eosin （HE） and oil red O staining； reactive oxygen species （ROS） were detected by enzyme-linked immunosorbent assay （ELISA）； Cu²⁺， superoxide dismutase （SOD）， and malondialdehyde （MDA） levels were measured by colorimetry； mRNA and protein expression of genes related to cuproptosis and oxidative stress pathways were analyzed by Real-time polymerase chain reaction（Real-time PCR） and Wes automated protein expression system. ResultsCompared with 12MCON， the 15MCON group showed significantly increased hepatic TG， Cu²⁺， ROS， and MDA levels （P<0.01）， decreased SOD （P<0.01）， hepatocyte swelling， and disordered arrangement. The mRNA and protein levels of ferredoxin 1 （FDX1）， dihydrolipoamide S-acetyltransferase （DLAT）， heat shock protein 70 （HSP70）， dihydrolipoamide dehydrogenase （DLD）， pyruvate dehydrogenase E1 subunit-β （PDHB）， nuclear factor erythroid 2-related factor 2 （Nrf2）， and peroxisome proliferator-activated receptor γ （PPARγ） were significantly elevated （P<0.05， P<0.01）. Compared with 15MCON group， the 15MHFD and 20MCON groups exhibited further increases in TG， Cu²⁺， ROS， and MDA （P<0.01）， reduced SOD （P<0.01）， and aggravated hepatocyte swelling and disorder. There were increased lipid droplets with mild vacuolization in the 15MHFD group， and no significant lipid deposition was observed in the 20MCON group. FDX1， DLAT， HSP70， DLD， PDHB， Nrf2， and PPARγ mRNA and protein levels were significantly increased （P<0.05， P<0.01）. Compared with 20MCON group， the model group demonstrated markedly elevated TG， Cu²⁺， ROS， and MDA （P<0.01）， reduced SOD （P<0.01）， severe hepatic steatosis， and upregulated expression of FDX1， DLAT， HSP70， DLD， PDHB， Nrf2， and PPARγ mRNA and proteins （P<0.05， P<0.01）. All abnormalities were significantly reversed after Liangyi Paste treatment. ConclusionLiangyi paste can ameliorate hepatic lipid deposition in naturally aged mice with a high-fat diet by modulating the cuproptosis/oxidative stress pathway.

Objective To evaluate the safety and efficacy of a self-developed micro-normothermic machine perfusion (NMP) system (micro-perfusion device) for preserving isolated porcine limbs. Methods Five healthy Landrace pigs were selected, and their left and right forelimbs were randomly divided into the NMP group and static cold storage (SCS) group. The NMP group was perfused with the self-developed micro-perfusion device and polymerized hemoglobin perfusate for 32 hours at normothermia, while the SCS group was preserved at 4 ℃. Hemodynamic parameters such as perfusion pressure and flow were monitored. The pH value, partial pressure of oxygen (PO₂), lactic acid (Lac), creatine kinase (CK) and lactate dehydrogenase (LDH) in the perfusate were measured. Hematoxylin-eosin staining was used to assess the muscle tissue structure, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was employed to evaluate muscle cell apoptosis, and immunohistochemistry staining was applied to detect the expressions of tumor necrosis factor (TNF)-α and interleukin (IL)-6. A mixed-effects model was used to analyze the effects of time and treatment methods on tissue structure, cell apoptosis and inflammatory factors. Results The device could stably maintain a perfusion pressure of (69±15) mmHg and a flow rate of (117±42) mL/min. The pH value and electrolytes of the perfusate were generally stable, with PO₂ maintained at a high level. Lac was maintained at 5.38(3.81, 6.45) mmol/L, while CK and LDH increased over time. After 32 hours of perfusion in the NMP group, both the myocyte spacing and apoptosis rate were better than those in the SCS group. Mixed-effects model analysis showed that there were statistically significant differences in the effects of NMP treatment and SCS treatment on myocyte spacing and apoptosis rate per unit time (both P < 0.05). There were no statistically significant differences in TNF-α and IL-6 between the two groups, and mixed-effects model analysis showed no statistically significant differences in the effects of NMP treatment and SCS treatment on TNF-α and IL-6 per unit time (both P > 0.05). Conclusions The micro-perfusion device used in this study may achieve 32-hour normothermic preservation in a porcine limb amputation model, maintain basic metabolism and ionic homeostasis, reduce muscle structural damage and cell apoptosis without inducing additional inflammatory responses. This technology is expected to significantly extend the time window for replantation of amputated limbs in disaster rescue and long-distance transportation, providing an important technical basis for clinical translation and subsequent replantation research.

Background: Influenza A virus (IAV) is a negative-sense RNA virus of the Orthomyxoviridae family and is the etiological agent of a highly contagious acute respiratory disease that can lead to acute lung injury. Objective: To elucidate the molecular mechanisms of IAV infection, an integrative research approach combining gene expression profiling, multinetwork analysis, and in vivo experimental validations was employed. Methods: First, a series of network-based analyses were performed, including protein-protein interaction network construction, weighted gene co-expression network analysis, and subsequent gene set enrichment analysis, to identify the major underlying mechanisms of IAV infection. Following gene expression analysis, core targets, both direct and indirect regulators, were screened. An IAV (H1N1) strain A/PR/8/34-induced acute lung injury mouse model was constructed for in vivo validations. Batch one included two groups to evaluate findings from the multi-network analysis: Mock (n = 10; 5 males and 5 females) and IAV (n = 10; 5 males and 5 females). Batch two included three groups to assess the role of toll-like receptor 3 (TLR3) in IAV infection: Mock (n = 6; 3 males and 3 females), IAV (n = 6; 3 males and 3 females), and TLR3 inhibitor (n = 6; 3 males and 3 females). Body weight was measured on days 0, 3, and 5 after infection. On day 5, lung tissues were collected to assess viral load and histopathological changes. Key targets were examined using enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining, both in sera and lung tissues. Results: IAV infection was significantly associated with dysregulation of the immune-inflammation system, such as the LTR, nucle-otide-binding oligomerization domain-(NOD) like receptor, retinoic acid-inducible gene I-like receptor, and nuclear factor kappa-B signaling pathways. Gene set enrichment analysis further indicated that the TLR and necroptosis signaling pathways played crucial roles in the progression of IAV infection (TLR signaling pathway normalized enrichment score = 2.3941, P = 1.00 × 10 ⁻¹⁰; necroptosis normalized enrichment score = 1.9421, P = 6.21 × 10 ⁻⁷). Among the core targets, TLR3 and mixed lineage kinase domain-like protein (MLKL) may regulate gene expression at the transcriptional level (all P < 0.05). In vivo validation using an IAV (PR8) infected acute lung injury mouse model demonstrated increased viral load and lung index, alveolar structural damage, and inflammatory cell infiltration. Immunofluorescence staining exhibited large gaps in Lamin B1 staining and breaches in Emerin signals following IAV-PR8 infection. Expression levels of TLR3, p-receptor-interacting serine/threonine-protein kinase 3 (RIPK3)/RIPK3, and p-mixed lineage kinase domain-like protein (MLKL)/MLKL proteins in lung tissues, as well as proinflammatory factors and mediators in sera, were significantly elevated after IAV infection. Moreover, enhanced neutrophil infiltration (myeloperoxidase) and citrullinated histone H3 (a neutrophil extracellular trap-specific marker), both established indicators of neutrophil extracellular trap formation, were observed. Notably, treatment with a TLR3 inhibitor significantly ameliorated IAV-induced acute lung injury by regulating necroptosis-related targets. Conclusion: Our study provides network-based in vivo evidence that TLR3-receptor-interacting serine/threonine-protein kinase 3-MLKL-mediated necroptosis may underlie IAV-induced acute lung injury and could serve as a potential therapeutic target in severe influenza cases.

ObjectiveTo investigate the antidepressant effects and mechanisms of total flavonoids from Cuscutae Semen （TFCC） in the mouse model of chronic unpredictable mild stress （CUMS）. MethodsFifty male 4-week-old ICR mice were randomized into five groups （n=10 per group）： blank control， model， Cuscutae Semen decoction （10.2 g·kg^-1·d^-1）， paroxetine （2.6 mg·kg^-1·d^-1）， and TFCC （173.2 mg·kg^-1·d^-1）. The other groups except the blank control group underwent chronic unpredictable mild stress （CUMS） for 4 weeks. Behavioral assessments were conducted post-modeling. Then， the model group received distilled water （10 mL·kg^-1·d^-1）， while treatment groups were administrated with respective agents via oral gavage （10 mL·kg^-1） for 4 weeks. Depression-like behaviors were evaluated by the sucrose preference test （SPT）， forced swimming test （FST）， and tail suspension test （TST）. Hippocampal neuronal morphology was observed via hematoxylin-eosin staining， and apoptosis in the brain tissue was assessed via terminal- deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL）. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the hippocampal levels of inflammatory cytokines ［interleukin （IL）-1β， IL-6， and TNF-α）］ and neurotransmitters ［5-hydroxytryptamine （5-HT）， dopamine （DA）， and brain-derived neurotrophic factor （BDNF）］， while the reactive oxygen species （ROS） levels were quantified via the DCFH-DA probe. Real-time PCR was performed to measure the mRNA levels of NOD-like receptor protein 3 （NLRP3）， apoptosis-associated Speck-like protein containing a CARD （ASC）， cysteinyl aspartate-specific proteinase-1 （Caspase-1）， IL-1β， and inducible nitric oxide synthase （iNOS）. Western blot was employed to evaluate the protein levels of NLRP3， ASC， Caspase-1， uncoupling protein 2 （UCP2）， and thioredoxin-interacting protein （TXNIP）. ResultsCompared with the blank control group， the model group exhibited weight loss （P<0.01）， reduced sucrose preference （P<0.01）， prolonged immobility time in FST and TST （P<0.01）， neuron disarrangement with nuclear pyknosis in hippocampal CA3 region， increased apoptosis in the brain tissue， elevated levels of IL-1β， IL-6， and TNF-α （P<0.01）， declined levels of 5-HT， DA， and BDNF （P<0.01）， increased ROS accumulation （P<0.01）， upregulated mRNA levels of NLRP3， ASC， Caspase-1， IL-1β， and iNOS （P<0.01）， down-regulated protein level of UCP2 （P<0.01）， and up-regulated protein levels of NLRP3， ASC， Caspase-1， and TXNIP （P<0.01）. Compared with the model group， the interventions restored sucrose preference （P<0.01）， shortened immobility time （P<0.01）， repaired hippocampal neuronal structure， reduced apoptosis， lowered the levels of inflammatory cytokines （P<0.01）， restored the levels of neurotransmitters （P<0.01）， alleviated ROS accumulation （P<0.01）， downregulated the mRNA levels of NLRP3， ASC， Caspase-1， IL-1β， and iNOS （P<0.01）， upregulated the protein level of UCP2 （P<0.01）， and reduced the protein levels of NLRP3， ASC， Caspase-1， and TXNIP （P<0.01）. Moreover， TFCC outperformed Cuscutae Semen decoction in ameliorating depressive behaviors. TFCC excelled in neuronal repair， neurotransmitter regulation， anti-inflammatory effects， and modulation of the UCP2/TXNIP/NLRP3 pathway （P<0.05）. ConclusionTFCC modulates the hippocampal UCP2/TXNIP/NLRP3 pathway to inhibit inflammasome activation， reduce oxidative stress， restore neurotransmitters， thus suppressing neuronal apoptosis and promoting the rearrangement and morphology recovery of hippocampal cells. It outperforms Cuscutae Semen decoction in the antidepressant efficacy.

ObjectiveTo investigate the antidepressant effects and mechanisms of total flavonoids from Cuscutae Semen （TFCC） in the mouse model of chronic unpredictable mild stress （CUMS）. MethodsFifty male 4-week-old ICR mice were randomized into five groups （n=10 per group）： blank control， model， Cuscutae Semen decoction （10.2 g·kg^-1·d^-1）， paroxetine （2.6 mg·kg^-1·d^-1）， and TFCC （173.2 mg·kg^-1·d^-1）. The other groups except the blank control group underwent chronic unpredictable mild stress （CUMS） for 4 weeks. Behavioral assessments were conducted post-modeling. Then， the model group received distilled water （10 mL·kg^-1·d^-1）， while treatment groups were administrated with respective agents via oral gavage （10 mL·kg^-1） for 4 weeks. Depression-like behaviors were evaluated by the sucrose preference test （SPT）， forced swimming test （FST）， and tail suspension test （TST）. Hippocampal neuronal morphology was observed via hematoxylin-eosin staining， and apoptosis in the brain tissue was assessed via terminal- deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL）. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the hippocampal levels of inflammatory cytokines ［interleukin （IL）-1β， IL-6， and TNF-α）］ and neurotransmitters ［5-hydroxytryptamine （5-HT）， dopamine （DA）， and brain-derived neurotrophic factor （BDNF）］， while the reactive oxygen species （ROS） levels were quantified via the DCFH-DA probe. Real-time PCR was performed to measure the mRNA levels of NOD-like receptor protein 3 （NLRP3）， apoptosis-associated Speck-like protein containing a CARD （ASC）， cysteinyl aspartate-specific proteinase-1 （Caspase-1）， IL-1β， and inducible nitric oxide synthase （iNOS）. Western blot was employed to evaluate the protein levels of NLRP3， ASC， Caspase-1， uncoupling protein 2 （UCP2）， and thioredoxin-interacting protein （TXNIP）. ResultsCompared with the blank control group， the model group exhibited weight loss （P<0.01）， reduced sucrose preference （P<0.01）， prolonged immobility time in FST and TST （P<0.01）， neuron disarrangement with nuclear pyknosis in hippocampal CA3 region， increased apoptosis in the brain tissue， elevated levels of IL-1β， IL-6， and TNF-α （P<0.01）， declined levels of 5-HT， DA， and BDNF （P<0.01）， increased ROS accumulation （P<0.01）， upregulated mRNA levels of NLRP3， ASC， Caspase-1， IL-1β， and iNOS （P<0.01）， down-regulated protein level of UCP2 （P<0.01）， and up-regulated protein levels of NLRP3， ASC， Caspase-1， and TXNIP （P<0.01）. Compared with the model group， the interventions restored sucrose preference （P<0.01）， shortened immobility time （P<0.01）， repaired hippocampal neuronal structure， reduced apoptosis， lowered the levels of inflammatory cytokines （P<0.01）， restored the levels of neurotransmitters （P<0.01）， alleviated ROS accumulation （P<0.01）， downregulated the mRNA levels of NLRP3， ASC， Caspase-1， IL-1β， and iNOS （P<0.01）， upregulated the protein level of UCP2 （P<0.01）， and reduced the protein levels of NLRP3， ASC， Caspase-1， and TXNIP （P<0.01）. Moreover， TFCC outperformed Cuscutae Semen decoction in ameliorating depressive behaviors. TFCC excelled in neuronal repair， neurotransmitter regulation， anti-inflammatory effects， and modulation of the UCP2/TXNIP/NLRP3 pathway （P<0.05）. ConclusionTFCC modulates the hippocampal UCP2/TXNIP/NLRP3 pathway to inhibit inflammasome activation， reduce oxidative stress， restore neurotransmitters， thus suppressing neuronal apoptosis and promoting the rearrangement and morphology recovery of hippocampal cells. It outperforms Cuscutae Semen decoction in the antidepressant efficacy.

Signal detection is a critical task in drug safety regulation. However, it inevitably generates irrelevant or false signals, posing challenges for resource allocation by marketing authorization holders. To reasonably assess these signals, different countries have established various principles for prioritizing the evaluation of risk signals. This study systematically compares these principles and finds that the U.S. Food and Drug Administration(FDA) focuses on practical issues, such as identifying drug confusion or drug interactions. However, China's Good Pharmacovigilance Practices and the European Medicines Agency(EMA) emphasize a comprehensive evaluation framework. The Council for International Organizations of Medical Sciences(CIOMS) emphasizes the consistency of multiple data sources, highlighting the reliability of signal evaluation. China practices a multidisciplinary approach combining traditional Chinese and western medicine, and the risk signals related to traditional Chinese medicine(TCM) have unique characteristics, including complex components, cumulative toxicity, specific theoretical foundations, and drug interactions. The different priorities in risk signal evaluation principles across countries suggest that China should strengthen clinical trial research, emphasize corroboration with evidence of multiple sources, and pay particular attention to the risks of drug interactions in the TCM regulatory science. Establishing the risk signal prioritization principles that align with the characteristics of TCM enables more precise and efficient scientific regulation of TCM.
Humans ; Medicine, Chinese Traditional/standards* ; China ; Drugs, Chinese Herbal/adverse effects* ; United States ; United States Food and Drug Administration

This study investigated the functions and regulatory mechanism of Portulacae Herba and its chemical components on the Toll-like receptor 4(TLR4)/myeloid differentiation primary response 88(MyD88)/nuclear factor kappa B(NF-κB) inflammatory signaling pathway in the colon tissue of mice with dextran sodium sulfate(DSS)-induced ulcerative colitis(UC). A total of 35 mice were randomly divided into groups, including a blank group, a model group, a mesalazine group(0. 5 g·kg~(-1)), and low, medium,and high dose alkaloids from Portulacae Herba groups(9, 18, 36 mg·kg~(-1)), and a combination treatment group, with 5 mice in each group. The blank group was given purified water, while the other groups were continuously given a 3% DSS solution for 7 days to induce the UC model. From day 8 onwards, the treatment group received oral gavage according to the prescribed doses for 14 days. The overall condition, body weight, stool characteristics, and presence of blood in the stool were recorded daily. After the experiment, the disease activity index(DAI) was assessed for each group, and colon length was measured. Histopathological changes in colon tissue were examined using hematoxylin-eosin(HE) staining. The levels of pro-inflammatory cytokines, tumor necrosis factor-α(TNF-α),and interleukin-1β( IL-1β) in serum were measured by enzyme-linked immunosorbent assay( ELISA). The protein and m RNA expression of TLR4, MyD88, and NF-κB in colon tissue were measured using Western blot and quantitative real-time PCR(qPCR).Compared to the blank group, the model group showed a significant decrease in body weight, a notable increase in DAI scores, a significant shortening of colon length, and evident histopathological damage. The levels of inflammatory cytokines TNF-α and IL-1β in the serum were significantly elevated, and the protein and m RNA expression of TLR4, MyD88, and NF-κB in colon tissue were significantly up-regulated. In contrast, the alkaloids from Portulacae Herba treatment groups significantly improved symptoms and reduced body weight loss in mice, decreased DAI scores, alleviated colon shortening, lowered serum levels of TNF-α and IL-1β,significantly down-regulated the expression levels of TLR4, MyD88, and NF-κB proteins and genes in colon tissue, as well as reduced histopathological damage. Therefore, the study suggests that alkaloids from Portulacae Herba can alleviate intestinal inflammation damage in DSS-induced UC mice, with its mechanism involving the TLR4/MyD88/NF-κB signaling pathway.
Animals ; Colitis, Ulcerative/immunology* ; Toll-Like Receptor 4/immunology* ; Myeloid Differentiation Factor 88/metabolism* ; Mice ; NF-kappa B/metabolism* ; Signal Transduction/drug effects* ; Male ; Alkaloids/administration & dosage* ; Drugs, Chinese Herbal/administration & dosage* ; Humans ; Female ; Colon/metabolism* ; Disease Models, Animal

Diterpenoid ferruginol is a key intermediate in biosynthesis of active ingredients such as tanshinone and carnosic acid.However, the traditional process of obtaining ferruginol from plants is often cumbersome and inefficient. In recent years, the increasingly developing gene editing technology has been gradually applied to the heterologous production of natural products, but the production of ferruginol in microbe is still very low, which has become an obstacle to the efficient biosynthesis of downstream chemicals, such as tanshinone. In this study, miltiradiene was produced by integrating the shortened diterpene synthase fusion protein,and the key genes in the MVA pathway were overexpressed to improve the yield of miltiradiene. Under the shake flask fermentation condition, the yield of miltiradiene reached about(113. 12±17. 4)mg·L~(-1). Subsequently, this study integrated the ferruginol synthase Sm CYP76AH1 and Sm CPR1 to reconstruct the ferruginol pathway and thereby realized the heterologous synthesis of ferruginol in Saccharomyces cerevisiae. The study selected the best ferruginol synthase(Il CYP76AH46) from different plants and optimized the expression of pathway genes through redox partner engineering to increase the yield of ferruginol. By increasing the copy number of diterpene synthase, CYP450, and CPR, the yield of ferruginol reached(370. 39± 21. 65) mg·L~(-1) in the shake flask, which was increased by 21. 57-fold compared with that when the initial ferruginol strain JMLT05 was used. Finally, 1 083. 51 mg·L~(-1) ferruginol was obtained by fed-batch fermentation, which is the highest yield of ferruginol from biosynthesis so far. This study provides not only research ideas for other metabolic engineering but also a platform for the construction of cell factories for downstream products.
Saccharomyces cerevisiae/genetics* ; Diterpenes/metabolism* ; Metabolic Engineering ; Fermentation ; Abietanes