Frontiers in Bioengineering and Biotechnology
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2021
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IF 5.89
Fu Xueqing,Zhang Fangyuan,Ma Yanan,Hassani Danial,Peng Bowen,Pan Qifang,Zhang Yuhua,Deng Zhongxiang,Liu Wenbo,Zhang Jixiu,Han Lei,Chen Dongfang,Zhao Jingya,Li Ling,Sun Xiaofen,Tang Kexuan
Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 μg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 μg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.
研究领域:
植物学
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合成生物学
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药用植物遗传工程
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高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2023
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IF 6.1
Guangjuan Luo,Ying Lin,Shuting Chen,Ruiming Xiao,Jiaxin Zhang,Cheng Li,Anthony J. Sinskey,Lei Ye,Shuli Liang
Patchoulol, a plant-derived sesquiterpene compound, is widely used in perfumes, cosmetics, and pharmaceuticals. Microbial production provides a promising alternative approach for the efficient and sustainable production of patchoulol. However, there are no systematic engineering studies on Komagataella phaffii aimed at achieving high-yield patchoulol production. Herein, by fusion overexpression of FPP synthase and patchoulol synthase (ERG20LPTS), increasing the precursor supply, adjusting the copy number of ERG20LPTS and PTS, and combined with adding auxiliary carbon source and methanol concentration optimization, we constructed a high-yield patchoulol-producing strain P6H53, which produced 149.64 mg/L patchoulol in shake-flask fermentation with methanol as the substrate. In fed-batch fermentation, strain P6H53 achieved the highest production (2.47 g/L, 21.48 mg/g DCW, and 283.25 mg/L/d) to date in a 5 L fermenter. This study will lay a good foundation for the development of K. phaffii as a promising chassis microbial cell for the synthesis of patchoulol and other sesquiterpenes with methanol as the carbon source.
研究领域:
合成生物学
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微生物学
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生物工程
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PubMed
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ACS Synthetic Biology
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2021
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IF 5.11
Min Liu,Yang-Chen Lin,Jiao-Jiao Guo,Meng-Meng Du,Xinyi Tao,Bei Gao,Ming Zhao,Yushu Ma,Feng-Qing Wang,Dong-Zhi Wei
Patchoulol is a tricyclic sesquiterpene widely used in perfumes and cosmetics. Herein, comprehensive engineering strategies were employed to construct an efficient yeast strain for patchoulol production. First, a platform strain was constructed via pathway modification. Second, three off-pathway genes were deleted, which led to significant physiological changes in yeast. Further, strengthening of the ergosterol pathway, enhancement of the energy supply, and a decrease in intracellular reactive oxygen species were implemented to improve the physiological status of yeast, demonstrating a new promotive relationship between ergosterol biosynthesis and synthesis of patchoulol. Moreover, patchoulol synthase was improved through protein modification and Mg2+ addition, reaching a final titer of 141.5 mg/L in a shake flask. Finally, a two-stage fermentation with dodecane addition was employed to achieve the highest production (1632.0 mg/L, 87.0 mg/g dry cell weight, 233.1 mg/L/d) ever reported for patchoulol in a 5 L bioreactor. This work lays a foundation for green and efficient patchoulol production.
研究领域:
生物技术
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代谢工程
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2019
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IF 3.571
Bin Ma,Min Liu,Zhen-Hai Li,Xinyi Tao,Dong-Zhi Wei,Feng-Qing Wang
Patchoulol, a natural sesquiterpene compound, is widely used in perfumes and cosmetics. Several strategies were adopted to enhance patchoulol production in Saccharomyces cerevisiae: (i) farnesyl pyrophosphate (FPP) synthase and patchoulol synthase were fused to increase the utilization of FPP precursor; (ii) expression of the limiting genes of the mevalonate pathway was enhanced; (iii) squalene synthase was weakened by a glucose-inducible promoter of HXT1 (promoter for hexose transporter) to reduce metabolic flux from FPP to ergosterol; and (iv) farnesol biosynthesis was inhibited to decrease the consumption of FPP. Glucose was used to balance the trade-off between the competitive squalene and patchoulol pathways. The patchoulol production was 59.2 ± 0.7 mg/L in a shaken flask with a final production of 466.8 ± 12.3 mg/L (20.5 ± 0.5 mg/g dry cell weight) combined with fermentation optimization, which was 7.8-fold higher than the reported maximum production. The work significantly promoted the industrialization process of patchoulol production using biobased microbial platforms.
研究领域:
生物技术
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代谢工程
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生物过程工程
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Fermentation-Basel
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2024
·Vol. 10
·期 4
·211
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IF 3.3
Qiu Tao,Guocheng Du,Jian Chen,Juan Zhang,Zheng Peng
Patchoulol is a natural sesquiterpene alcohol with extensive applications in cosmetics and pharmaceuticals. In this study, we first constructed the synthesis pathway of patchoulol inSaccharomyces cerevisiaeby expressing the patchoulol synthasePTSgene using the strong promoterGAL1. Afterward, the metabolic flux of the precursor was enhanced by strengthening the mevalonate pathway and balancing the precursor competition pathway, resulting in a 32.74-fold increase in patchoulol production. Subsequently, the supply of acetyl-CoA in yeast was increased by modifying transcriptional regulators and modulating the acetyl-CoA pathway, and the titer of patchoulol reached 155.94 mg/L. Finally, optimization of the fermentation conditions resulted in a titer of 195.96 mg/L in the shake flasks. Further, batch-fed fermentation in a 5 L bioreactor yielded 1.95 g/L. This work accelerated the development of a microbial cell factory for the production of patchoulol.
研究领域:
生物化学
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代谢工程
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合成生物学
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BIOCHEMICAL ENGINEERING JOURNAL
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2024
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IF 3.7
Guangjuan Luo,Ying Lin,Shupeng Ruan,Ruiming Xiao,Xinying Zhang,Shuli Liang
ABSTRACT Mitochondria are a potential sub-organelle for the synthesis of terpenoids due to its abundant acetyl-CoA, cofactors, and compact compartment for targeting metabolic pathways and enzymes. Hence, conducting systematic investigations and harnessing the subcellular compartment of mitochondria may offer a viable avenue for enhancing terpenoids biosynthesis in yeast and other eukaryotic cells. Previous endeavors to boost terpenoids production in Komagataella phaffii mainly focused on diverse metabolic engineering strategies in the cytoplasm. Numerous studies have demonstrated that reconstructing specific metabolic pathways within mitochondria can lead to higher production yields than engineering the same pathways in the cytoplasm. In our study, we initially identified 6 well-performed mitochondrial targeting sequences (MTSs) through colocalization observation of EGFP and a mitochondrial red fluorescent probe via fluorescence microscopy. These MTSs were selected for the assembly of complete patchoulol biosynthesis pathways in the mitochondria, which resulted in a 1.7-fold increase in patchoulol production compared to the original strain. Furthermore, dual regulation of the cytoplasm and mitochondria was implemented in the P6H4＋PM2 strain to further enhance patchoulol synthesis to 109 mg/L, representing a 1.0-fold and 10.7-fold greater yield than that of the strains engineered solely in the cytoplasm or mitochondria, respectively. This study offers valuable guidance for effectively utilizing mitochondria to achieve higher patchoulol and other sesquiterpenes through dual cytoplasmic-mitochondrial engineering in K. phaffii .
研究领域:
生物化学
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合成生物学
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Synthetic and Systems Biotechnology
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2026
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IF 4.4
Xiaochun Zheng,Yajun Li,Zhenhua Liu,Peng Sun,Yaqi Dong,Xue Wang,Yanan Wang,Xiaobing Yang
Patchoulol is a widely used sesquiterpenoid in perfumes, cosmetics, foods and pharmaceuticals. The plant-dependent production is suffering from limited growing area, long seasonal cycle, etc. Microbial production represents a sustainable alternative as it is featured with mild operating conditions and eco-friendliness. Herein, we engineered the oleaginous Rhodotorula toruloides toward patchoulol production. First, the patchoulol biosynthesis baseline was constructed by employing a chimeric enzyme of the Pogostemon cablin originated patchoulol synthase and the native FPPS. Second, the supply of essential intermediates was streamlined by redeploying the mevalonate (MVA) pathway while the recycling of NADPH was enhanced through over-expressing related enzymes. Third, the patchoulol production was further enhanced to 724.8 mg/L, 6.0 mg/L/h and 36.2 mg/g glucose by down-regulating the squalene biosynthesis and tuning the cultivation condition in shake flask. Finally, the production of patchoulol was increased to 1.31 g/L and 13.8 mg/g glucose in the minimal medium in a 3-L bioreactor. Our study demonstrated the potential of R. toruloides in producing patchoulol, and should shed light on the microbial synthesis of other sesquiterpenes.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2023
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IF 6.1
Qian-Qian Peng,Qi Guo,Cheng Chen,Ping Song,Yue-Tong Wang,Xiao-Jun Ji,Chao Ye,Tian-Qiong Shi
Patchoulol is an important sesquiterpene alcohol with a strong and lasting odor, which has led to prominent applications in perfumes and cosmetics. In this study, systematic metabolic engineering strategies were adopted to create an efficient yeast cell factory for patchoulol overproduction. First, a baseline strain was constructed by selecting a highly active patchoulol synthase. Subsequently, the mevalonate precursor pool was expanded to boost patchoulol synthesis. Moreover, a method for downregulating squalene synthesis based on Cu2+-repressible promoter was optimized, which significantly improved the patchoulol titer by 100.9% to 124 mg/L. In addition, a protein fusion strategy resulted in a final titer of 235 mg/L in shake flasks. Finally, 2.864 g/L patchoulol could be produced in a 5 L bioreactor, representing a remarkable 1684-fold increase compared to the baseline strain. To our knowledge, this is the highest patchoulol titer reported so far.
研究领域:
生物技术
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代谢工程
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合成生物学
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PubMed
高影响因子
Synthetic and Systems Biotechnology
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2026
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IF 4.8
Danni Fu,Yongyi Zhou,Ziying Wu,Shihan Wang,Yongmin Lao,Hong-Bin Wang,Hong-Lei Jin
Patchoulol is a valuable sesquiterpene for fragrance and cosmetics, but plant extraction is slow and resource-intensive. Here, we combined enzyme, pathway and transporter engineering to build a yeast chassis for high-titer patchoulol production. Semi-rational engineering of patchoulol synthase (PTS) increased catalytic efficiency; together with multi-copy PTS expression, the shake-flask titer rose from 0.40 ± 0.05 mg/L (PT01) to 258.72 ± 27.35 mg/L. Strengthening glycolysis and the mevalonate pathway and down-regulating ERG9 redirected flux to FPP. Guided by docking and transcript analysis, we identified PDR12 as an effective efflux pump; inducible overexpression increased the titer to 372.80 ± 14.92 mg/L. In a 5-L two-stage fed-batch with a dodecane overlay, the best strain reached 2.86 g/L (2856.25 mg/L). This work provides a general framework to couple metabolic with transporter engineering for hydrophobic terpenoids.
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BIORESOURCE TECHNOLOGY
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2026
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IF 8.2
Li Li,Yasen Wang,Buqing Wang,Liqun Shen,Yahui Gao,Wei Lin,Zijie Li
Numerous organisms have evolved the ability to utilize light through photoreceptor proteins that mediate diverse biological processes. Currently, several optogenetic sensor systems are widely used in yeast. However, when these systems are applied for gene repression to regulate endogenous yeast gene expression, they typically require the insertion of corresponding target sites near the native promoter of the gene of interest to achieve precise modulation. To address these constraints, a novel blue light-inducible optogenetic tool designated iLight9 was developed, a single-component optogenetic biosensor integrated with the CRISPR-dCas9 platform. The stability of the iLight9 system was further enhanced by employing a strategy involving the addition of a protein degradation tag. The resulting system was designated as iLight9O, which facilitated programmable regulation of distinct genes through the introduction of specific sgRNAs. Subsequently, systematic metabolic engineering strategies were employed to construct an efficient patchoulol-producing cell factory in Saccharomyces cerevisiae . Moreover, a two-step isoprenol utilization (IU) pathway was introduced into the recombinant strain to enhance its capacity for patchoulol biosynthesis. Crucially, the iLight9O system was adopted to dynamically downregulate squalene synthase, a key enzyme in the competing squalene biosynthetic pathway. This optogenetic flux control strategy increased patchoulol titers by 66 % in the IU-optimized strain and 24 % in the MVAIU2 strain, demonstrating significant improvements over static engineering approaches.
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